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https://openalex.org/W3133636318	https://munin.uit.no/bitstream/10037/21727/2/article.pdf	English	null	Maximal Strength, Sprint, and Jump Performance in High-Level Female Football Players Are Maintained With a Customized Training Program During the COVID-19 Lockdown	Frontiers in physiology	2,021	cc-by	6,867	ORIGINAL RESEARCH published: 26 February 2021 doi: 10.3389/fphys.2021.623885 Keywords: resistance training, sprint, soccer, counter movement jump, squat, COVID-19 Sigurd Pedersen1, Dag Johansen2, Andrea Casolo3,4, Morten B. Randers1,5, Edvard H. Sagelv1, Boye Welde1, Andreas Kjæreng Winther1 and Svein Arne Pettersen1 1 School of Sport Sciences, Faculty of Health Sciences, UiT The Arctic University of Norway, Tromsø, Norway, 2 Department of Computer Science, Faculty of Natural Sciences and Technology, UiT The Arctic University of Norway, Tromsø, Norway, 3 Department of Bioengineering, Imperial College London, London, United Kingdom, 4 Department of Biomedical Sciences, University of Padova, Padua, Italy, 5 Department of Sport Sciences and Clinical Biomechanics, Faculty of Health Sciences, University of Southern Denmark, Odense, Denmark Edited by: Edited by: Solfrid Bratland-Sanda, University of South-Eastern Norway, Norway Introduction: The COVID-19 outbreak with partial lockdown has inevitably led to an alteration in training routines for football players worldwide. Thus, coaches had to face with the novel challenge of minimizing the potential decline in ﬁtness during this period of training disruption. Reviewed by: Stephen Seiler, University of Agder, Norway Souhail Hermassi, Qatar University, Qatar Methods: In this observational pre- to posttest study involving Norwegian female football players (18.8 ± 1.9 years, height 1.68 ± 0.4 m, mass 61.3 ± 3.7 kg), we investigated the effects of a prescribed home-based and group-based intervention, implemented during the COVID-19 lockdown, on maximal muscular force production and high velocity variables. Speciﬁcally, maximal partial squat strength one repetition maximum (1RM), counter movement jump (CMJ) and 15 m sprint time were assessed 1 week prior to the lockdown and 12 weeks after the onset of lockdown. We also collected training content and volume from the prescribed training program and self-reported perceived training quality and motivation toward training. Correspondence: Sigurd Pedersen Sigurd.pedersen@uit.no Specialty section: This article was submitted to Exercise Physiology, a section of the journal Frontiers in Physiology Specialty section: This article was submitted to Exercise Physiology, a section of the journal Frontiers in Physiology Received: 30 October 2020 Accepted: 09 February 2021 Published: 26 February 2021 Received: 30 October 2020 Accepted: 09 February 2021 Published: 26 February 2021 Results: We observed no change in 1RM [pretest: 104 ± 12 kg, posttest: 101 ± 11 kg (P = 0.28)], CMJ height [pretest: 28.1 ± 2.3 cm, posttest: 26.8 ± 1.9 (P = 0.09)], and 15 m sprint time [pretest: 2.60 ± 0.08 s, posttest: 2.61 ± 0.07 s (P = 0.52)]. Maximal Strength, Sprint, and Jump Performance in High-Level Female Football Players Are Maintained With a Customized Training Program During the COVID-19 Lockdown Sigurd Pedersen1*, Dag Johansen2, Andrea Casolo3,4, Morten B. Randers1,5, Edvard H. Sagelv1, Boye Welde1, Andreas Kjæreng Winther1 and Svein Arne Pettersen1 Design, Subjects, Procedure, and Questionnaires Football ﬁtness includes both aerobic and anaerobic-capacity, and explosive muscle actions (Bangsbo et al., 2006). For example, dynamic muscle strength such as partial squat one repetition maximum (1RM) is suggested to reﬂect functional strength of football players (Wisløﬀet al., 1998), which is associated with muscular power (Stølen et al., 2005) and the ability to perform football speciﬁc actions (Wing et al., 2020). Mimicking the movement patterns in football during training was challenged during the lockdown period, which may have led to declines in football-speciﬁc physical ﬁtness (Mohr et al., 2020). Our study is a longitudinal 12-week observational study with a pretest posttest design. Two female football teams playing at level three in Norway were originally invited to another study, whose main aim was to investigate the association between high-force/power tests and physical performance derived from tracking data during football match play. Since the COVID-19 lockdown was imposed by national authorities 1 week following pretest start, and 1 day prior to pretest the second team (n = 13) (Figure 1), the team that completed the lab tests (n = 13) involved in that study were invited to participate in a new data collection (posttest) 12 weeks after the ﬁrst visit to the laboratory. Four players had left Tromsø for personal reasons during the lockdown period, thus nine players attended the follow-up measurements and were eligible for this study (Table 1). The lockdown was introduced 4 weeks prior to the planned start of the competitive season for the team. Data for eight players is reported for the force data acquisition in the CMJ test and eight players for 1RM, due to incomplete force data recording in the CMJ for one player at pretest and a hand injury at posttest for another player. Eight players are included in the dexa-scan data. All the other variables are reported for pre- and posttest for all nine players. The training plan information was retrieved from the team’s coaches (Table 2). One of the main intentions of the pre-season period in football is to optimize physical performance including jumping and sprinting ability, and maximal strength (Rønnestad et al., 2011). However, how these abilities are aﬀected when the pre- season is unexpectedly interrupted, is mostly unknown. INTRODUCTION lockdown (home-based, group based, and without normal football play), could preserve 1RM partial squat strength, counter movement jump (CMJ) and 15 m sprint time. To the authors’ knowledge, the eﬀect of COVID-19 related training adjustments on strength and strength derivatives is only available in male football players (Cohen et al., 2020; Moreno-Pérez et al., 2020). Thus, the aim of our study was to assess the eﬀects of a prescribed unsupervised 12-week home- and group- based training program without gym facilities on 1RM partial squat strength, CMJ and 15 m sprint time in female high-level football players during a period without full contact football training. In 2019 and 2020, the COVID-19 outbreak resulted in country lockdowns where both the general population and athletes were exposed to unexpected behavioral restrictions (e.g., social distancing, closure and/or limitation of non-essential activities such as gyms and training grounds, and ultimately, self- isolation). Strict quarantine rules were introduced following national/international travel, after direct exposure to the virus or if showing COVID-19 symptoms. Consequently, athletes were enforced to cancel and/or postpone their competitions and to abruptly adjust their training routines (Sarto et al., 2020). In Norway, these regulations were imposed for all sports and were introduced during football teams’ pre-season preparations. This led to a rapid shift in training plans and training practice with for example, some teams prescribing home-based training for their players (Sarto et al., 2020). Citation: Pedersen S, Johansen D, Casolo A, Randers MB, Sagelv EH, Welde B, Winther AK and Pettersen SA (2021) Maximal Strength, Sprint, and Jump Performance in High-Level Female Football Players Are Maintained With a Customized Training Program During the COVID-19 Lockdown. Front. Physiol. 12:623885. doi: 10.3389/fphys.2021.623885 Conclusion: Our ﬁndings suggest that a prescribed home-based and group-based intervention with increased training time devoted to strength, jump, and sprint ability, and regulated to obtain a sufﬁcient infection control level is feasible and effective to preserve strength, jumping, and sprinting abilities of high-level female football players during a ∼3-month period of a pandemic-induced lockdown. February 2021 \| Volume 12 \| Article 623885 1 Frontiers in Physiology \| www.frontiersin.org Female Football During COVID-19 Pedersen et al. Frontiers in Physiology \| www.frontiersin.org Design, Subjects, Procedure, and Questionnaires Several researchers have suggested negative eﬀects of self-isolation following the COVID-19 lockdown (Mohr et al., 2020; Sarto et al., 2020), suggesting that it may result in lower training volume and quality, and in turn, decreased physical ﬁtness (Girardi et al., 2020; Sarto et al., 2020). Indeed, a number of studies have reported reduced physical activity (Xiang et al., 2020; Zheng et al., 2020) and training hours during the COVID- 19 lockdown (Mon-López et al., 2020; Zinner et al., 2020), and there are already ﬁndings of decreased cycling performance in cyclists (Muriel et al., 2020) and reduced hamstring strength in football players (Moreno-Pérez et al., 2020). However, some have presented the potential of maintaining physical ﬁtness in multidisciplinary sports such as football, by performing circuit- based training (Latella and Haﬀ, 2020). This was recently shown in a male football team, where jump height was preserved following 15 weeks of isolated training (Cohen et al., 2020). According to the Helsinki declaration, all participants were informed of the potential beneﬁts, risks, and procedures of the study, both orally and in writing, before signing an informed consent. The original study and the data storage protocol was approved by the Norwegian Center for Research Data (NSD) (approval reference number: 989024), and approved our changed aim and a new data collection period in June (correction reference number: 768380), without any further ethical approval per institutional and national guidelines for research on sport and exercise science. Longer periods without strength training (12 weeks) may lead to reduced strength of 7–12% in strength trained individuals (Mujika and Padilla, 2000). Importantly, small quantities of training can attenuate the strength loss following complete training cessation in high levels athletes (García-Pallarés et al., 2010). Our planned data collection involving female football players was abruptly interrupted by the pandemic. Thus, we had the opportunity to investigate whether a change in prescribed training designed to limit COVID-19 infection during At pretest, the players answered one custom-made question about their experience with strength training in the squat exercise with answering option 1, No experience; 2, Some experience (<1 year); and 3, A lot of experience (>1 year). Two months after posttest in June 2020, the players received an individual custom- made questionnaire about their pre-season training habits before February 2021 \| Volume 12 \| Article 623885 2 Female Football During COVID-19 Pedersen et al. Design, Subjects, Procedure, and Questionnaires TABLE 1 \| Baseline participant characteristics (mean ± SD). where the ﬁnal version was developed with consensus from all four researchers. Prior to pretest, all the participants underwent 3–4 supervised familiarization sessions to the partial squat exercise over 2 weeks, in order to perform the movement safely and technically sound (Ploutz-Snyder and Giamis, 2001). All tests and familiarization sessions started with the same standardized warm-up routine consisting of 5 min of self-selected low intensity cycling on an ergometer bike (Pro/Trainer, Wattbike Ltd., Nottingham, United Kingdom) and followed by 5 min low intensity running of self-selected speed in the gym. The participants were recommended to not perform strenuous physical exercise 24 h prior to testing. Pretests of physical ﬁtness (e.g., 1RM, CMJ, and 15 m sprint) were conducted in the laboratory between 17:00 and 19:00 on two separate occasions, separated by 3 days. During the ﬁrst day, the sprint tests were carried out ﬁrst followed by the CMJ test; thereafter half of the players underwent the dexa-scan. On the second day, the players underwent a 1RM test, followed by a dexa-scan for the second half of the players. TABLE 2 \| Changes in prescribed training from pre- to posttest, before and during lockdown. TABLE 2 \| Changes in prescribed training from pre- to posttest, before and during lockdown. During normal pre-season training before lockdown During lockdown %-change Total training (min) 291 ± 77 233 ± 47 −20 Football training (min) 207 ± 55 95 ± 106 −54 Strength training (min) 28 ± 27 40 ± 39 43 Speed and jump training (min) 6 ± 13 26 ± 19 333 Endurance training, running (min) 0 ± 0 45 ± 39 NA Individual training (sessions) 0.3 ± 0.5 2.0 ± 2.0 567 Group/team training (sessions) 3.6 ± 0.7 1.8 ± 1.7 −50 Weekly training prescribed by the coaches to their players during the spring 2020. The data is presented as average minutes ± SD. Design, Subjects, Procedure, and Questionnaires FIGURE 1 \| Timeline of COVID-19 regulations affecting the team in the current study. The chart displays both the governmental and football associations’ regulations (upper side of the line) and the teams’ prescribed training to these regulations (lower side of the line). Pretest was carried out prior to the ﬁrst date in the scheme, while posttest was performed immediately after the last date. FIGURE 1 \| Timeline of COVID-19 regulations affecting the team in the current study. The chart displays both the governmental and football associations’ regulations (upper side of the line) and the teams’ prescribed training to these regulations (lower side of the line). Pretest was carried out prior to the ﬁrst date in the scheme, while posttest was performed immediately after the last date. TABLE 1 \| Baseline participant characteristics (mean ± SD). Age (year) 18.8 ± 1.9 Body mass (kg) 61.3 ± 3.7 Height (m) 1.68 ± 0.4 Lean mass (kg) 44.5 ± 2.0 Bone mass (kg) 2.66 ± 2.0 Body fat (%) 24.88 ± 0.04 Fat mass (kg) 14.9 ± 3.5 Leg lean mass (kg) 15.4 ± 8.6 Body composition is measured by a dual-energy X-ray absorptiometry machine (dexa-scan) (Lunar Prodigy; GE Medical Systems, Buckinghamshire, United Kingdom). TABLE 2 \| Changes in prescribed training from pre- to posttest, before and during lockdown. During normal pre-season training before lockdown During lockdown %-change Total training (min) 291 ± 77 233 ± 47 −20 Football training (min) 207 ± 55 95 ± 106 −54 Strength training (min) 28 ± 27 40 ± 39 43 Speed and jump training (min) 6 ± 13 26 ± 19 333 Endurance training, running (min) 0 ± 0 45 ± 39 NA Individual training (sessions) 0.3 ± 0.5 2.0 ± 2.0 567 Group/team training (sessions) 3.6 ± 0.7 1.8 ± 1.7 −50 Weekly training prescribed by the coaches to their players during the spring 2020. The data is presented as average minutes ± SD. TABLE 1 \| Baseline participant characteristics (mean ± SD). Age (year) 18.8 ± 1.9 Body mass (kg) 61.3 ± 3.7 Height (m) 1.68 ± 0.4 Lean mass (kg) 44.5 ± 2.0 Bone mass (kg) 2.66 ± 2.0 Body fat (%) 24.88 ± 0.04 Fat mass (kg) 14.9 ± 3.5 Leg lean mass (kg) 15.4 ± 8.6 Body composition is measured by a dual-energy X-ray absorptiometry machine (dexa-scan) (Lunar Prodigy; GE Medical Systems, Buckinghamshire, United Kingdom). DISCUSSION Our study describes the eﬀects on partial squat 1RM, CMJ, and sprint performance in female football players during a period of a global pandemic, which resulted in comprehensive adjustments of players’ training routines. Our main ﬁndings were that maximal strength, sprint time and jump performance did not decrease during lockdown. Counter Movement Jump Standing on the force platform (Hur-Labs, ALU4, Finland), the participants were instructed to perform a CMJ with the aim to jump as high as possible, with the hands ﬁxed on the hips during the entire movement. Force data were recorded with bespoke software (Force platform software suite, HURlabs oy, Kokkola, Finland). The software calculates jump height as the center of mass displacement calculated from the force developed and measured body mass. Each player performed two trials with a minimum 3 min rest between the two trials. The highest jump was taken for analysis. Change in perceived adherence to the prescribed training and perceived level of motivation toward training were both non- signiﬁcant (P > 0.05), while perceived quality of training was signiﬁcantly reduced from pre- to posttest (P < 0.05) (Figure 3). Sprint the direction of the data from pre-post lockdown (Roberson et al., 1995). All data are presented as mean ± standard deviation (SD), or 95% conﬁdence intervals (CI). Alpha level was set to 0.05. The Statistical Package for the Social Sciences (SPSS, Version 26, IBM, Armonk, NY, United States) was used for the statistical analyses. p Prior to the sprint test, in addition to the general standardized warm up, the participants performed three 15 m strides at their subjective eﬀort of 85–90% of maximal acceleration speed. Thereafter, the participants performed three, 15 m sprints with 3 min rest between each attempt. The sprint times were measured by single-beam electronic photocells (ATU-X, IC control AB, Stockholm, Sweden) mounted to the ﬂoor and walls. The starting photocell was placed 20 cm above the ground, while the 5, 10, and 15 m photocells were placed 100 cm above the ground. Self- initiated, the players started the sprint in a static position placing their front foot 30 cm behind the starting line. The fastest 5, 10, and 15 m split times were included in the analyses. RESULTS Six of the players reported to have some experience with strength training using the squat exercise, while the three remaining players reported to have a lot of experience. The pre- and posttest results are presented in Table 3, with the individual percentage changes illustrated in Figure 2. No signiﬁcant changes were observed for absolute- and relative 1RM partial squat strength (P > 0.05), CMJ jump height (P > 0.05), force production variables in the CMJ test (all P > 0.05), or in 15 m sprint times (P > 0.05, in all cases) from pre- to posttest. Strength The participants underwent a 1RM test in the partial back squat exercise for the assessment of maximal dynamic strength. An Olympic barbell (T-100G; Eleiko, Halmstad, Sweden) was used for both familiarization and the main experimental testing. During the 1RM trials, the participants were instructed to lift the bar from the rack, step one step back, go slow into the descending phase of the movement, followed by a maximal intended velocity during the concentric phase. The participants initiated the concentric phase as response to an orally “go” by the researcher measuring the 90◦knee angle at the knee joint with a goniometer (Pedersen et al., 2019). The same researcher measured knee angle for all the participants during both pre- and posttest. The participants warmed up with 10 repetitions by lifting the bar (20 kg), followed by 10 repetitions of the participants’ perceived ∼50% 1RM. The starting 1RM attempt consisted of one repetition at a high load, which the participants knew they could manage. Each following attempt was completed with 5–10 kg additional load until failure. All players had a minimum of 3 min rest between each lift. The highest load lifted by the participants was deﬁned as their 1RM. and during the lockdown. This consisted of six questions where the ﬁrst reﬂected their adherence to the training prescribed by the coaches during the lockdown, in a bipolar ﬁve-unit Likert-scale. The second and third question were about their perceived quality of training and motivation to training, respectively, answered on a bipolar three-unit Likert scale. These three questions were ﬁrst addressed for the pre-season period prior to the lockdown, and subsequently for the period after the lockdown. The questionnaire was designed by two researchers (SP and SAP), and later discussed by additional two researchers (BW and TH) February 2021 \| Volume 12 \| Article 623885 Frontiers in Physiology \| www.frontiersin.org 3 Female Football During COVID-19 Pedersen et al. J, counter movement jump; 1RM, one repetition maximum. P, the P-value from the paired sample t-test from pre- to posttest. Statistical Analyses We rather expected that no access to the team’s- and commercial gyms might have a negative inﬂuence on maximal strength during the COVID-19 period. Recently, one study found that a group of semi-professional male football players reduced hamstring muscle strength following 25 days of home conﬁnement due to the COVID-19 lockdown (Moreno-Pérez et al., 2020). However, these ﬁndings are not directly comparable as the muscles, exercise modality and muscular contraction type (Nordic hamstring, an eccentric contraction exercise) were diﬀerent for the strength test in the study by Moreno-Pérez et al. (2020) than in our study. Eccentric strength is more susceptible to decline compared to other neuromuscular factors (Mujika and Padilla, 2001). This may explain the discrepancy between our study and the study by Moreno-Pérez et al. (2020), as the partial squats in our study emphasize concentric strength. Highly strength trained athletes are more susceptible to decrements in strength following training restriction compared to less trained (Rønnestad et al., 2011). The prescribed training by the team in our study prior to the lockdown consisted of little time devoted to strength training (Table 2). Hence, pretest 1RM was likely not inﬂuenced by systematic maximal strength training in the players in our study. For example, our study’s players’ 1RM was similar to the baseline 1RM partial squat strength in another study of female football players, which increased their 1RM squat strength by 30% following 5 weeks of strength training (Pedersen et al., 2019). However, although the players in our study reported to have some experience with the squat exercise, it may be that their exercise modalities were ineﬀective or non-speciﬁc, thus their maximal partial squat strength was relatively low compared to the levels expected following systematic strength training (Pedersen et al., 2019). FIGURE 2 \| Percentage changes for each individual participant from pre- to posttest for absolute one repetition maximum in the partial squat exercise (1RM) (kg), 15 m sprint time (s) and counter movement jump (CMJ) height (cm). The sprint time for 15 m accompanied by split times at 5 and 10 m did not change during the lockdown. A study by Sporis et al. (2011) showed that starters in oﬃcial matches improved sprint performance more than non-starters over a season. Speciﬁc small-sided games are typically used as training drills during normal football training. Statistical Analyses The Shapiro–Wilk test and visual inspection of Q–Q plots were used to assess normality distribution of data. For 1RM, CMJ, and 15 m sprint times we used Student paired sample t-tests to determine the change from pre- to posttest. For training and questionnaire data, non-parametric tests were used for analyses as these variables were considered non-normally distributed. For the questionnaire data (e.g., training adherence, quality, and motivation), we used a non-parametric signed rank tests to assess g Researchers have speculated that COVID-19 induced restrictions will lead to training cessation and consequently physiological detraining during (Girardi et al., 2020), where football players ﬁrst trained in solitude, followed by a second TABLE 3 \| Changes in selected parameters from pre- to posttest. Pre (mean ± SD) Post (mean ± SD) Change (mean and 95% CI) P-value Partial squat 1RM 1RM (kg) 104 ± 12 101 ± 11 3 (3; 8) 0.28 1RM (kg·mb−1) 1.69 ± 0.24 1.65 ± 0.23 0.03 ( −0.05; 0.12) 0.39 15 m sprint test 5 m (s) 1.03 ± 0.04 1.04 ± 0.05 0.01 ( −0.05; 0.03) 0.52 10 m (s) 1.86 ± 0.05 1.88 ± 0.05 0.01 ( −0.04; 0.01) 0.28 15 m (s) 2.60 ± 0.08 2.61 ± 0.07 0.01 ( −0.04; 0.03) 0.68 CMJ Jump height (cm) 28.1 ± 2.3 26.8 ± 1.9 1.4 ( −0.26; 2.98) 0.09 Takeoff velocity (m s−1) 2.34 ± 0.10 2.29 ± 0.08 0.05 ( −0.03; 0.13) 0.17 Maximum force (N) 1274 ± 70 1265 ± 73 9 ( −58; 77) 0.75 Maximum power (W) 2665 ± 167 2605 ± 109 60 ( −96; 216) 0.40 Flight time (ms) 490 ± 26 485 ± 18 5.6 ( −15; 26) 0.57 Average power (W) 628 ± 89 591 ± 76 36 ( −8; 81) 0.10 Average force (N) 763 ± 37 747 ± 37 16 ( −8; 40) 0.15 CMJ, counter movement jump; 1RM, one repetition maximum. P, the P-value from the paired sample t-test from pre- to posttest. TABLE 3 \| Changes in selected parameters from pre- to posttest. February 2021 \| Volume 12 \| Article 623885 4 Female Football During COVID-19 Pedersen et al. FIGURE 2 \| Percentage changes for each individual participant from pre- to posttest for absolute one repetition maximum in the partial squat exercise (1RM) (kg), 15 m sprint time (s) and counter movement jump (CMJ) height (cm). inﬂuence maximal strength (Rønnestad et al., 2008). Statistical Analyses Small-sided games are shown to both provoke high maximal acceleration distance during play (Ade et al., 2014) and improve sprint performance (Hammami et al., 2018). However, sprinting is an uncomplicated movement pattern to carry out outside the football ﬁeld where especially short sprints can be trained almost anywhere without the use of equipment, and consequently lead to improvements in acceleration (Spinks et al., 2007), or sprint performance maintenance, as found in our study, being in line with earlier ﬁndings (Mujika et al., 2009). The sprint training eﬀect is usually not superior when a supervising coach is included compared with individual unsupervised sprint training (Haugen et al., 2015). Thus, sprint-training eﬀect solely relies on the players themselves, where the players in our study performed suﬃcient amounts to preserve sprint performance. The prescribed volume of sprint and jump training time showed a tendency to increase during the lockdown (Table 2), and may have compensated for the reduced speciﬁc football training. stage in which training was performed in small groups with contact restrictions (Mohr et al., 2020). The team in our study had a total weekly training volume of ∼5 h prior and ∼4 h during the lockdown, which was a decreased total training time (∼1 h). This is less than the ∼3.5 h reduced training time recently reported for female football players in the three highest leagues in Spain during the lockdown (Mon-López et al., 2020). The participants in our study tended to perform more strength training during the lockdown, potentially compensating for the loss of sports speciﬁc training during this period. This increase in strength training time did not increase strength, jump, or sprint performance, but rather maintained it. Importantly, our results reﬂect the prescribed training from the coaches, and not the reported training performed by the players, as in the study by Mon-López et al. (2020). However, their initial training volume was probably much higher than in the current study, as their self-reported weekly training pre-lockdown was 12 h. The training volume in our study is much lower than for elite players. Self-reported training data from the ﬁrst and second placing team in the highest division in Norway last season, showed that the players trained 9.2 ± 2.5 weekly hours during the preseason (unpublished results). Statistical Analyses We did not expect that reduced football training per se would lead to a marked strength loss, as football training do not seem to The CMJ height was preserved during the lockdown. This is in line with recent ﬁndings in male football players February 2021 \| Volume 12 \| Article 623885 Frontiers in Physiology \| www.frontiersin.org 5 Female Football During COVID-19 Pedersen et al. FIGURE 3 \| (A) Perceived adherence to proportion of the prescribed training plan [y-axis, 1. to a little degree (0–20%), 2. to some degree (21–40%), 3. approximately half (41–60%), 4. to a large degree (61–80%), 5. almost all (81–100%)]. (B) Perceived degree of motivation for training (y-axis, 1. highly motivated, 2. average motivated, 3. low motivated). (C) Perceived level of quality of the training conducted (y-axis, 1. high quality, 2. average quality, 3. low quality). The ﬁgure displays the mean ± SD. ∗Indicates a signiﬁcant difference (P < 0.05) from pre lockdown to lockdown, derived from the Signed rank test. FIGURE 3 \| (A) Perceived adherence to proportion of the prescribed training plan [y-axis, 1. to a little degree (0–20%), 2. to some degree (21–40%), 3. approximately half (41–60%), 4. to a large degree (61–80%), 5. almost all (81–100%)]. (B) Perceived degree of motivation for training (y-axis, 1. highly motivated, 2. average motivated, 3. low motivated). (C) Perceived level of quality of the training conducted (y-axis, 1. high quality, 2. average quality, 3. low quality). The ﬁgure displays the mean ± SD. ∗Indicates a signiﬁcant difference (P < 0.05) from pre lockdown to lockdown, derived from the Signed rank test. (Cohen et al., 2020). Jump performances as during heading situations are observed in large sided football games (Owen et al., 2014). In order to follow the regulations by the authorities, both large sided and small-sided games were not performed by the team in the current study during the lockdown. With total training cessation, jump height decrements are reported following 4 weeks in adult males (Izquierdo et al., 2007), but not following 3 weeks in adolescent males (Gavanda et al., 2020). Maximal power is likely better preserved than maximal force (Bosquet et al., 2013). Importantly, the reduction is shown to be more profound as the training cessation duration increases, where maximal force declines are reported following 3 weeks of training cessation (Bosquet et al., 2013). Future Steps and Recommendations Future Steps and Recommendations Furthermore, as football is a complex sport in its requirements for diﬀerent physical aspects, coaches should consider breaks like those that we have experienced due to COVID-19 as opportunities, and prioritize endurance, speed and power training in lieu of speciﬁc football training. Statistical Analyses to our knowledge this is the ﬁrst study to report pre- and posttest prior and following the COVID-19 lockdown in female football players. Due to the uncontrolled design of our study, it should be considered descriptive and thus report changes in acceleration and jump height over the COVID-19 training period. Unfortunately, this design does not allow for causality inference (i.e., why we observed no change in performance). This information is highly relevant for football coaches. Second, although the training was described by the coaches, we do not know whether the players adhered to the training regimen, which we measured in retrospect with a subjective rating on the Likert scale. Others have found reduced jump height after an oﬀ- season training program (Koundourakis et al., 2014). The same is shown for sprint performance (Clemente et al., 2021). However, if the players are given an exercise plan during the oﬀ-season with gym sessions implemented, sprint and jump maintenance is possible (Requena et al., 2017). In our study, the players had not access to gyms, and were thus no able to implement proper maximal strength training in line with previous recommendations (Rønnestad et al., 2008; Helgerud et al., 2011). However, they still managed to implement a feasible training regimen suﬃcient to preserve strength levels. In total, the preserved strength, jump, and sprint performance during the COVID-19 lockdown in the team in our study is likely an eﬀect of adhering to a well-designed physical training program, which according to our questionnaire was well implemented by the participants. For players of this level, strength training in the gym or full contact football trainings are not necessary to maintain maximal strength, running speed or jump ability. CONCLUSION Maximal partial squat strength, CMJ, and sprint performance were preserved in female football players during a 12-week period of absence from normal football training and gym facilities during the COVID-19-induced lockdown. Thus, all the variables reﬂecting maximal and rapid muscular force production remained unchanged, likely due to adherence to a well-designed home-based and group-based training program by the team’s coaches. REFERENCES doi: 10.1123/ijspp. 2020-0501 Gavanda, S., Geisler, S., Quitmann, O. J., Bauhaus, H., and Schiﬀer, T. (2020). Three weeks of detraining does not decrease muscle thickness, strength or sport performance in adolescent athletes. Int. J. Exerc. Sci. 13, 633–644. Owen, A. L., Wong, D. P., Paul, D., and Dellal, A. (2014). Physical and technical comparisons between various-sided games within professional soccer. Int. J. Sports Med. 35, 286–292. doi: 10.1055/s-0033-1351333 Girardi, M., Casolo, A., Nuccio, S., Gattoni, C., and Capelli, C. (2020). Detraining eﬀects prevention: A new rising challenge for athletes. Front. Physiol. 11:588784. doi: 10.3389/fphys.2020.588784 Pedersen, S., Heitmann, K. A., Sagelv, E. H., Johansen, D., and Pettersen, S. A. (2019). Improved maximal strength is not associated with improvements in sprint time or jump height in high-level female football players: a cluster- randomized controlled trial. BMC Sports Sci. Med. Rehabil. 11:20. doi: 10.1186/ s13102-019-0133-9 Hammami, A., Gabbett, T. J., Slimani, M., and Bouhlel, E. (2018). Does small- sided games training improve physical ﬁtness and team-sport-speciﬁc skills? A systematic review and meta-analysis. J. Sports Med. Phys. Fitness 58, 1446–1455. Haugen, T., Tonnessen, E., Oksenholt, O., Haugen, F. L., Paulsen, G., Enoksen, E., et al. (2015). Sprint conditioning of junior soccer players: eﬀects of training intensity and technique supervision. PLoS One 10:e0121827. doi: 10.1371/ journal.pone.0121827 Ploutz-Snyder, L. L., and Giamis, E. L. (2001). Orientation and familiarization to 1RM strength testing in old and young women. J. Strength Cond. Res. 15, 519–523. doi: 10.1519/00124278-200111000-00020 Helgerud, J., Rodas, G., Kemi, O. J., and Hoﬀ, J. (2011). Strength and endurance in elite football players. Int. J. Sports Med. 32, 677–682. doi: 10.1055/s-0031- 1275742 Requena, B., García, I., Suárez-Arrones, L., Sáez de Villarreal, E., Naranjo Orellana, J., and Santalla, A. (2017). Oﬀ-season eﬀects on functional performance, body composition, and blood parameters in top-level professional soccer players. J. Strength Cond. Res. 31, 939–946. doi: 10.1519/jsc.0000000000001568 Izquierdo, M., Ibañez, J., González-Badillo, J. J., Ratamess, N. A., Kraemer, W. J., Häkkinen, K., et al. (2007). Detraining and tapering eﬀects on hormonal responses and strength performance. J. Strength Cond. Res. 21, 768–775. doi: 10.1519/00124278-200708000-00019 Roberson, P. K., Shema, S. J., Mundfrom, D. J., and Holmes, T. M. (1995). Analysis of paired Likert data: how to evaluate change and preference questions. Family Med. 27, 671–675. of paired Likert data: how to evaluate change and preference questions. Family Med. 27, 671–675. Koundourakis, N. E., Androulakis, N. REFERENCES Mohr, M., Nassis, G. P., Brito, J., Randers, M. B., Castagna, C., Parnell, D., et al. (2020). Return to elite football after the COVID-19 lockdown. Managing Sport Leisure 2020, 1–9. doi: 10.1080/23750472.2020.1768635 Ade, J. D., Harley, J. A., and Bradley, P. S. (2014). Physiological response, time- motion characteristics, and reproducibility of various speed-endurance drills in elite youth soccer players: small-sided games versus generic running. Int. J. Sports Physiol. Perform. 9, 471–479. doi: 10.1123/ijspp.2013-0390 Mon-López, D., García-Aliaga, A., Ginés Bartolomé, A., and Muriarte Solana, D. (2020). How has COVID-19 modiﬁed training and mood in professional and non-professional football players? Physiol. Behav. 227, 113148–113148. doi: 10.1016/j.physbeh.2020.113148 Bangsbo, J., Mohr, M., Poulsen, A., Perez-Gomez, J., and Krustrup, P. (2006). Training and testing the elite athlete. J. Exerc. Sci. Fitness 4, 1–14. Moreno-Pérez, V., Del Coso, J., Romero-Rodríguez, D., Marcé-Hernández, L., Peñaranda, M., and Madruga-Parera, M. (2020). Eﬀects of home conﬁnement due to COVID-19 pandemic on eccentric hamstring muscle strength in football players. Scand. J. Med. Sci. Sports 30, 2010–2012. doi: 10.1111/sms.13768 Bosquet, L., Berryman, N., Dupuy, O., Mekary, S., Arvisais, D., Bherer, L., et al. (2013). Eﬀect of training cessation on muscular performance: A meta-analysis. Scand. J. Med. Sci. Sports 23, 140–149e. doi: 10.1111/sms.12047 Clemente, F. M., Ramirez-Campillo, R., and Sarmento, H. (2021). Detrimental eﬀects of the oﬀ-season in soccer players: A systematic review and meta- analysis. Sports Med. doi: 10.1007/s40279-020-01407-4 Mujika, I., and Padilla, S. (2000). Detraining: loss of training-induced physiological and performance adaptations. Part II Sports Med. 30, 145–154. doi: 10.2165/ 00007256-200030030-00001 Mujika, I., and Padilla, S. (2001). Muscular characteristics of detraining in humans. Med. Sci. Sports Exerc. 33, 1297–1303. doi: 10.1097/00005768-200108000- 00009 Cohen, D. D., Restrepo, A., Richter, C., Harry, J. R., Franchi, M. V., Restrepo, C., et al. (2020). Detraining of speciﬁc neuromuscular qualities in elite footballers during COVID-19 quarantine. Sci. Med. Football 2020, 1–6. doi: 10.1080/ 24733938.2020.1834123 Mujika, I., Santisteban, J., and Castagna, C. (2009). In-season eﬀect of short-term sprint and power training programs on elite junior soccer players. J. Strength Condit. Res. 23, 2581–2587. doi: 10.1519/JSC.0b013e3181bc1aac García-Pallarés, J., Sanchez-Medina, L., Pérez, C. E., Izquierdo-Gabarren, M., and Izquierdo, M. (2010). Physiological eﬀects of tapering and detraining in world-class kayakers. Med. Sci. Sports Exerc. 42, 1209–1214. doi: 10.1249/MSS. 0b013e3181c9228c Muriel, X., Courel-Ibáñez, J., Cerezuela-Espejo, V., and Pallarés, J. G. (2020). Training load and performance impairments in professional cyclists during COVID-19 lockdown. Int. J. Sports Physiol. Perform. 1, 1–4. DATA AVAILABILITY STATEMENT The main limitation in the current study is the low sample size, which increases the possibility of a statistical type 2 error, and such a consequence cannot be conclusively ruled out. However, The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. February 2021 \| Volume 12 \| Article 623885 Frontiers in Physiology \| www.frontiersin.org 6 Female Football During COVID-19 Pedersen et al. AUTHOR CONTRIBUTIONS This research was funded by RDA and Tromsø Research Foundation. The funding body had no role in designing the study and data collection, analysis, interpretation, and in writing of the study. This research was funded by RDA and Tromsø Research Foundation. The funding body had no role in designing the study and data collection, analysis, interpretation, and in writing of the study. SP: in charge of the writing process, conceptualization and design, and data collection. SAP and DJ: conceptualization and design, SP: in charge of the writing process, conceptualization and design, and data collection. SAP and DJ: conceptualization and design, and critical manuscript revision. AW and ES: contribution to and data collection. SAP and DJ: conceptualization and design, and critical manuscript revision. AW and ES: contribution to and critical manuscript revision. AW and ES: contribution to ETHICS STATEMENT data collection, statistical analyses, and manuscript revision. BW: conceptualization and design, contribution to statistical analyses, and critical manuscript revision. MR and AC: contribution to critical manuscript revision. All authors contributed to ﬁnal approval of the version to be published. Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study. Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study. REFERENCES E., Malliaraki, N., Tsatsanis, C., Venihaki, M., and Margioris, A. N. (2014). Discrepancy between exercise performance, body composition, and sex steroid response after a six-week detraining period in professional soccer players. PLoS One 9:e87803. doi: 10.1371/journal.pone. 0087803 Rønnestad, B. R., Kvamme, N. H., Sunde, A., and Raastad, T. (2008). Short-term eﬀects of strength and plyometric training on sprint and jump performance in professional soccer players. J. Strength Condit. Res. 22, 773–780. doi: 10.1519/ JSC.0b013e31816a5e86 Rønnestad, B. R., Nymark, B. S., and Raastad, T. (2011). Eﬀects of in-season strength maintenance training frequency in professional soccer players. J. Strength Cond. Res. 25, 2653–2660. doi: 10.1519/JSC.0b013e31822dcd 96 Latella, C., and Haﬀ, G. G. (2020). Global challenges of being a strength athlete during a pandemic: impacts and sports-speciﬁc training considerations and recommendations. Sports 8:100. doi: 10.3390/sports8070100 96 February 2021 \| Volume 12 \| Article 623885 Frontiers in Physiology \| www.frontiersin.org 7 Female Football During COVID-19 Pedersen et al. Zheng, C., Huang, W. Y., Sheridan, S., Sit, C. H., Chen, X. K., and Wong, S. H. (2020). COVID-19 pandemic brings a sedentary lifestyle in young adults: A cross-sectional and longitudinal study. Int. J. Environ. Res. Public Health 17:6035. doi: 10.3390/ijerph171760 35 Sarto, F., Impellizzeri, F. M., Sporri, J., Porcelli, S., Olmo, J., Requena, B., et al. (2020). Impact of potential physiological changes due to COVID-19 home conﬁnement on athlete health protection in elite sports: a call for awareness in sports programming. Sports Med. 50, 1417–1419. doi: 10.1007/s40279-020- 01297-6 Zinner, C., Matzka, M., Leppich, R., Kounev, S., Holmberg, H.-C., and Sperlich, B. (2020). The impact of the german strategy for containment of coronavirus SARS-CoV-2 on training characteristics, physical activity and sleep of highly trained kayakers and canoeists: a retrospective observational study. Front. Sports Active Living 2:579830. doi 10.3389/fspor.2020.5798 30 Spinks, C. D., Murphy, A. J., Spinks, W. L., and Lockie, R. G. (2007). The eﬀects of resisted sprint training on acceleration performance and kinematics in soccer, rugby union, and Australian football players. J. Strength Condit. Res. 21, 77–85. doi: 10.1519/00124278-200702000-00015 Sporis, G., Jovanovic, M., Omrcen, D., and Matkovic, B. (2011). Can the oﬃcial soccer game be considered the most important contribution to player’s physical ﬁtness level? J. Sports Med. Phys. Fitness 51, 374–380. 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. Frontiers in Physiology \| www.frontiersin.org REFERENCES 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. Stølen, T., Chamari, K., Castagna, C., and Wisloﬀ, U. (2005). Physiology of soccer: an update. Sports Med. 35, 501–536. doi: 10.2165/00007256-200535060-00004 Wing, C. E., Turner, A. N., and Bishop, C. J. (2020). Importance of strength and power on key performance indicators in elite youth soccer. J. Strength Condit. Res. 34, 2006–2014. doi: 10.1519/JSC.0000000000002446 Copyright © 2021 Pedersen, Johansen, Casolo, Randers, Sagelv, Welde, Winther and Pettersen. 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. Copyright © 2021 Pedersen, Johansen, Casolo, Randers, Sagelv, Welde, Winther and Pettersen. 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. Wisløﬀ, U., Helgerud, J., and Hoﬀ, J. (1998). Strength and endurance of elite soccer players. Med. Sci. Sports Exerc. 30, 462–467. doi: 10.1097/00005768-199803000- 00019 Xiang, M., Zhang, Z., and Kuwahara, K. (2020). Impact of COVID-19 pandemic on children and adolescents’ lifestyle behavior larger than expected. Prog. Cardiovasc. Dis. 63, 531–532. doi: 10.1016/j.pcad.2020.04.013 February 2021 \| Volume 12 \| Article 623885 Frontiers in Physiology \| www.frontiersin.org 8
https://openalex.org/W2134514009	https://europepmc.org/articles/pmc2918549?pdf=render	English	null	Proteomic analysis of proteins expressing in regions of rat brain by a combination of SDS-PAGE with nano-liquid chromatography-quadrupole-time of flight tandem mass spectrometry	Proteome science	2,010	cc-by	9,092	RESEARCH Open Access Katagiri et al. Proteome Science 2010, 8:41 http://www.proteomesci.com/content/8/1/41 Katagiri et al. Proteome Science 2010, 8:41 http://www.proteomesci.com/content/8/1/41 Katagiri et al. Proteome Science 2010, 8:41 Abstract Background: Most biological functions controlled by the brain and their related disorders are closely associated with activation in specific regions of the brain. Neuroproteomics has been applied to the analysis of whole brain, and the general pattern of protein expression in all regions has been elucidated. However, the comprehensive proteome of each brain region remains unclear. Results: In this study, we carried out comparative proteomics of six regions of the adult rat brain: thalamus, hippocampus, frontal cortex, parietal cortex, occipital cortex, and amygdala using semi-quantitative analysis by Mascot Score of the identified proteins. In order to identify efficiently the proteins that are present in the brain, the proteins were separated by a combination of SDS-PAGE on a C18 column-equipped nano-liquid chromatograph, and analyzed by quadrupole-time of flight-tandem-mass spectrometry. The proteomic data show 2,909 peptides in the rat brain, with more than 200 identified as region-abundant proteins by semi-quantitative analysis. The regions containing the identified proteins are membrane (20.0%), cytoplasm (19.5%), mitochondrion (17.1%), cytoskeleton (8.2%), nucleus (4.7%), extracellular region (3.3%), and other (18.0%). Of the identified proteins, the expressions of glial fibrillary acidic protein, GABA transporter 3, Septin 5, heat shock protein 90, synaptotagmin, heat shock protein 70, and pyruvate kinase were confirmed by immunoblotting. We examined the distributions in rat brain of GABA transporter 3, glial fibrillary acidic protein, and heat shock protein 70 by immunohistochemistry, and found that the proteins are localized around the regions observed by proteomic analysis and immunoblotting. IPA analysis indicates that pathways closely related to the biological functions of each region may be activated in rat brain. Conclusions: These observations indicate that proteomics in each region of adult rat brain may provide a novel way to elucidate biological actions associated with the activation of regions of the brain. Proteomic analysis of proteins expressing in regions of rat brain by a combination of SDS-PAGE with nano-liquid chromatography- quadrupole-time of flight tandem mass spectrometry Tomoki Katagiri1,6, Naoya Hatano2, Masamune Aihara1, Hiroo Kawano3, Mariko Okamoto1, Ying Liu4, Tomonori Izumi5, Tsuyoshi Maekawa5, Shoji Nakamura4, Tokuhiro Ishihara3, Mutsunori Shirai6, Yoichi Mizukami1* * Correspondence: mizukami@yamaguchi-u.ac.jp 1Center for Gene Research, Yamaguchi University, Yamaguchi, 755-8505, Japan © 2010 Katagiri 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. © 2010 Katagiri 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. Background of all components expressed in each region is essential to understand the mechanisms of higher actions and their functional properties. Recently, methods for the compre- hensive analyses of gene expression such as DNA array and serial analysis gene expression have been developed in addition to the disclosure of genomic sequence infor- mation [4-7]. These applications have provided much information about gene expression in the brain [8-13]. A comprehensive account of the products of gene The mammalian central nervous system regulates higher biological actions such as feelings and behaviors, which are known to associate with the activation of specific regions of brain as determined by positron-emission topography and MRI techniques [1-3]. A characterization Katagiri et al. Proteome Science 2010, 8:41 http://www.proteomesci.com/content/8/1/41 Page 2 of 12 extracted from the gel pieces were applied to nano-LC- Q-TOF-MS/MS and analyzed by the Mascot search engine to identify amino acid sequences (Fig. 1). All together in all brain regions, 2,909 redundant peptides were assigned, and 515 proteins were identified with more than 95% confidence based on the amino acid sequences deduced from the MS/MS peaks (Additional file 1, Table S1). The proteins assigned by a single pep- tide were confirmed manually. Representative MS/MS spectra of the identified proteins GABA transporter 3 (GAT 3) and Latrophilin 2 are shown in Fig. 2. GAT 3 was identified based on the amino acid sequence GTI- SAITEK deduced by both b type ions and y type ions on the MS/MS spectrum, the sequence of which corre- sponds to 1.8% of the whole sequence. For the Latrophi- lin 2 precursor, the amino acid sequence deduced from the MS spectrum is LGADFIGR, which covers 0.6% of the full sequence. The peaks used for the identification of Latrophillin 2 were observed more prominently than other peaks on the spectrum, and the confidence level calculated from the peak data was greater than 95% by Mascot search. The numbers of proteins identified in each region without redundancy were 250, 225,149, 273, 202, and 198 for thalamus, hippocampus, frontal cortex, parietal cortex, occipital cortex, and amygdala, respec- tively (Fig. 3). Among the identified proteins, the num- ber of region-abundant proteins was 63 in thalamus, 38 in hippocampus, 14 in frontal cortex, 66 in parietal cor- tex, 24 in occipital cortex, and 36 in amygdala (Fig. 3) by semi-quantitative analysis. Results Identification of proteins in each region of rat brain To identify proteins expressing in each region of rat brain, the removed brains were divided into six regions, thalamus, hippocampus, frontal cortex, parietal cortex, occipital cortex, and amygdala, according to the atlas of Paxinos and Watson. To test the efficiency of protein extraction by lysis buffer, we determined the protein concentrations extracted by lysis buffers included Triton X-100, CHAPS, NP-40, or SDS. The extracted proteins were determined by EZQ protein quantification kit (Molecular Probe), which is able to determine the pro- tein concentrations in the presence of the detergents. The proteins concentration in the solution extracted by SDS sample buffer was significantly high compared with those in other lysis buffers (data not shown). The pro- teins in each region were extracted by lysis buffer including SDS, and separated by electrophoresis. Each gel lane was sliced into 24 pieces, and the proteins Background Several functional mole- cules were observed in the specific regions, such as G protein coupled receptors (thyroid stimulating hormone receptor 1 and Latrophilin 2) in the parietal cortex, and olfactory receptors (olfactory receptor family 10 and olfactory Olr 436) in the occipital cortex (Table 1). Next we analyzed the intracellular localization of the identi- fied proteins based on the component section in the NCBI Entrez Gene. In all regions, most proteins loca- lized in the membrane fraction (20.0%), with other pro- teins classified into cytoplasm (19.5%), mitochondrion (17.1%), cytoskeleton (8.2%), nucleus (4.7%), extracellular region (3.3%), and other (18.0%) (Fig. 4). expression through protein profiling is needed even more than genome information to fully understand higher bio- logical actions. Neuroproteomics has been applied to the analysis of whole brain, and the general pattern of protein expres- sion in all regions has been elucidated [14-16]. In speci- fic regions, such as the hippocampus, thalamus, and striatum, the relationships between protein stimulations or diseases have been examined by proteomic analysis [17-20]. However, the comprehensive proteome of each brain region remains unclear despite the attention paid to the biological functions of each region, although comprehensive investigations of gene expression in each region of the brain have been undertaken using DNA array and in situ hybridization techniques [8]. We have undertaken proteomic analysis using a vari- ety of protocols according to the research targets, such as the combination of two-dimensional electrophoresis with matrix-assisted laser desorption/ionization time-of- flight mass spectrometry and nano-liquid chromatogra- phy (LC)-quadrupole-time of flight (Q-TOF)-tandem mass spectrometry (MS/MS) after column concentration [21-27]. Here, we have divided the rat brain into six regions, thalamus, hippocampus, frontal cortex, parietal cortex, occipital cortex, and amygdala, and compared the proteome of each region by a combination of sodium dodecyl sulfate (SDS)-polyacrylamide gel elec- trophoresis (PAGE) and nano-LC-Q-TOF-MS/MS. We have identified a total of 2,909 peptides in all regions of the rat brain, and found proteins specifically expressed in each region of brain: 63 proteins in the thalamus, 38 in the hippocampus, 14 in the frontal cortex, 66 in the parietal cortex, 24 in the occipital cortex, and 36 in the amygdala by semi-quantitative analysis. Confirmation of protein expression in each region of rat brain The identified sequence within the entire amino acid sequence of GAT 3 is indicated by the underline (lower panel). The MS/MS spectrum for the peptide derived from the Latrophilin 2 precursor (B), identified only in the parietal cortex of the rat brain, is shown; the amino acid sequence GADFIGR deduced from the 2 b type ions (blue) and 7 y type ions (red) were assigned by Mascot search (upper panel). The identified sequence within the entire amino acid sequence of Latrophilin 2 is indicated by the underline (lower panel). Figure 1 Flow diagram of the experimental design. Rat brains were divided into six regions: thalamus, hippocampus, frontal cortex, parietal cortex, occipital cortex, and amygdala. The divided samples were lysed in lysis buffer containing SDS, and subjected to SDS-PAGE with Coomassie Brilliant Blue staining. The gel lane was divided into 24 slices, and the slices were pre-treated by in-gel trypsin digestion. The amino acid sequences of all detected proteins were determined by nano-LC-Q-TOF-MS/MS. Figure 1 Flow diagram of the experimental design. Rat brains were divided into six regions: thalamus, hippocampus, frontal cortex, parietal cortex, occipital cortex, and amygdala. The divided samples were lysed in lysis buffer containing SDS, and subjected to SDS-PAGE with Coomassie Brilliant Blue staining. The gel lane was divided into 24 slices, and the slices were pre-treated by in-gel trypsin digestion. The amino acid sequences of all detected proteins were determined by nano-LC-Q-TOF-MS/MS. regions by immunoblotting using specific antibodies. Consistent with these blotting data, the proteins were detected ubiquitously in all regions with constant values in the Mascot scores (Fig. 5E and 5F). Furthermore, we carried out a distribution analysis with antibodies for GFAP, GAT 3, and HSP 70, useful for immunohisto- chemistry, to confirm the data obtained by the immuno- blotting and MS/MS analysis. Immunohistochemical observations indicated that GAT 3 shows preferential staining in the thalamus, hypothalamus, medulla, and olfactory bulb, consistent with the MASCOT score data (Fig. 6A and 6B). GFAP staining was observed in all regions, and was enhanced in part of the hippocampus and thalamus (Fig. 6A and 6C). HSP 70 was ubiquitously detected in all regions of the brain by immunohistochem- istry (Fig. 6A and 6D). These data are mostly consistent with the data obtained by immunoblotting and MS/MS analysis. Together with all of the observations, the data that is almost the same as that obtained using Mascot scores (Fig. 5A). Confirmation of protein expression in each region of rat brain To confirm the brain distribution of proteins identified in the six regions by Q-TOF-MS/MS, we carried out immu- noblotting using specific antibodies, and the results were compared with Mascot scores, since there is a correlation between the protein amounts on the gels and Mascot score [22]. Immunoblotting using the anti-glial fibrillary acidic protein (GFAP) antibody demonstrated that bands were observed in all regions, and the densities were increased in the thalamus and hippocampus, a finding Katagiri et al. Proteome Science 2010, 8:41 http://www.proteomesci.com/content/8/1/41 Page 3 of 12 Figure 2 Representative MS/MS spectra of proteins identified in rat brain. The MS/MS spectrum for the peptide derived from GAT 3 (A), identified only in the thalamus of the rat brain, is shown; the amino acid sequence GTISAITEK deduced from the 5 b type ions (blue) and 8 y type ions (red) were assigned by Mascot search (upper panel). The identified sequence within the entire amino acid sequence of GAT 3 is indicated by the underline (lower panel). The MS/MS spectrum for the peptide derived from the Latrophilin 2 precursor (B), identified only in the parietal cortex of the rat brain, is shown; the amino acid sequence GADFIGR deduced from the 2 b type ions (blue) and 7 y type ions (red) were assigned by Mascot search (upper panel). The identified sequence within the entire amino acid sequence of Latrophilin 2 is indicated by the underline (lower panel). Figure 1 Flow diagram of the experimental design. Rat brains were divided into six regions: thalamus, hippocampus, frontal cortex, parietal cortex, occipital cortex, and amygdala. The divided samples were lysed in lysis buffer containing SDS, and subjected to SDS-PAGE with Coomassie Brilliant Blue staining. The gel lane was divided into 24 slices, and the slices were pre-treated by in-gel trypsin digestion. The amino acid sequences of all detected proteins were determined by nano-LC-Q-TOF-MS/MS. Figure 2 Representative MS/MS spectra of proteins identified in rat brain. The MS/MS spectrum for the peptide derived from Figure 2 Representative MS/MS spectra of proteins identified in rat brain. The MS/MS spectrum for the peptide derived from GAT 3 (A), identified only in the thalamus of the rat brain, is shown; the amino acid sequence GTISAITEK deduced from the 5 b type ions (blue) and 8 y type ions (red) were assigned by Mascot search (upper panel). Confirmation of protein expression in each region of rat brain By region, 250 proteins were identified in the thalamus, 225 in the hippocampus, 149 in the frontal cortex, 273 in the parietal cortex, 202 in the occipital cortex, and 198 in the amygdala without redundancy. Sixty-three proteins in the thalamus, 38 in the hippocampus, 14 in the frontal cortex, 66 the in parietal cortex, 24 in the occipital cortex, and 36 in the amygdala were found in only that region of the brain. Figure 3 Schematic representation of proteins identified in six regions of rat brain. In total, 2,909 peptides including redundant peptides were identified by nano-LC-Q-TOF-MS/MS in all regions of the brain, leaving a total of 515 proteins. By region, 250 proteins were identified in the thalamus, 225 in the hippocampus, 149 in the frontal cortex, 273 in the parietal cortex, 202 in the occipital cortex, and 198 in the amygdala without redundancy. Sixty-three proteins in the thalamus, 38 in the hippocampus, 14 in the frontal cortex, 66 the in parietal cortex, 24 in the occipital cortex, and 36 in the amygdala were found in only that region of the brain. p [ ] By comparing the proteins found in each region, pro- teins closely related to the function of each region have been identified in this study. Of the identified proteins, GAT 3 was found only in the thalamus, and this specific expression was confirmed by immunoblotting. In the mammalian thalamus, GABA is a major inhibitory neu- rotransmitter, and the GABA transporter mediates GABA uptake into presynaptic terminals to terminate the effects of GABA [28,29]. Consistent with our data, ultrastructural investigations show that GABA transpor- ter 3 is expressed most prominently in the thalamus [8,30]. The immunohistochemical investigation also detected GAT 3 in the olfactory bulb and hypothalamus, and GAT 3 may be expressed except for the regions used in this study [31]. In samples extracted from the occipital cortex, two types of olfactory receptors were specifically identified. Olfactory receptors are under- stood to function as molecular sensors for odorants [32]. Olfactory receptors are expressed mostly in pyra- midal cells of the occipital cortex, which is consistent with our data [33]. Olfactory stimulation results in the activation of the bilateral occipital cortex as detected by positron-emission topography [34]. Improvements in extra-conversion and regional cerebral blood flow might be induced by smell stimulation through the activation of the occipital cortex [35]. Confirmation of protein expression in each region of rat brain GAT 3 was only detected in the thala- mus by immunoblotting, which is completely consistent with the results obtained by MS/MS analysis (Fig. 5B). Septin 5 detected in only Parietal cortex by mass analysis, was mainly observed as single band on the immunoblots in Parietal cortex fraction (Fig. 5C). The small amount of Septin 5 was observed in other regions. The difference of data might be due to the sensitivity between immuno- blotting and mass analysis. The anti-heat shock protein (HSP) 90 antibody recognized a protein with an approxi- mate molecular mass of 90 kDa in all regions of the brain on the immunoblots, but the band densities in the parie- tal cortex and occipital cortex were slightly reduced com- pared with other regions (Fig. 5D). The Mascot scores for HSP 90 were also low in both regions (Fig. 5D). The amount of synaptotagmin (Fig. 5E), HSP 70 (Fig. 5F), and pyruvate kinase (data not shown) were similar in all Katagiri et al. Proteome Science 2010, 8:41 http://www.proteomesci.com/content/8/1/41 Katagiri et al. Proteome Science 2010, 8:41 http://www.proteomesci.com/content/8/1/41 Page 4 of 12 Membrane proteins including ion channels, receptors, and ion transporters play important roles in brain func- tions, with nerve cells in particular utilizing diverse functional proteins on the membrane for receiving, con- ducting and transmitting signals. In this report, the sam- ples extracted directly by SDS from each region of the brain were separated using SDS-PAGE. The approach has advantages in identifying many proteins including membrane proteins in the brain because the SDS essen- tial for electrophoresis is a strong detergent that lyses most proteins, and can be used directly for the prepara- tion of brain samples, although the resolution cannot be improved. Using this protocol, proteins expressed in the brain were efficiently identified, and over 20% of the identified proteins were deduced to localize in mem- branes. In hippocampal samples prepared by two- dimensional electrophoresis, over 70% of the identified proteins were cytoplasm-localized, while only 7% were detected as membrane proteins [17]. Figure 3 Schematic representation of proteins identified in six regions of rat brain. In total, 2,909 peptides including redundant peptides were identified by nano-LC-Q-TOF-MS/MS in all regions of the brain, leaving a total of 515 proteins. Confirmation of protein expression in each region of rat brain The olfactory receptors expressed in the occipital cortex may be involved in bio- logical functions, resulting in smell stimulation. Septin 5 participating in neuronal development was mainly detected in occipital cortex and thalamus. Septin 5 is colocalized in synaptic vesicles with SNARE proteins, and plays a role in neurotransmitter release [36]. How- ever, Septin 5 mice showed no changes in synaptic transmission in hippocampus [37]. In our data, the pro- tein amount of Septin 5 in hippocampus was much less determined by nano-LC-Q-TOF-MS/MS are reliable for the identification of brain proteins. IPA analysis in proteins identified in each region of rat brain We carried out the pathway analysis by IPA analysis using the data of proteins identified in each region of rat brain. The representative networks in each region were shown in Fig.7 with Additional file 2, Table S2 and the canonical pathways were also revealed in Additional file 3, Fig. S1. The pathway through corticotropin releas- ing hormone receptor was detected in thalamus releas- ing corticotropin-releasing hormone under stress (Fig.7). In hippocampus, the pathways through ERK1 and S6 kinase were detected n Fig.7 and Additional file 3, Fig. S1, suggesting that the development and functions in nervous system may be activated through growth factors such as BDNF in the region. The pathway related to citrate cycle was observed in frontal cortex and the pathway involving in mitochondria dysfunction in amyg- dala was shown in Additional file 3, Fig. S1. Discussion We have conducted a comprehensive analysis of the proteins expressing in six regions of rat brain following a proteomic approach using SDS-PAGE and nano-LC- Q-TOF-MS/MS. As a result, we identified 250 proteins in thalamus, 225 in hippocampus, 149 in frontal cortex, 273 in parietal cortex, 202 in occipital cortex, and 198 in amygdala. Furthermore, the localizations of several proteins identified by proteomics were confirmed by immunoblotting and immunohistochemistry using speci- fic antibodies. Page 5 of 12 Katagiri et al. Discussion Proteome Science 2010, 8:41 http://www.proteomesci.com/content/8/1/41 Table 1 Representative membrane proteins identified in each region of adult rat brain by Q-TOF-MS/MS NCBI ID Protein Name Region Localization Function Receptor gi\|94380294 PREDICTED: similar to Carcinoembryonic antigen-related cell adhesion molecule 1 precursor (Biliary glycoprotein 1) (BGP-1) (Murine hepatitis virus receptor) (MHV-R) T ND gi\|32401457 opsin 5 H, A ND G-protein coupled receptor gi\|38259186 adiponectin receptor 1 F M fatty acid metabolism gi\|62990176 thyroid stimulating hormone receptor P M G-protein coupled receptor gi\|6912464 latrophilin 2 precursor P M G-protein coupled receptor gi\|52317184 olfactory receptor, family 10, subfamily X, member 1 O M olfactory receptor gi\|47577367 olfactory receptor Olr436 O M olfactory receptor Ion channel gi\|6755963 voltage-dependent anion channel 1 T, H, F, P, O, A M, Mt voltage-gated ion- selective channel activity Na Pump gi\|21450321 Na+/K+ -ATPase alpha 3 subunit T, H, F, P, O, A ND gi\|30409956 ATPase, Na+/K+ transporting, alpha 2 polypeptide T, H, F, P, O M sodium:potassium- exchanging ATPase gi\|6978543 ATPase, Na+/K+ transporting, alpha 1 polypeptide T, F, P, O, A M sodium:potassium- exchanging ATPase gi\|6978549 ATPase, Na+/K+ transporting, beta 1 polypeptide T, H, F, P, O, A M sodium:potassium- exchanging ATPase Ca Pump gi\|62234487 plasma membrane calcium ATPase 1 T, H, P, O M calcium-transporting ATPase gi\|48255951 plasma membrane calcium ATPase 2 isoform a P, O M calcium-transporting ATPase Proton Pump gi\|34856315 PREDICTED: similar to ATPase, H+ transporting, V1 subunit B, isoform 1 T, H, O, A ND gi\|62665162 PREDICTED: ATPase, H+ transporting, V0 subunit D isoform 1 (predicted) T, H, F, P, A ND gi\|34869154 PREDICTED: similar to ATPase, H+ transporting, V1 subunit A, isoform 1 T, H, F, P ND gi\|12025532 ATPase, H+ transporting, lysosomal V0 subunit a isoform 1 T, F, P Cy, M, N hydrogen ion transporter activity gi\|47717102 ATPase, H+ transporting, lysosomal 50/57kDa, V1 subunit H isoform 2 T Cy, M hydrogen-transporting ATP synthase activity gi\|19913426 ATPase, H+ transporting, lysosomal 56/58kDa, V1 subunit B1 H, F, O, A Cy, M, I hydrogen-transporting ATPase gi\| 124244102 ATPase, H+ transporting, lysosomal V0 subunit a isoform 2 P Cy, Ex, M Transporter gi\|78126167 solute carrier family 1 (glial high affinity glutamate transporter), member 2 isoform a T, H, F, P, O, A M glutamate transport gi\|9507115 solute carrier family 1 (glial high affinity glutamate transporter), member 3 P, A M glutamate transport gi\|400626 Sodium- and chloride-dependent GABA transporter 3 T M neurotransmitter transport T, thalamus; H, hippocampus; F, frontal cortex; P, parietal cortex; O, occipital cortex; A, amygdala M, membrane; Ex, extracellular region; Mt, mitochondrion; N, nucleus; Cy, cytoplasm; I, intracellular; ND, not described Katagiri et al. Discussion Proteome Science 2010, 8:41 http://www.proteomesci.com/content/8/1/41 Page 6 of 12 than other regions as shown in Fig. 5 and mass analysis, which was consistent with data with Septin 5-/-mice. In the hippocampal region related to memory we identi hybridization [8]. The specific expression of Glyoxalase 1, as indicated in this study, suggests the involvement of Glyoxalase 1 in the biological functions of the hippo Figure 4 Intracellular localization of proteins identified by Q-TOF-MS/MS in rat brain. The intracellular localizations of all proteins identified by nano-LC-Q-TOF-MS/MS were classified based on the component section in NCBI Entrez Gene. hybridization [8]. The specific expression of Glyoxalase 1, as indicated in this study, suggests the involvement of Glyoxalase 1 in the biological functions of the hippo- campus such as memory and long-term potentiation. In fact, Chen et al. indicate that Glyoxalase 1 plays a novel than other regions as shown in Fig. 5 and mass analysis, which was consistent with data with Septin 5-/-mice. In the hippocampal region related to memory, we identi- fied Glyoxalase 1, the localization of which was found to be mostly consistent with that determined by in situ than other regions as shown in Fig. 5 and mass analysis, which was consistent with data with Septin 5-/-mice. In the hippocampal region related to memory, we identi- fied Glyoxalase 1, the localization of which was found to be mostly consistent with that determined by in situ Page 7 of 12 Katagiri et al. Proteome Science 2010, 8:41 http://www.proteomesci.com/content/8/1/41 Katagiri et al. Proteome Science 2010, 8:41 role in Alzheimer’s disease and frontotemporal dementia [38]. N t k l sis f th p t i s p ss d i h associated with amino acid metabolism, molecular trans- port, and small molecular biochemistry in most regions, hi h is sist t ith th p i s bs ti s th t Figure 5 Immunoblotting analysis of proteins identified in six regions of rat brain. Rat brains were divided into six regions (thalamus, hippocampus, frontal cortex, parietal cortex, occipital cortex, and amygdala), and were extracted with lysis buffer. The samples (2.0 μg protein) were subjected to immunoblotting with anti-GFAP antibody (A), anti-GAT 3 antibody (B), anti-Septin 5 antibody (C), anti-HSP 90 antibody (D), anti-synaptotagmin antibody (E), and anti-HSP 70 antibody (F). Discussion Immunoblotting data using the indicated antibodies are shown in the upper panels; Mascot scores of the proteins determined by Q-TOF-MS/MS are shown in the lower panels. The immunoblotting data are shown as representative blots obtained in independent experiments from 3 rats. Data represent the means ± SE. Statistical significance was determined by ANOVA followed by Bonferroni’s test. 1 p < 0.05 vs Hip, Fro, Par, Occ, and Amy, 2 p < 0.05 vs Amy, 3 p < 0.05 Amy and Hip. Katagiri et al. Proteome Science 2010, 8:41 http://www.proteomesci.com/content/8/1/41 Page 7 of 12 Figure 5 Immunoblotting analysis of proteins identified in six regions of rat brain. Rat brains were divided into six regions (thalamus, hippocampus, frontal cortex, parietal cortex, occipital cortex, and amygdala), and were extracted with lysis buffer. The samples (2.0 μg protein) were subjected to immunoblotting with anti-GFAP antibody (A), anti-GAT 3 antibody (B), anti-Septin 5 antibody (C), anti-HSP 90 antibody (D), anti-synaptotagmin antibody (E), and anti-HSP 70 antibody (F). Immunoblotting data using the indicated antibodies are shown in the upper panels; Mascot scores of the proteins determined by Q-TOF-MS/MS are shown in the lower panels. The immunoblotting data are shown as representative blots obtained in independent experiments from 3 rats. Data represent the means ± SE. Statistical significance was determined by ANOVA followed by Bonferroni’s test. 1 p < 0.05 vs Hip, Fro, Par, Occ, and Amy, 2 p < 0.05 vs Amy, 3 p < 0.05 Amy and Hip. Figure 5 Immunoblotting analysis of proteins identified in six regions of rat brain. Rat brains were divide Figure 5 Immunoblotting analysis of proteins identified in six regions of rat brain. Rat brains were divided into six regions (thalamus, hippocampus, frontal cortex, parietal cortex, occipital cortex, and amygdala), and were extracted with lysis buffer. The samples (2.0 μg protein) were subjected to immunoblotting with anti-GFAP antibody (A), anti-GAT 3 antibody (B), anti-Septin 5 antibody (C), anti-HSP 90 antibody (D), anti-synaptotagmin antibody (E), and anti-HSP 70 antibody (F). Immunoblotting data using the indicated antibodies are shown in the upper panels; Mascot scores of the proteins determined by Q-TOF-MS/MS are shown in the lower panels. The immunoblotting data are shown as representative blots obtained in independent experiments from 3 rats. Data represent the means ± SE. Statistical significance was determined by ANOVA followed by Bonferroni’s test. Experimental Procedures Materials Anti-GFAP, anti-GAT 3, and anti-synaptophysin antibo- dies were from Sigma-Aldrich (St. Louis, MO). Anti- HSP 70, anti-HSP 90, and anti-a-enolase antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The anti-synaptotagmin antibody was from BD Transduction Laboratories (Mississauga, ON). The anti- Septin 5 antibody was from Abcam (Cambridge, MA). The anti-pyruvate kinase antibody was from Chemicon International, Inc (Temecula, CA). Sequencing grade- modified trypsin was from Promega Corp. (Madison, WI). All other chemicals were commercially available. Figure 6 Immunohistochemical analysis of proteins identified by nano-LC-Q-TOF-MS/MS in rat brain. Rat brains were removed, fixed with 4% paraformaldehyde, and stained with hematoxylin and eosin (A), anti-GAT 3 antibody (B), anti-GFAP antibody (C), or anti- HSP 70 antibody (D) as described in “Experimental Procedures”. The panels show representative photographs from independent experiments from 3 rats. Final magnification × 10. Animals Serum- and glucocorticoid-inducible kinase1 increased the acetylation and activation of NF- kB through phosphorylation of p300, and this also leads to the expression of N-methyl-d-aspartate receptor, NR2A and NR2B that is implicated in neuronal plasticity in hippocampus [40]. Dynamic chromatin remodeling in hippocampal neurons are associated with N-methyl-d-aspartate receptor-mediated activation of Bdnf gene promoter 1 [41]. BDNF activates ERK path- way in hippocampus neurons. The pathways shown by IPA analysis based on the data are closely related to the biological functions reported in each region of brain, suggesting that proteomic profiling of regions may be useful for the elucidation of biological functions. Conclusions A total of 2,909 peptides in all regions of the rat brain were identified, and we displayed proteins expressed in each region of brain: 250 proteins in the thalamus, 225 in the hippocampus, 149 in the frontal cortex, 273 in the parietal cortex, 202 in the occipital cortex, and 198 in the amygdala. Of the identified proteins, the expres- sions of GFAP, GAT3, Septin 5, HSP 90, synaptotagmin, HSP 70, and pyruvate kinase were confirmed by immu- noblotting, and localizations of GAT3, GFAP, and HSP 70 were confirmed by immunohistochemistry. Proteo- mics analysis in each region of the brain reveals that Animals Sprague-Dawley rats were housed in individual plastic cages (40 × 25 × 25 cm) with wood chip bedding in a room with a 12 h light cycle (12:12 light-dark) main- tained at 22°C. Animals had free access to food pellets and tap water. All experiments were accordance with the standards of the Committee for Ethics on Animal Experiments at Yamaguchi University School of Medi- cine. Rats were randomly assigned from a group, and were anesthetized with Nembutal (40 mg/kg, i.p.). After the anesthetization, rats were transcardially perfused with 0.9% chilled saline, and the brains were removed. In rat brains, cortex, hippocampus, thalamus, and amyg- dala were dissected, and the cortex were divided into 3 sections, frontal, parietal, and occipital cortexes. The division into six regions: thalamus, hippocampus, frontal cortex, parietal cortex, occipital cortex, and amygdala was carried out according to the atlas of Paxinos and Watson. Samples were homogenized individually in lysis buffer [150 mM Tris (pH 6.8), 12% (w/v) SDS, 36% (v/v) glycerol, and 6% (v/v) 2-mercaptoethanol]. The samples were centrifuged (15,000 × g, 30 min, 4°C), and the supernatants were stored at -20°C. In hippocampus playing an important role in long-term memory, the pathways involving in signal transduction such as ERK, NF-kB, S6 kinase, and mTOR were observed by IPA analysis. Long-term depression in hip- pocampus requires rapid protein synthesis, which is associated with mTOR, ERK, and S6-dependent signal- ing pathways [39]. Serum- and glucocorticoid-inducible kinase1 increased the acetylation and activation of NF- kB through phosphorylation of p300, and this also leads to the expression of N-methyl-d-aspartate receptor, NR2A and NR2B that is implicated in neuronal plasticity in hippocampus [40]. Dynamic chromatin remodeling in hippocampal neurons are associated with N-methyl-d-aspartate receptor-mediated activation of Bdnf gene promoter 1 [41]. BDNF activates ERK path- way in hippocampus neurons. The pathways shown by IPA analysis based on the data are closely related to the biological functions reported in each region of brain, suggesting that proteomic profiling of regions may be useful for the elucidation of biological functions. In hippocampus playing an important role in long-term memory, the pathways involving in signal transduction such as ERK, NF-kB, S6 kinase, and mTOR were observed by IPA analysis. Long-term depression in hip- pocampus requires rapid protein synthesis, which is associated with mTOR, ERK, and S6-dependent signal- ing pathways [39]. Discussion 1 p < 0.05 vs Hip, Fro, Par, Occ, and Amy, 2 p < 0.05 vs Amy, *3 p < 0.05 Amy and Hip. role in Alzheimer’s disease and frontotemporal dementia [38]. associated with amino acid metabolism, molecular trans- port, and small molecular biochemistry in most regions, which is consistent with the previous observations that neuron activity in the brain was regulated by small molecules such as neurotransmitter and ion transport. Network analysis of the proteins expressed in each region of rat brain indicates that these proteins were linked in a pathway. Many of these proteins have Page 8 of 12 Katagiri et al. Proteome Science 2010, 8:41 http://www.proteomesci.com/content/8/1/41 protein compositions differ among the regions, and these differences in protein expression may be involved in distinct biological actions. Further investigations are needed to elucidate the molecules involved in the biolo- gical actions that take place in each region of the brain. Figure 6 Immunohistochemical analysis of proteins identified by nano-LC-Q-TOF-MS/MS in rat brain. Rat brains were removed, fixed with 4% paraformaldehyde, and stained with hematoxylin and eosin (A), anti-GAT 3 antibody (B), anti-GFAP antibody (C), or anti- HSP 70 antibody (D) as described in “Experimental Procedures”. The panels show representative photographs from independent experiments from 3 rats. Final magnification × 10. In-gel digestion with trypsin Samples were subjected to SDS-PAGE, and stained lightly with Coomassie Brilliant Blue. In-gel digestion with trypsin was performed as described previously [22,42]. The protein bands in the lanes for samples from the six regions were excised from the gel and divided equally into 24 slices. Each gel slice was diced into small pieces and destained by rinsing in 30% acetonitrile con- taining 25 mM NH4HCO3. After the gel pieces were dehydrated in 100% acetonitrile, they were dried Katagiri et al. Proteome Science 2010, 8:41 http://www.proteomesci.com/content/8/1/41 Page 9 of 12 Figure 7 Network analysis of proteins identified in each region of rat brain by Ingenuity pathway analysis. The networks were analyzed based on the data of proteins identified in the indicated regions of rat brain. The networks were revealed as circles (genes) and lines (biological relationship). Solid lines mean direct interaction, and dotted lines show indirect interactions between the genes. Figure 7 Network analysis of proteins identified in each region of rat brain by Ingenuity pathway analysis. The networks were analyzed based on the data of proteins identified in the indicated regions of rat brain. The networks were revealed as circles (genes) and lines (biological relationship). Solid lines mean direct interaction, and dotted lines show indirect interactions between the genes. Figure 7 Network analysis of proteins identified in each region of rat brain by Ingenuity pathway analysis. The networks were analyzed based on the data of proteins identified in the indicated regions of rat brain. The networks were revealed as circles (genes) and lines (biological relationship). Solid lines mean direct interaction, and dotted lines show indirect interactions between the genes. Page 10 of 12 Page 10 of 12 Katagiri et al. Proteome Science 2010, 8:41 http://www.proteomesci.com/content/8/1/41 naturally at room temperature for 30 min. The proteins in the gel pieces were reduced by incubation with 10 mM dithiothreitol in 25 mM NH4HCO3 at 56°C for 1 hr, and alkylated with 55 mM iodoacetamide in 25 mM NH4HCO3 at room temperature for 45 min in the dark. The gel pieces were dehydrated in 50% acetonitrile containing 25 mM NH4HCO3 twice for 30 min, and then in 100% acetonitrile once for 5 min. After drying for 30 min at room temperature, the gel pieces were rehydrated in TPCK-treated trypsin solution (Trypsin Gold, Mass spectrometry grade, Promega; 10 ng/μl in 25 mM NH4HCO3) on ice for 30 min. Mass spectrometric analysis The digested peptides were subjected to LC-MS/MS analysis as described previously [22,24]. Briefly, LC-MS/ MS analysis was performed on a Q-Tof Micro (Micro- mass, Manchester, UK) interfaced with a capillary reverse-phase liquid chromatograph (Micromass CapLC™system) as described previously [24]. A linear gradient of acetonitrile in 0.1% formic acid was pro- duced and split at a 1:20 ratio. The gradient solution was then injected into a nano LC column (PepMap C18, 75 μm × 150 mm, LC Packings, Amsterdam, Holland) at 100 nl/min. The eluted peptides were sprayed directly into the mass spectrometer. The MS/MS data were acquired by MassLynx software (ver. 4.0; Micromass) using automatic switching between MS and MS/MS modes, and converted to a single text file (containing the observed m/z of the precursor peptide, the fragment ion m/z, and intensity values) by ProteinLynx software (ver. 2.0; Micromass). The files were analyzed by Mascot MS/MS Ions Search (ver. 3.5; Matrix Science Ltd.) to search and assign the obtained peptides to the NCBI Reference Sequence database (RefSeQ 20060317; 119764 sequences; selected species for the database were Homo sapiens, Mus musculus, and Rattus norvegicus). Peaks from porcine trypsin, human keratins and bovine serum proteins were removed. We set the parameters as fol- lows: Parent mass error tolerance, ± 0.4 Da; Fragment mass error tolerance, ± 0.2 Da; Enzyme, trypsin; Maxi- mum number of missed cleavages, 1; Fixed post-transla- tional modifications, none; Variable post-translational modifications, oxidation (Met), carbamidomethyl (Cys), propionamide (Cys); Mass values, Monoisotopic. For peptide and protein identification, the search results were processed using a STEM software (STrategic Extractor for Mascot results) as follows [43]. (i) The Electrophoresis and immunoblotting Electrophoresis and immunoblotting were carried out as described previously [21,44,45]. The brain extracts (400 ng) and molecular mass standards were sub- jected to electrophoresis in 10% (w/v) polyacrylamide gels in the presence of SDS, and transferred to nitro- cellulose membranes. The blots were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.05% (w/v) Tween-20, and incubated with antibody. The blots were then washed, and the antigens were visualized by enhanced chemiluminescent detection reagents. Histochemistry and immunohistochemistry Rat brains were removed, washed with phosphate-buf- fered solution, and fixed with 4% paraformaldehyde. Then the paraffin-embedded samples were sliced into 4 μm pieces, and stained with hematoxylin and eosin [46,47]. For immunohistochemical observation, the specimens were incubated with 3% H2O2 in phos- phate-buffered saline to quench the endogenous perox- idase activity, and then with blocking solution (Dako, Foster City, CA) to inhibit non-specific binding after deparaffinization. Antigen retrieval was performed in 100% formic acid for 1 min at room temperature, and the samples were incubated with antibodies in 1% bovine serum albumin in phosphate-buffered saline for 1 hr and immunostained by the avidin-biotin peroxi- dase complex method using a Vectastatin kit (Vector Laboratories, Burlingame, CA). The peroxidase label was visualized by exposing the sections to diaminobenzidine. In-gel digestion with trypsin After removing excess solution, digestion was performed overnight at 37°C. The resulting peptides were extracted twice with 50% acetonitrile containing 0.1% trifluoroacetic for 30 min. The extracts were dried in a vacuum concentra- tor and dissolved in a solution of 0.1% formic acid for mass spectrometric analysis. candidate peptide sequences were screened with the probability-based MOWSE scores that exceeded their thresholds (p < 0.05) and with MS/MS signals for y- or b-ions > 3. (ii) Redundant peptide sequences were removed. (iii) Each peptide sequence was assigned to a protein that gave the maximal number of peptide assignments among the candidates. In this study, visual inspection was applied to individual MS/MS spectra for reliable identification of peptide sequences. Gene names followed the NCBI Gene ID. Ingenuity network analysis The proteins identified in each region were carried out network analysis using Ingenuity Pathways analysis ver.8.6 (IPA, http://www.ingenuity.com). IPA analysis discerns molecular and cellular functions and canonical pathways on the basis of millions findings reported in the literatures, and the software is weekly updated. IPA uses a Fisher’s exact test to determine whether the input genes were significantly related to pathways compared to the whole ingenuity knowledge base. Page 11 of 12 Page 11 of 12 Katagiri et al. Proteome Science 2010, 8:41 http://www.proteomesci.com/content/8/1/41 6. Caron H, van Schaik B, van der Mee M, Baas F, Riggins G, van Sluis P, Hermus MC, van Asperen R, Boon K, Voute PA, et al: The human transcriptome map: clustering of highly expressed genes in chromosomal domains. Science 2001, 291:1289-1292. Author details 1Center for Gene Research, Yamaguchi University, Yamaguchi, 755-8505, Japan. 2Department of Rare Sugar Research Center, Kagawa University, Kagawa, 761-0793, Japan. 3First Department of Pathology, Yamaguchi University Graduate School of Medicine, Yamaguchi, 755-8505, Japan. 4Department of Neuroscience Yamaguchi University Graduate School of Medicine, Yamaguchi, 755-8505, Japan. 5Department of Stress and Bio- response Medicine, Yamaguchi University Graduate School of Medicine, Yamaguchi, 755-8505, Japan. 6Department of Microbiology and Immunology, Yamaguchi University Graduate School of Medicine, Yamaguchi, 755-8505, Japan. 15. Schindler J, Lewandrowski U, Sickmann A, Friauf E, Nothwang HG: Proteomic analysis of brain plasma membranes isolated by affinity two- phase partitioning. Mol Cell Proteomics 2006, 5:390-400. 16. Kim SY, Chudapongse N, Lee SM, Levin MC, Oh JT, Park HJ, Ho IK: Proteomic analysis of phosphotyrosyl proteins in morphine-dependent rat brains. Brain Res Mol Brain Res 2005, 133:58-70. 17. Fountoulakis M, Tsangaris GT, Maris A, Lubec G: The rat brain hippocampus proteome. J Chromatogr B Analyt Technol Biomed Life Sci 2005, 819:115-129. g , , p p gy gy, Yamaguchi University Graduate School of Medicine, Yamaguchi, 755-8505, Japan. 18. Pan S, Shi M, Jin J, Albin RL, Lieberman A, Gearing M, Lin B, Pan C, Yan X, Kashima DT, Zhang J: Proteomics identification of proteins in human cortex using multidimensional separations and MALDI tandem mass spectrometer. Mol Cell Proteomics 2007, 6:1818-1823. Received: 25 February 2010 Accepted: 27 July 2010 Published: 27 July 2010 22. Kikuchi M, Hatano N, Yokota S, Shimozawa N, Imanaka T, Taniguchi H: Proteomic analysis of rat liver peroxisome: presence of peroxisome- specific isozyme of Lon protease. J Biol Chem 2004, 279:421-428. Abbreviations C l d h 11. Ibrahim SM, Mix E, Bottcher T, Koczan D, Gold R, Rolfs A, Thiesen HJ: Gene expression profiling of the nervous system in murine experimental autoimmune encephalomyelitis. Brain 2001, 124:1927-1938. LC: liquid chromatography; Q-TOF: quadrupole-time of flight; MS/MS: tandem mass spectrometry; SDS: sodium dodecyl sulfate; PAGE: LC: liquid chromatography; Q-TOF: quadrupole-time of flight; MS/MS: tandem mass spectrometry; SDS: sodium dodecyl sulfate; PAGE: polyacrylamide gel electrophoresis; GFAP: glial fibrillary acidic protein; GAT 3: GABA transporter 3; HSP: heat shock protein; p y y polyacrylamide gel electrophoresis; GFAP: glial fibrillary acidic protein; GAT 3: GABA transporter 3; HSP: heat shock protein; 12. Mycko MP, Papoian R, Boschert U, Raine CS, Selmaj KW: cDNA microarray analysis in multiple sclerosis lesions: detection of genes associated with disease activity. Brain 2003, 126:1048-1057. Authors’ contributions TK, NH, TIzumi, TM, MS, and YM have made substantial contribution to the data acquisition and interpretation in proteomics. HK and TIshihara have participated in the acquisition of the immunohistochemical data. TK, MA, MO, and YM have carried out the experiments of western blotting. YL and SN have contributed to the sample preparations of rat brain. YM have preformed the design of the experiments and have been involved in writing the manuscript. All of authors have read and approved the final manuscript. 19. Paulson L, Martin P, Nilsson CL, Ljung E, Westman-Brinkmalm A, Blennow K, Davidsson P: Comparative proteome analysis of thalamus in MK-801- treated rats. Proteomics 2004, 4:819-825. 20. Yeom M, Shim I, Lee HJ, Hahm DH: Proteomic analysis of nicotine- associated protein expression in the striatum of repeated nicotine- treated rats. Biochem Biophys Res Commun 2005, 326:321-328. Acknowledgements 13. Sturzebecher S, Wandinger KP, Rosenwald A, Sathyamoorthy M, Tzou A, Mattar P, Frank JA, Staudt L, Martin R, McFarland HF: Expression profiling identifies responder and non-responder phenotypes to interferon-beta in multiple sclerosis. Brain 2003, 126:1419-1429. This work was supported in part by grants from the Ministry of Education, Science and Culture of Japan, Takeda Science Foundation, and NOVARTIS Foundation (Japan) for the Promotion of Science. 14. Schuchhardt J, Glintschert A, Hartl D, Irmler M, Beckers J, Stephan C, Marcus K, Klose J, Meyer HE, Malik A: BrainProfileDB - a platform for integration of functional genomics data. Proteomics 2008, 8:1162-1164. Additional material chromosomal domains. Science 2001, 291:1289-1292. Additional file 1: Table S1. Identification of proteins in each region of adult rat brain. Rat brains were divided into six regions, and extracted, separated by SDS-PAGE. The samples were subjected to nano-LC-Q-TOF- MS/MS, and analyzed by Mascot search. Mascot scores were subtracted cut off scores, and the localizations and biological functions were searched by NCBI Entrez Gene. 7. Sun T, Patoine C, Abu-Khalil A, Visvader J, Sum E, Cherry TJ, Orkin SH, Geschwind DH, Walsh CA: Early asymmetry of gene transcription in embryonic human left and right cerebral cortex. Science 2005, 308:1794-1798. 8. Lein ES, Hawrylycz MJ, Ao N, Ayres M, Bensinger A, Bernard A, Boe AF, Boguski MS, Brockway KS, Byrnes EJ, et al: Genome-wide atlas of gene expression in the adult mouse brain. Nature 2007, 445:168-176. Additional file 2: Table S2. Lists of ingenuity networks generated by proteins identified in each region of rat brain. Additional file 2: Table S2. Lists of ingenuity networks generated by proteins identified in each region of rat brain. 9. Enard W, Khaitovich P, Klose J, Zollner S, Heissig F, Giavalisco P, Nieselt- Struwe K, Muchmore E, Varki A, Ravid R, et al: Intra- and interspecific variation in primate gene expression patterns. Science 2002, 296:340-343 Additional file 3: Figure S1. Canonical pathways analyzed by proteins identified in each region of rat brain. 10. Pomeroy SL, Tamayo P, Gaasenbeek M, Sturla LM, Angelo M, McLaughlin ME, Kim JY, Goumnerova LC, Black PM, Lau C, et al: Prediction of central nervous system embryonal tumour outcome based on gene expression. Nature 2002, 415:436-442. Competing interests 21. Mizukami Y, Iwamatsu A, Aki T, Kimura M, Nakamura K, Nao T, Okusa T, Matsuzaki M, Yoshida K, Kobayashi S: ERK1/2 regulates intracellular ATP levels through alpha-enolase expression in cardiomyocytes exposed to ischemic hypoxia and reoxygenation. J Biol Chem 2004, 279:50120-50131. The authors declare that they have no competing interests. Received: 25 February 2010 Accepted: 27 July 2010 Published: 27 July 2010 26. Nunomura K, Nagano K, Itagaki C, Taoka M, Okamura N, Yamauchi Y, Sugano S, Takahashi N, Izumi T, Isobe T: Cell surface labeling and mass References 1. Ethofer T, Pourtois G, Wildgruber D: Investigating audiovisual integration of emotional signals in the human brain. Prog Brain Res 2006, 156:345-361. 1. Ethofer T, Pourtois G, Wildgruber D: Investigating audiovisual integration of emotional signals in the human brain. Prog Brain Res 2006, 156:345-361. 2. Taylor SF, Welsh RC, Wager TD, Phan KL, Fitzgerald KD, Gehring WJ: A functional neuroimaging study of motivation and executive function. Neuroimage 2004, 21:1045-1054. 3. Allman JM, Hakeem A, Erwin JM, Nimchinsky E, Hof P: The anterior cingulate cortex. The evolution of an interface between emotion and cognition. Ann N Y Acad Sci 2001, 935:107-117. 4. Ross-Macdonald P, Coelho PS, Roemer T, Agarwal S, Kumar A, Jansen R, Cheung KH, Sheehan A, Symoniatis D, Umansky L, et al: Large-scale analysis of the yeast genome by transposon tagging and gene disruption. Nature 1999, 402:413-418. 5 Young RA: Biomedical discovery with DNA arrays Cell 2000 102:9 15 23. Kawakami T, Hoshida Y, Kanai F, Tanaka Y, Tateishi K, Ikenoue T, Obi S, Sato S, Teratani T, Shiina S, et al: Proteomic analysis of sera from hepatocellular carcinoma patients after radiofrequency ablation treatment. Proteomics 2005, 5:4287-4295. 2. Taylor SF, Welsh RC, Wager TD, Phan KL, Fitzgerald KD, Gehring WJ: A functional neuroimaging study of motivation and executive function. Neuroimage 2004, 21:1045-1054. 2. Taylor SF, Welsh RC, Wager TD, Phan KL, Fitzgerald KD, Gehring WJ: A functional neuroimaging study of motivation and executive function. Neuroimage 2004, 21:1045-1054. 24. Tokumitsu H, Hatano N, Inuzuka H, Yokokura S, Nozaki N, Kobayashi R: Mechanism of the generation of autonomous activity of Ca2 +/calmodulin-dependent protein kinase IV. J Biol Chem 2004, 279:40296-40302. g 3. Allman JM, Hakeem A, Erwin JM, Nimchinsky E, Hof P: The anterior cingulate cortex. The evolution of an interface between emotion and cognition. Ann N Y Acad Sci 2001, 935:107-117. 25. Tokumitsu H, Hatano N, Inuzuka H, Sueyoshi Y, Yokokura S, Ichimura T, Nozaki N, Kobayashi R: Phosphorylation of Numb family proteins. Possible involvement of Ca2+/calmodulin-dependent protein kinases. J Biol Chem 2005, 280:35108-35118. 4. Ross-Macdonald P, Coelho PS, Roemer T, Agarwal S, Kumar A, Jansen R, Cheung KH, Sheehan A, Symoniatis D, Umansky L, et al: Large-scale analysis of the yeast genome by transposon tagging and gene disruption. Nature 1999, 402:413-418. 26. Nunomura K, Nagano K, Itagaki C, Taoka M, Okamura N, Yamauchi Y, Sugano S, Takahashi N, Izumi T, Isobe T: Cell surface labeling and mass 26. spectrometry reveal diversity of cell surface markers and signaling molecules expressed in undifferentiated mouse embryonic stem cells. Mol Cell Proteomics 2005, 4:1968-1976. Vaidya JG, Paradiso S, Andreasen NC, Johnson DL, Boles Ponto LL, Hichwa RD: Correlation between extraversion and regional cerebral blood flow in response to olfactory stimuli. Am J Psychiatry 2007, 164:339-341. 36. Amin ND, Zheng YL, Kesavapany S, Kanungo J, Guszczynski T, Sihag RK, Rudrabhatla P, Albers W, Grant P, Pant HC: Cyclin-dependent kinase 5 phosphorylation of human septin SEPT5 (hCDCrel-1) modulates exocytosis. J Neurosci 2008, 28:3631-3643. 37. Peng XR, Jia Z, Zhang Y, Ware J, Trimble WS: The septin CDCrel-1 is dispensable for normal development and neurotransmitter release. Mol Cell Biol 2002, 22:378-387. 38. Chen F, Wollmer MA, Hoerndli F, Munch G, Kuhla B, Rogaev EI, Tsolaki M, Papassotiropoulos A, Gotz J: Role for glyoxalase I in Alzheimer’s disease. Proc Natl Acad Sci USA 2004, 101:7687-7692. 39. Antion MD, Hou L, Wong H, Hoeffer CA, Klann E: mGluR-dependent long- term depression is associated with increased phosphorylation of S6 and synthesis of elongation factor 1A but remains expressed in S6K-deficient mice. Mol Cell Biol 2008, 28:2996-3007. 40. Tai DJ, Su CC, Ma YL, Lee EH: SGK1 phosphorylation of IkappaB Kinase alpha and p300 Up-regulates NF-kappaB activity and increases N- Methyl-D-aspartate receptor NR2A and NR2B expression. J Biol Chem 2009, 284:4073-4089. 41. Tian F, Hu XZ, Wu X, Jiang H, Pan H, Marini AM, Lipsky RH: Dynamic chromatin remodeling events in hippocampal neurons are associated with NMDA receptor-mediated activation of Bdnf gene promoter 1. J Neurochem 2009, 109:1375-1388. 42. Shevchenko A, Jensen ON, Podtelejnikov AV, Sagliocco F, Wilm M, Vorm O, Mortensen P, Shevchenko A, Boucherie H, Mann M: Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. Proc Natl Acad Sci USA 1996, 93:14440-14445. 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: Submit your next manuscript to BioMed Central and take full advantage of: 43. Shinkawa T, Taoka M, Yamauchi Y, Ichimura T, Kaji H, Takahashi N, Isobe T: STEM: a software tool for large-scale proteomic data analyses. J Proteome Res 2005, 4:1826-1831. 44. spectrometry reveal diversity of cell surface markers and signaling molecules expressed in undifferentiated mouse embryonic stem cells. Mol Cell Proteomics 2005, 4:1968-1976. affected by systemic Alambda amyloidosis with antibodies against three different regions of immunoglobulin lambda light chain. Pathol Int 2007, 57:343-350. affected by systemic Alambda amyloidosis with antibodies against three different regions of immunoglobulin lambda light chain. Pathol Int 2007, 57:343-350. spectrometry reveal diversity of cell surface markers and signaling molecules expressed in undifferentiated mouse embryonic stem cells. Mol Cell Proteomics 2005, 4:1968-1976. spectrometry reveal diversity of cell surface markers and signaling molecules expressed in undifferentiated mouse embryonic stem cells. Mol Cell Proteomics 2005, 4:1968-1976. 47. Hoshii Y, Kiyama M, Cui D, Kawano H, Ishihara T: Immunohistochemical study of immunoglobulin light chain amyloidosis with antibodies to the immunoglobulin light chain variable region. Pathol Int 2006, 56:324-330. 27. Nagano K, Taoka M, Yamauchi Y, Itagaki C, Shinkawa T, Nunomura K, Okamura N, Takahashi N, Izumi T, Isobe T: Large-scale identification of proteins expressed in mouse embryonic stem cells. Proteomics 2005, 5:1346-1361. doi:10.1186/1477-5956-8-41 Cite this article as: Katagiri et al.: Proteomic analysis of proteins expressing in regions of rat brain by a combination of SDS-PAGE with nano-liquid chromatography-quadrupole-time of flight tandem mass spectrometry. Proteome Science 2010 8:41. 28. Crunelli V, Leresche N: A role for GABAB receptors in excitation and inhibition of thalamocortical cells. Trends Neurosci 1991, 14:16-21. 29. Ulrich D, Huguenard JR: GABAB receptor-mediated responses in GABAergic projection neurones of rat nucleus reticularis thalami in vitro. J Physiol 1996, 493(Pt 3):845-854. 30. De Biasi S, Vitellaro-Zuccarello L, Brecha NC: Immunoreactivity for the GABA transporter-1 and GABA transporter-3 is restricted to astrocytes in the rat thalamus. A light and electron-microscopic immunolocalization. Neuroscience 1998, 83:815-828. 31. Kawamoto M, Ohno K, Kuriyama K, Kubo T, Sato K: Developmental changes in GABA transporter (GAT1 and GAT3) mRNA expressions in the rat olfactory bulb. Brain Res Dev Brain Res 2001, 126:137-145. 32. Wensley CH, Stone DM, Baker H, Kauer JS, Margolis FL, Chikaraishi DM: Olfactory marker protein mRNA is found in axons of olfactory receptor neurons. J Neurosci 1995, 15:4827-4837. 33. Otaki JM, Yamamoto H, Firestein S: Odorant receptor expression in the mouse cerebral cortex. J Neurobiol 2004, 58:315-327. 34. Qureshy A, Kawashima R, Imran MB, Sugiura M, Goto R, Okada K, Inoue K, Itoh M, Schormann T, Zilles K, Fukuda H: Functional mapping of human brain in olfactory processing: a PET study. J Neurophysiol 2000, 84:1656-1666. 35. References Nunomura K, Nagano K, Itagaki C, Taoka M, Okamura N, Yamauchi Y, Sugano S, Takahashi N, Izumi T, Isobe T: Cell surface labeling and mass 5. Young RA: Biomedical discovery with DNA arrays. Cell 2000, 102:9-15. Katagiri et al. Proteome Science 2010, 8:41 http://www.proteomesci.com/content/8/1/41 Katagiri et al. Proteome Science 2010, 8:41 http://www.proteomesci.com/content/8/1/41 Page 12 of 12 affected by systemic Alambda amyloidosis with antibodies against three different regions of immunoglobulin lambda light chain. Pathol Int 2007, 57:343-350. 47. Hoshii Y, Kiyama M, Cui D, Kawano H, Ishihara T: Immunohistochemical study of immunoglobulin light chain amyloidosis with antibodies to the immunoglobulin light chain variable region. Pathol Int 2006, 56:324-330. doi:10.1186/1477-5956-8-41 Cite this article as: Katagiri et al.: Proteomic analysis of proteins expressing in regions of rat brain by a combination of SDS-PAGE with nano-liquid chromatography-quadrupole-time of flight tandem mass spectrometry. Proteome Science 2010 8:41. affected by systemic Alambda amyloidosis with antibodies against three different regions of immunoglobulin lambda light chain. Pathol Int 2007, 57:343-350. 47. Hoshii Y, Kiyama M, Cui D, Kawano H, Ishihara T: Immunohistochemical study of immunoglobulin light chain amyloidosis with antibodies to the immunoglobulin light chain variable region. Pathol Int 2006, 56:324-330. spectrometry reveal diversity of cell surface markers and signaling molecules expressed in undifferentiated mouse embryonic stem cells. Mol Cell Proteomics 2005, 4:1968-1976. Mizukami Y, Kobayashi S, Uberall F, Hellbert K, Kobayashi N, Yoshida K: Nuclear mitogen-activated protein kinase activation by protein kinase czeta during reoxygenation after ischemic hypoxia. J Biol Chem 2000, 275:19921-19927. 45. Mizukami Y, Yoshioka K, Morimoto S, Yoshida K: A novel mechanism of JNK1 activation. Nuclear translocation and activation of JNK1 during ischemia and reperfusion. J Biol Chem 1997, 272:16657-16662. 46. Kiyama M, Hoshii Y, Cui D, Kawano H, Kanda T, Ishihara T: Immunohistochemical and immunochemical study of amyloid in liver 46. Kiyama M, Hoshii Y, Cui D, Kawano H, Kanda T, Ishihara T: 46. Kiyama M, Hoshii Y, Cui D, Kawano H, Kanda T, Ishihara T: Immunohistochemical and immunochemical study of amyloid in liver Immunohistochemical and immunochemical study of amyloid in liver
https://openalex.org/W1785161218	https://www.cienciasmarinas.com.mx/index.php/cmarinas/article/download/1470/1789	Spanish; Castilian	null	Sediment variations and littoral transport at La Victoria Beach, Cádiz, Spain	Ciencias marinas	2,009	cc-by	9,592	Sediment variations and littoral transport at La Victoria Beach, Cádiz, Spain Variaciones sedimentarias y transporte litoral en Playa de la Victoria, Cádiz, España GE Avila-Serrano1, MA Santa Rosa del Río1, G Anfuso-Melfi2, J Benavente-González2, R Guardado-France1, O González-Yajimovich1, EK Velázquez-González3 GE Avila-Serrano1, MA Santa Rosa del Río1, G Anfuso-Melfi2, J Benavente-González2, R Guardado-France1, O González-Yajimovich1, EK Velázquez-González3 1 Departamento de Geología, Facultad de Ciencias Marinas, Universidad Autónoma de Baja California, Carretera Tijuana- Ensenada Km 103, Apartado postal 453, Ensenada, CP 22870, Baja California, México. * E-mail: gavila@uabc.mx 2 Departamento de Geología, Facultad de Ciencias del Mar y Ambientales, Universidad de Cádiz, Puerto Real, España. 3División de Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana, México. Resumen Se evaluó la evolución de Playa de la Victoria, localizada en la provincia de Cádiz (España), para el periodo de febrero a octubre de 2004, comparando dos secciones de playa, la sección norte o Final Victoria (FV) y la sección central u Hotel Victoria (HV), su litología y pendiente. Se determinaron los parámetros texturales y contenido de CaCO3 de 24 muestras de sedimento y se obtuvo el transporte litoral por medio de trazadores fluorescentes. Se midieron perfiles topográficos con una estación total durante la bajamar viva cada 15 días. Las muestras de sedimento fueron analizadas por el método de momentos y se calcularon sus parámetros texturales (media, asimetría, clasificación y curtosis). Los resultados muestran que, durante el periodo estudiado, el sedimento en la sección FV estuvo moderadamente bien clasificado y extremadamente leptocúrtico, mientras que la sección HV el análisis mostró dos grupos de sedimento: arenas finas cerca de la rompiente y guijarros muy finos en la berma; sin embargo, en ambas secciones fue evidente el periodo de invierno. El contenido de CaCO3 varió entre 3% y 9%, lo que indica inestabilidad de la playa. La dirección del transporte litoral fue NW–SE, coincidiendo con el comportamiento de la región, y se encontró un desplazamiento de 130 m a una velocidad de 0.29 m s–1. Palabras clave: parámetros texturales, perfiles de playa, periodo de invierno. Abstract The evolution of La Victoria Beach, located in the province of Cádiz (Spain), was evaluated from February to October 2004 by comparing the lithology and slope of two beach sections: the northern or Final Victoria (FV) section and the southern or Hotel Victoria (HV) section. Textural parameters and CaCO3 content of 24 sediment samples were obtained, and littoral transport was determined by fluorescent tracers. Topographic profiles were measured every 15 days using a total station during low spring tides. The sediment samples were analyzed by the method of moments and their textural parameters (mean, asymmetry, sorting, and kurtosis) calculated. The textural analysis showed that the sediment in section FV during the study period was moderately well classified and extremely leptokurtic, whereas section HV had two sediment groups: fine sands near the breaker zone and very fine pebbles at the berm; however, the winter period was evident in both sections. The CaCO3 content varied between 3% and 9%, indicating beach instability. The direction of the littoral transport, in agreement with the region’s behavior, was NW–SE, and showed a displacement of 130 m at 0.29 m s–1. Key words: beach profiles, textural parameter, winter period. Ciencias Marinas (2009), 35(3): 259–269 Ciencias Marinas (2009), 35(3): 259–269 http://dx.doi.org/10.7773/cm.v35i3.1470 Introducción Esta situación ha provocado que el 40% de la costa esté urbanizada o sea urbanizable (MOPU 1991). Los ejemplos más evidentes de infraestructuras en la línea de costa son las marinas y los puertos, que se utilizan para satisfacer necesidades económicas de las ciudades costeras. Asimismo, la regeneración o realimentación artificial de las playas permite contar con mayores extensiones de arena, lo cual es un gran atractivo turístico y propicia la entrada de divi- sas al país (Anfuso 2001). Entre 1983 y 1993, se regeneró el 14% de las playas españolas y casi la tercera parte de estas labores fueron realizadas en las costas de Andalucía (CMAJA 1995). El retroceso de la línea de costa en España se ha comba- tido con obras de regeneración, muchas veces acompañadas de la construcción de estructuras costeras, como sucede en la pro- vincia de Cádiz. En la década de 1990 se realizaron inversiones de €63,158.00 anuales para frenar o revertir estos procesos (Muñoz et al. 2001). Many beaches have a special morphology, such as those on rocky platforms that limit wave height and energy, which in turn reduces the volume of sediment deposited (Muñoz-Pérez 1996). Such beaches are found in the province of Cádiz (e.g., Regla Beach in Chipiona, Fuentebravía Beach in the port of Santa María, and La Victoria Beach in the city of Cádiz). The rocky-shore platform of these beaches is composed of sand- stone and calcareous conglomerates of Pliocene age and its upper limit is around mean sea level (Muñoz-Pérez 1996). Several regeneration projects have been implemented in recent years to increase beach width (berm and upper beach face) in several areas of Cádiz, especially those important for tourism. Among the most important beaches are La Victoria (Cádiz), La Barrosa (Chiclana), Camposoto (San Fernando), and Regla (Chipiona). These projects were undertaken after short-term point studies conducted by private companies under the supervision of the Ministry of the Environment’s Demarca- tion of Andalusian Atlantic Coasts program (Anfuso 2001). Existen playas con una morfología especial, tales como las que están sobre lajas rocosas que limitan la altura de las olas así como su energía, produciendo una disminución en el volu- men de sedimento depositado (Muñoz-Pérez 1996). Introducción Hence, beach profiles are an important indicator of shoreline variations since they allow us to reconstruct the morphology of the region, as well as infer the volume of sediment that is eroded or deposited in a certain time. Para poder entender los cambios morfológicos de las pla- yas, se han hecho diferentes estudios en los cuales se conside- ran los fenómenos perpendiculares y paralelos a la playa de manera independiente, lo cual puede funcionar para tramos de costa lo suficientemente largos y homogéneos (Muñoz-Pérez 1996). Por ello los perfiles de playa son un indicador impor- tante de las variaciones de la línea de costa, ya que nos permi- ten reconstruir la morfología de la región, así como inferir el volumen de sedimento que es erosionado o depositado en un determinado tiempo. As a result of Spain’s benign climate, both its Atlantic and Mediterranean coasts (especially in Andalusia) are popular destinations during several months of the year. The country has more than 8000 km of littoral, and the coastal zone (considered to be about 5 km wide) shelters 30% of the population, increas- ing to 82% in the summer (June–August). This means that 40% of the coastline has been urbanized or can be developed (MOPU 1991). Marinas and ports are the most evident examples of coastal infrastructures that play a key role in supporting the economy of nearby communities. The artificial regeneration or restoration of sandy beaches is also important to promote tourism and thus increase the input of foreign currency (Anfuso 2001). Between 1983 and 1993, 14% of Spanish beaches were regenerated and almost a third of them were in Andalusia (CMAJA 1995). Coastline regression has been checked by regeneration projects, oftentimes accompanied by the construction of coastal structures, as occurs in the province of Cádiz. During the 1990s, these projects yielded annual investments of €63,158.00 (Muñoz et al. 2001). p En España, las buenas condiciones climáticas hacen que el ambiente costero resulte atractivo durante varios meses del año, tanto en la costa mediterránea como en la vertiente atlán- tica, especialmente en Andalucía. Las costas españolas cuentan con 8000 km de litoral y, si se considera un ancho de 5 km de la zona costera, éstas zonas albegan actualmente al 30% de la población, incrementándose temporalmente al 82% en el verano (junio a agosto). Introducción Desde siempre la zona costera ha atraído la atención del ser humano, por lo que muchas de las grandes ciudades de la anti- güedad se hallaban ubicadas en puertos naturales, como es el caso de Cádiz, en España. Las causas de dicha ocupación son muy diversass y se han ido multiplicando con el transcurrir de los siglos. En un principio ésta se ajustaba más a necesidades eminentemente prácticas (pesca, industria, transporte), a las que más recientemente se han sumado las de tipo lúdico y esté- tico (Benavente 2000). Esto ha llevado a una alta concentra- ción de población en las áreas más próximas a la costa (Short 1979, CERC 1984, Komar 1998). Coastal areas have always attracted humans. Many of the large ancient cities were located in natural harbours; such is the case of Cádiz (Spain). The motives for settling such areas have differed and changed over the centuries, from pri- marily practical purposes (fishing, industry, transport) in the beginning to recreational or scenic reasons in more recent times (Benavente 2000). This has led to a large concentration of population along the coastline (Short 1979, CERC 1984, Komar 1998). Beaches are gently sloping stretches of sand or gravel located along the shoreline, subject to wind, tide and wave 259 Ciencias Marinas, Vol. 35, No. 3, 2009 action, among other elements (Anfuso 2001). These energetic agents control the morphology of beaches, causing them to undergo constant changes (Carter 1988). Predicting possible future beach behaviour is therefore important in order to deter- mine how it will affect the contiguous infrastructure. Las playas son extensiones de arena o grava de poca pen- diente que se encuentran ubicadas a un costado del mar. Estas extensiones de arena se ven sometidas a la acción del viento, mareas y oleaje, entre las más importantes (Anfuso 2001). Dichos agentes energéticos son los que controlan la morfología de las playas, ocasionando que éstas se mantengan en cons- tante cambio (Carter 1988). Por las consecuencias que puedan tener sobre la infraestructura construida adyacente a la línea de costa, es de gran importancia poder predecir los posibles cam- bios que a futuro pueden presentar las playas. To be able to understand changes in beach morphology, several studies have been conducted considering longshore and onshore drifts independently, which is feasible for sufficiently long and homogeneous coastal sections (Muñoz-Pérez 1996). Materiales y métodos Playa de la Victoria (36º52′ N, 6º27′ W) se localiza en la zona suratlántica española (fig. 1), en el municipio de Cádiz, donde se extiende desde la zona urbana ocupando la parte occi- dental de la ciudad. Para este estudio se consideraron dos sec- ciones de la playa: (1) la sección norte o Final Victoria (FV), limita con el espigón sur de Playa Santa María del Mar y des- cansa sobre laja rocosa que sirve de protección natural al oleaje incidente; y (2) la sección central u Hotel Victoria (HV), que es una playa disipativa expuesta al oleaje frente al hotel del mismo nombre. Figure 1. Map of the study area showing the location and extension of the coastline at La Victoria Beach in SW Spain. Figura 1. Mapa de localización del área de que muestra la ubicación y extensión de la línea de costa en Playa de la Victoria al SO de Espana. Spain Chipiona Regla Tres Piedras La Ballena La Costilla Vistahermosa * El Puerto de Santa María Guadalete River Rota Puerto Real Atlantic Ocean 36° 35’ 36° 40’ 36° 45’ 36° 50’ 36° 55’ 37° 05’ 37° 00’ 37° 15’ 37° 10’ 6° 45’ 6° 40’ 6° 35’ 6° 30’ 6° 25’ 6° 20’ Cádiz San Fernando Chiclana 2.5 5 km 0 Los perfiles se realizaron de manera perpendicular a la costa utilizando una estación total Leica TC407 y un prisma con base graduada de 5 m. Esta estación permite medir ángulos verticales y horizontales, y está provista de una cruz filial con un par de hilos estadimétricos con los cuales se calcula la dife- rencia en la altura del terreno (distancias y desnivel). Se empleó la nivelación diferencial (King 1972), que es la meto- dología clásica usada en los estudios de morfodinámica. Los perfiles topográficos fueron espaciados homogéneamente a lo largo de toda el área de estudio, cada 30 m. La longitud de los perfiles dependía del ancho de la playa, de la pendiente y del nivel del mar en el momento de realizar la campaña, de manera que en la sección HV los perfiles tuvieron una longitud aproxi- mada de 180 m y un error en z de ± 0.02 m, mientras que en la sección FV su longitud aproximada fue de 70 m y un error en z de ± 0.02 m. Material and methods En los últimos años se han llevado a cabo varios proyectos de regeneración de playas con el fin de ampliar su ancho (berma y parte superior de la cara de la playa) en diferentes áreas de Cádiz, particularmente en áreas de importancia turística como las playas de la Victoria (Cádiz), La Barrosa (Chiclana), Camposoto (San Fernando) y Regla (Chipiona), entre las más importantes. Estas obras de regeneración han sido efectuadas tras estudios puntuales de corta duración realizados por empresas privadas bajo la supervisión de la Demarcación de Costas Andalucía-Atlántico (Anfuso 2001). La Victoria Beach (36º52′ N, 6º27′ W) is located on the south Atlantic coast of Spain (fig. 1), in the southwestern part of the city of Cádiz. Two sections of the beach were considered for this study: (1) the northern section denominated Final Victoria (FV), which delimits the southern pier of Santa María del Mar Beach and has a rocky platform that serves as natural protection against incident waves; and (2) the central section denominated Hotel Victoria (HV), which is a wave-exposed dissipative beach located in front of a hotel of the same name. Como una contribución al conocimiento aplicable a la pla- neación y manejo de las playas, el presente trabajo se centró en determinar la geomorfología y los procesos costeros actuales en Playa de la Victoria. Profiles were made perpendicular to the beach using a Leica TC407 total station and a prism with a graduated (5 m) base. This station allows measurement of vertical and horizon- tal angles, and has a filial cross with a pair of measuring lines that are used to calculate the difference in beach height (dis- tance and slope). Differential leveling (King 1972), the classi- cal method employed in morphodynamic studies, was used. The topographic profiles were spaced homogeneously, every Introducción En la pro- vincia de Cádiz se pueden encontrar playas de este tipo, como son las playas de Regla en Chipiona, Fuentebravía en el Puerto de Santa María y de la Victoria en la ciudad de Cádiz. Estas playas presentan en su base una laja rocosa constituida por are- niscas y conglomerados calcáreos del Plioceno, cuya cota superior oscila alrededor del nivel medio del mar (Muñoz- Pérez 1996). To contribute to the knowledge of beach planning and use, the purpose of this study was to determine the current geomor- phology and coastal processes at La Victoria Beach. 260 Avila-Serrano et al.: Sediment variations and littoral transport at La Victoria Beach Materiales y métodos Las campañas se realizaron con una periodicidad de 15 días de febrero a octubre de 2004. Rota El Puerto de Santa María Guadalete River El Puerto de Santa María Guadalete Riv Vistahermosa z Puerto Real San Fernando El seguimiento topográfico permite reconstruir las caracte- rísticas morfológicas de las playas cuyo volumen se estima por medio del largo y ancho medido en los perfiles. Estos resulta- dos se compararon mediante el programa Surfer (versión 8) usando el método de interpolación de Kriging, y obteniendo así el sedimento depositado y/o erosionado en las distintas campa- ñas de muestreo. Chiclana La granulometría se realizó con muestras de sedimento recolectadas de la zona intermareal, por ser ésta la parte donde se registran las mayores variaciones granulométricas (Benavente 2000). La toma de muestras fue simultánea a la Figure 1. Map of the study area showing the location and extension of the coastline at La Victoria Beach in SW Spain. Figura 1. Mapa de localización del área de que muestra la ubicación y extensión de la línea de costa en Playa de la Victoria al SO de Espana. Figure 1. Map of the study area showing the location and extension of the coastline at La Victoria Beach in SW Spain. Figura 1. Mapa de localización del área de que muestra la ubicación y extensión de la línea de costa en Playa de la Victoria al SO de Espana. 261 Ciencias Marinas, Vol. 35, No. 3, 2009 30 m, in order to cover all the study area. Profile length depended on beach width, slope, and sea level at the time of the survey. The approximate length of the profiles in section HV was 180 m (with a z error of ±0.02 m), while that of the profiles in section FV was 70 m (z error of ±0.02 m). Profiles were taken every 15 days from February to October 2004. realización de los perfiles y en forma equidistante, tomando sedimento superficial con una pala pequeña hasta aproximada- mente 30 cm de profundidad (Benavente 1997). También se tomaron alrededor de 200 g para analizar el contenido de car- bonatos (CaCO3) en cada muestra. El análisis textural se realizó con muestras de sedimento obtenidas en la sección HV, por ser la parte de la playa donde se registran las mayores variaciones granulométricas. Materiales y métodos Este se realizó en el laboratorio del Departamento de Cristalo- grafía y Mineralogía, Estratigrafía, Geodinámica, Petrología y Geoquímica de la Universidad de Cádiz, por medio de tami- zado, y los parámetros se obtuvieron con el método de los momentos descrito por Folk (1974). Continuous topographic measurements allow reconstruc- tion of the morphological characteristics of a beach, the length and width determined by the profiles providing volume data. The results were compared using Surfer Software (version 8) and the Kriging interpolation method, and the amount of sedi- ment deposited and/or eroded during the different surveys was determined. For grain size analysis, sediment samples were collected from the intertidal zone since most granulometric variations occur in this part (Benavente 2000). Surface sediments (approximately the top 30 cm) were collected with a small shovel at the same time as profiles were taken and at the same distances (Benavente 1997). Around 200 g were taken in order to also analyze the carbonate (CaCO3) content in each sample. Para determinar el contenido de CaCO3 se hizo un análisis composicional por medio del calcímetro de Bernard (Wiesmann y Nehring 1951). Con el resultado de las réplicas se sacó la media porcentual, misma que representó el porcen- taje para cada muestra. Posteriormente se realizó una gráfica en la cual se muestra la zona en la que predomina el CaCO3. El transporte litoral se estimó tiñendo sedimento con pin- tura fluorescente con disolvente que contenía tolueno para evi- tar la compactación de los granos de sedimento. Se pintaron 10 kg de sedimento de la cara de la playa con el fin de conservar sus características tanto granulométricas como hidrodinámicas (Ingle 1966, Teleki 1966, Yasso 1966). La arena pintada se vol- vió a sembrar en la cara de la playa de la sección HV, y al final del ciclo mareal se tomaron muestras a lo largo de toda la playa para hacer un análisis con el método integral espacial (spatial integral method, SIM) (Horikawa 1988). Para determinar la distribución de granos a lo largo del área de muestreo, se conta- ron granos pintados con ayuda de una lámpara fluorescente, y por medio del programa Surfer (ver. 8) se hizo una gráfica bidi- mensional donde se aprecia la distribución de trazadores a lo largo de la playa. Sediment samples collected from section HV were used for textural analysis, since greater granulometric variations occur along this part of the beach. Materiales y métodos Laboratory work was carried out at the Department of Crystallography, Mineralogy, Stratigraphy, Geodynamics, Petrology, and Geochemistry of Cádiz Univer- sity. Sediments were sieved and parameters were obtained using the method of moments described by Folk (1974). The CaCO3 content was determined using a Bernard calci- meter (Wiesmann and Nehring 1951). From the results of the replicas, the mean percentage was obtained, which was the representative percentage for each sample. A graph was then plotted to determine the area where CaCO3 predominated. Littoral transport was estimated by staining sediment with fluorescent paint containing toluene to prevent the compaction of sediment grains. About 10 kg of sediment from the beach face were painted to conserve both the granulometric and hydrodynamic characteristics (Ingle 1966, Teleki 1966, Yasso 1966). The painted sand was replaced on the beach face of sec- tion HV, and at the end of the tide cycle samples were collected from all along the beach for analysis using the spatial integral method (Horikawa 1988). To determine the distribution of grains in the study area, painted grains were counted with a flu- orescent lamp, and Surfer Software (version 8) was used to plot a two-dimensional graph that showed the distribution of the tracers along the beach. Para el análisis de transporte sedimentario, por medio del método de trazadores fluorescentes se calculó la distancia reco- rrida (y) considerando la distancia a partir del centro de masa de los trazadores. Para esto se utilizó la fórmula del método de integración propuesta por Komar (1969): y = ∑Pidi / ∑Pi donde Pi es la cantidad de granos teñidos que aparecen en la celda i, y di es la distancia a la que se encuentra dicha celda del punto de siembra. La velocidad de transporte sedimentario se calculó divi- diendo la distancia recorrida entre el tiempo utilizado para ello, Vt = y/t, en este caso la duración del ciclo mareal (12 h 16 min). To analyze sediment transport, the fluorescent tracer method was applied to calculate the distance covered (y) con- sidering the distance travelled from the center of mass of the tracers, using the equation of the integration method proposed by Komar (1969): Perfiles topográficos y = ∑Pidi / ∑Pi Volúmenes de sedimento Section HV is 180 m long. Its morphological characteristics were more homogenous during the study period, and there was less deposition and/or erosion. In March 2004, a soft slope and spilling breaker waves were observed. The most significant changes occurred in June and October, when bars were observed along the berm parallel to the coastline, with chan- nels perpendicular to the coast. A partir de los perfiles topográficos se obtuvieron los volú- menes de sedimento para las distintas campañas de muestreo (tabla 1). La sección FV presentó en la primera campaña un volumen de 112,311.76 m3, y en la última uno de 75,755.26 m3, mostrando una disminución de 36,556.5 m3. Para la sección HV, en cambio, en la primera campaña se obtuvo un volumen de 20,906.35 m3, y en la última uno de 122,187.81 m3, indi- cando un aumento de 101,281.46 m3. Avila-Serrano et al.: Sediment variations and littoral transport at La Victoria Beach Sediment transport velocity was calculated dividing the distance covered by the time selected for this analysis, in this case the length of the tide cycle (12 h 16 min): Vt = y/t. colapsada (surfing); en el norte de esta sección la altura del perfil disminuyó; al sur de la misma sección la altura en la cara de la playa también disminuyó presentando dos cordones de bermas que caracterizan su parte superior y un canal en la berma (fig. 2). En el centro, la cara de la playa era más extensa y tenía una pendiente abrupta hacia la zona de rompiente. Sediment volumes Based on the topographic profiles, sediment volumes were calculated for the different surveys (table 1). For section FV, a volume of 112,311.76 m3 was determined for the first survey and of 75,755.26 m3 for the last, thus showing a decrease of Sección central (Hotel Victoria) In June, the northern part continued to present stepping and the central part of the berm presented erosion. Two small accumulations were observed in the lower part of the beach face in the northern and southern parts (fig. 5). La sección HV tiene una longitud de 180 m. Sus caracte- rísticas morfológicas fueron más homogéneas durante el periodo de muestreo, sin tanta depositación y/o erosión. En marzo presentó una pendiente suave con rompiente tipo derrame (spilling). Los cambios más significativos fueron medidos en junio y octubre, cuando se presentaron barras en la berma paralelas a la línea de costa, con canales perpendiculares a la costa. In October, the characteristics were very similar to those of the first survey (March). Once again, in the northern part the beach face had a flat section that extended along the beach, with small variations in the southern part (fig. 6). Northern section (Final Victoria) Northern section (Final Victoria) Northern section (Final Victoria) In March 2004, the slope in the breaker zone (collapsing breaking waves) decreased homogeneously throughout the area. Profile height decreased in the northern part of this sec- tion; in the southern part, the height of the beach face also decreased presenting two berm ridges, characteristic of its upper part, and a berm channel (fig. 2). In the central part, the beach face was more extensive and had an abrupt slope towards the breaker zone. En mayo se presentó un canal en los primeros 50 m con dirección norte-sur, paralelo a la línea de costa, que se fue disi- pando hasta desaparecer por completo. Resalta el hundimiento de la parte sur debida al sistema de drenaje urbano (fig. 4). También se registró escalonamiento en la parte norte de la berma, así como depresiones en el resto de la cara de la playa y se modificó el hundimiento por el drenaje urbano. By April, accumulation had occurred in the northern part of FV. The central part showed slight depressions along the beach face and the two berm ridges were no longer noticeable parallel to the coast (fig. 3). This survey revealed accumulation on the beach face, a decreased bar in the first 50 m of the northern part, and erosion of the beach face in the southern part. En junio continúa el escalonamiento en la parte norte y la erosión de la berma en la parte central. En la parte baja de la cara de la playa hay dos pequeñas acumulaciones en las partes norte y sur (fig. 5). In May, a channel appeared in the first 50 m with north- south direction, parallel to the coastline, which slowly dissi- pated until disappearing completely. Sinking was observed in the southern part as a result of the urban drainage system (fig. 4). Stepping was also observed in the northern part of the berm, as well as depressions along the rest of the beach face and modification of the sinking caused by the urban drainage. En octubre las características son muy parecidas a la pri- mera campaña (marzo): en la parte norte nuevamente se notaba una parte plana en la cara de la playa que se extendía a lo largo de la misma, con pequeñas variaciones en el sur (fig. 6). Results Topographic profiles Para abril hubo una acumulación en la parte norte de FV. La parte central presentaba ligeras depresiones en la cara de la playa y, notablemente, ya no se estaban definidos los cordones de la berma que estaban al sur, paralelos a la costa (fig. 3). Esta campaña presentó acumulación en la cara de playa, y con la disminución de la barra en los primeros 50 m de la parte norte, también se presentó erosión en la cara de playa hacia el sur. Sección norte (Final Victoria) where Pi is the quantity of stained grains observed in cell i, and di is the distance at which that cell is from the original point. En marzo de 2004, en la zona de rompiente la pendiente decreció homogéneamente en toda el área con rompiente tipo 262 Avila-Serrano et al.: Sediment variations and littoral transport at La Victoria Beach Análisis textural La flecha roja indica la presencia de un canal ubicado en los primeros 50 m de la sección Final Victoria y la flecha negra el hundimiento provocado por el sistema de drenaje urbano. Figure 5. Three-dimensional section for June 2004. The red arrows indicate stepping along the berm and the black arrows accumulation in the lower part of the beach face in section Final Victoria?. Figura 5. Sección tridimensional durante junio de 2004 Las flechas rojas muestran el escalonamiento en la zona de berma y las flechas negras acumulación de la parte inferior de la cara de la playa en la sección Final Victoria. Figure 5. Three-dimensional section for June 2004. The red arrows indicate stepping along the berm and the black arrows accumulation in the lower part of the beach face in section Final Victoria?. Figure 5. Three-dimensional section for June 2004. The red arrows indicate stepping along the berm and the black arrows accumulation in the lower part of the beach face in section Final Victoria?. Figure 4. Three-dimensional section for May 2004. The red arrow indicates the presence of the channel in the first 50 m of section Final Victoria and the black arrow the sinking caused by the urban drainage system. Figure 4. Three-dimensional section for May 2004. The red arrow indicates the presence of the channel in the first 50 m of section Final Victoria and the black arrow the sinking caused by the urban drainage system. Figura 5. Sección tridimensional durante junio de 2004 Las flechas rojas muestran el escalonamiento en la zona de berma y las flechas negras acumulación de la parte inferior de la cara de la playa en la sección Final Victoria. Figura 5. Sección tridimensional durante junio de 2004 Las flechas rojas muestran el escalonamiento en la zona de berma y las flechas negras acumulación de la parte inferior de la cara de la playa en la sección Final Victoria. Figura 4. Sección tridimensional durante mayo de 2004. La flecha roja indica la presencia de un canal ubicado en los primeros 50 m de la sección Final Victoria y la flecha negra el hundimiento provocado por el sistema de drenaje urbano. 36,556.5 m3. For section HV, a volume of 20,906.35 m3 was recorded for the first survey and of 122,187.81 m3 for the last, indicating an increase of 101,281.46 m3. Análisis textural en tres transectos (V1, V2, V3) paralelos a la línea de costa (tabla 2). Su tamaño medio de grano durante el periodo de muestreo fue de 2 a 3 phi; esto es arena fina según la clasifica- ción de Wentworth (1922). La clasificación o desviación estándar (σ), en promedio 0.67, caracteriza a la playa como moderadamente bien clasificada (Folk 1966). La asimetría (sk) mostró un valor de –0.33, con una tendencia fuertemente asi- métrica hacia los gruesos. La kurtosis (k), de 4.99, corresponde a una distribución extremadamente leptocúrtica (Folk 1966). Por ende, la distribución está más sesgada a los finos, dado que Análisis textural Se analizaron un total de 24 muestras de sedimento recolec- tadas en la zona intermareal de la sección HV, la cual se dividió 263 Ciencias Marinas, Vol. 35, No. 3, 2009 36,556.5 m3. For section HV, a volume of 20,906.35 m3 was recorded for the first survey and of 122,187.81 m3 for the last, indicating an increase of 101,281.46 m3. Textural analysis en tres transectos (V1, V2, V3) paralelos a la línea de costa (tabla 2). Su tamaño medio de grano durante el periodo de muestreo fue de 2 a 3 phi; esto es arena fina según la clasifica- ción de Wentworth (1922). La clasificación o desviación estándar (σ) en promedio 0 67 caracteriza a la playa como Figure 4. Three-dimensional section for May 2004. The red arrow indicates the presence of the channel in the first 50 m of section Final Victoria and the black arrow the sinking caused by the urban drainage system. Figura 4. Sección tridimensional durante mayo de 2004. La flecha roja indica la presencia de un canal ubicado en los primeros 50 m de la sección Final Victoria y la flecha negra el hundimiento provocado por el sistema de drenaje urbano. Figure 5. Three-dimensional section for June 2004. The red arrows indicate stepping along the berm and the black arrows accumulation in the lower part of the beach face in section Final Victoria?. Figura 5. Sección tridimensional durante junio de 2004 Las flechas rojas muestran el escalonamiento en la zona de berma y las flechas negras acumulación de la parte inferior de la cara de la playa en la sección Final Victoria. Figure 2. Three-dimensional section for March 2004. The red arrow indicates the much steeper part and the black arrow the berm channel in section Final Victoria. Figura 2. Sección tridimensional durante marzo de 2004. La flecha roja indica la parte con mucho mayor pendiente y la flecha negra el canal en la berma en la sección Final Victoria. Figure 3. Three-dimensional section for April 2004. The red arrow indicates accumulation in section Final Victoria and the black arrow the erosion on the berm face. Figura 3. Sección tridimensional durante abril de 2004. La flecha roja indica acumulación en la sección Final Victoria y la flecha negra erosión en la berma. Ciencias Marinas, V Figure 2. Three-dimensional section for March 2004. Análisis textural The red arrow indicates the much steeper part and the black arrow the berm channel in section Final Victoria. Figura 2. Sección tridimensional durante marzo de 2004. La flecha roja indica la parte con mucho mayor pendiente y la flecha negra el canal en la berma en la sección Final Victoria. Ciencias Marinas, Vol. 35, No. 3, 2009 Figure 2. Three-dimensional section for March 2004. The red arrow indicates the much steeper part and the black arrow the berm channel in section Final Victoria. Figura 2. Sección tridimensional durante marzo de 2004. La flecha roja indica la parte con mucho mayor pendiente y la flecha negra el canal en la berma en la sección Final Victoria. Figure 3. Three-dimensional section for April 2004. The red arrow indicates accumulation in section Final Victoria and the black arrow the erosion on the berm face. Figura 3. Sección tridimensional durante abril de 2004. La flecha roja indica acumulación en la sección Final Victoria y la flecha negra erosión en la berma. Figure 3. Three-dimensional section for April 2004. The red arrow indicates accumulation in section Final Victoria and the black arrow the erosion on the berm face. Figura 3. Sección tridimensional durante abril de 2004. La flecha roja indica acumulación en la sección Final Victoria y la flecha negra erosión en la berma. Figure 2. Three-dimensional section for March 2004. The red arrow indicates the much steeper part and the black arrow the berm channel in section Final Victoria. Figure 3. Three-dimensional section for April 2004. The red arrow indicates accumulation in section Final Victoria and the black arrow the erosion on the berm face. Figura 2. Sección tridimensional durante marzo de 2004. La flecha roja indica la parte con mucho mayor pendiente y la flecha negra el canal en la berma en la sección Final Victoria. Figura 3. Sección tridimensional durante abril de 2004. La flecha roja indica acumulación en la sección Final Victoria y la flecha negra erosión en la berma. Figure 4. Three-dimensional section for May 2004. The red arrow indicates the presence of the channel in the first 50 m of section Final Victoria and the black arrow the sinking caused by the urban drainage system. Figura 4. Sección tridimensional durante mayo de 2004. Discussion The topographic surveys done in section FV show a clear reduction in the beach profile and a steeper slope than HV. This can be attributed to the rocky platform in this section, which is responsible for the erosion during the sampling period, increas- ing the beach slope and producing a morphological deficit after the 2003 winter period. This is because of the sudden increase in friction between the wave and bottom, causing the wave to increase in height and break with greater force, thus removing the sediment. Durante junio la sección HV presentó un aumento en el ancho de la playa y una pendiente suave, características de una playa disipativa con rompiente en derrame y con poca incidencia de oleaje. Komar (1998) relaciona este fenómeno con el efecto de oleaje constructivo que caracteriza a la temporada de verano. En octubre se registró un aumento en la extensión de la cara de la playa debido a la alimentación artificial de arena que se hizo durante los meses de verano (agosto a septiembre de 2004), en donde se vertieron 485,000 m3 de arena, teniendo un relleno para la sección FV de 435,000 m3. Es evidente que esto impidió determinar el cambio natural de manera precisa; sin embargo, debido a que el relleno de sedimento se realiza periódicamente, queda como un registro para su seguimiento y para futuros estudios. Como sucedió en la primavera de 1991, cuando se regeneró Playa de la Victoria y se comprobó que en la parte sobre laja rocosa (sección FV) la tasa de pérdida de sedimento es mayor que en la sección HV. En esta última el relleno fue sólo de 50,000 m3, vertidos a razón de 170 m3 por metro lineal, lo que representó un tercio de lo vertido entonces en la sección FV (510 m3 por metro lineal). In June 2004, section HV showed an increase in beach width and a soft slope. This is characteristic of a dissipative beach with spilling breakers and little wave incidence. Komar (1998) associated this with the effect of constructive waves that characterize the summer period. An increase in the extent of the beach face was recorded in October as a result of the volume of sand that was artificially introduced (485,000 m3) during the summer months (August–September 2004), result- ing in an increase in volume of 435,000 m3 for section FV. Carbonate content Carbonate content ranged from 3.5% to 9.5% in the 24 samples analyzed, and was more dominant in the part closest to the breaker zone, with a concentration of 9%, whereas it was only 3% at the berm. In section HV, CaCO3 content ranged from 5% to 6% (fig. 9). Discusión Los levantamientos topográficos en la sección FV muestran una clara disminución del perfil de playa con pendiente más abrupta que HV. Posiblemente esto se deba a que la playa está sobre laja rocosa que es la responsable de la erosión durante el periodo de estudio, aumentando la pendiente de la playa y pro- vocando un déficit en su morfología después de los temporales del invierno de 2003. Esto es porque el roce de la ola con el fondo se puede incrementar repentinamente, haciendo que ésta aumente su altura y colapse con mayor energía sobre la cara de la playa removiendo el sedimento. Contenido de carbonatos To determine the littoral transport, the distance travelled by the tracers was calculated (fig. 8). Dilution was not very high, and the tracers moved less than 200 m to the south of the initial injection point, which had the greatest concentration. In general, a southward, offshore transport was observed, with a displacement of 130 m at a speed of 0.29 m s–1. Se analizó el contenido de carbonatos de un total de 24 muestras, en las cuales se obtuvo una concentración entre 3.5% y 9.5% de CaCO3. Éste se presentó en mayor proporción en la parte más cercana a la rompiente, con una concentración de 9%, mientras que en la berma sólo se encontró un 3%, y en la sección HV se encontraron concentraciones alrededor del 5% al 6% de CaCO3 (fig. 9). Trazadores fluorescentes Con el objeto de obtener el transporte litoral, se determinó la distancia que habían recorrido los trazadores (fig. 8). No se observó mucha dilución, el movimiento no fue mayor a 200 m al sur del punto de siembra, donde se registró la mayor concen- tración. En general, el transporte se produjo hacia el sur con tendencia mar adentro (perpendicular a la costa), con un des- plazamiento de 130 m a una velocidad de 0.29 m s–1. Textural analysis A total of 24 sediment samples were collected from the intertidal zone of section HV, which was divided into three transects (V1, V2, V3) parallel to the coastline (table 2). Mean grain size was 2–3 phi during the study period, corresponding 264 Avila-Serrano et al.: Sediment variations and littoral transport at La Victoria Beach Avila-Serrano et al.: Sediment variations and littoral transport at La Victoria Beach el mayor porcentaje de sedimento fue retenido en los tamices de 2 y 3 phi (fig. 7). to fine sand according to the classification proposed by Went- worth (1922). The sorting or standard deviation (σ), with a mean of 0.67, indicated that the beach fell into the moderately well classified category (Folk 1966). A value of –0.33 was obtained for asymmetry (sk), indicating a strong asymmetric tendency towards thick grain sizes. Kurtosis (k) was 4.99, cor- responding to an extremely leptokurtic distribution (Folk 1966). This means that grain size distribution was biased towards fine sizes, with the highest percentage of sediments retained in the 2 and 3 phi sieves (fig. 7). Discussion La parte norte de la sección Final Victoria es plana (izquierda de las flechas) y se observa el desarrollo de berma (flechas); y hacia el sur hay poca variación en la berma. Figure 7. Spatial distribution of the grain size for La Victoria Beach. Figura 7. Distribución espacial del tamaño de grano en Playa de la Victoria. 0 20 40 60 80 100 120 140 160 180 200 -40 -20 0 20 40 1.6 1.65 1.7 1.75 1.8 1.85 1.9 1.95 2 2.05 2.1 2.15 2.2 2.25 2.3 2.35 2.4 Sea Land N Grain size (phi) Distance (m) Figure 9. Spatial distribution of the concentration of carbonates at La Victoria Beach. Figura 9. Distribución espacial de la concentración de carbonatos en Playa de la Victoria. Esto corrobora las diferencias dinámicas morfologícas entre las playas en ambas secciones, y confirma la mayor tasa de ero- sión de FV. Figure 7. Spatial distribution of the grain size for La Victoria Beach. Figura 7. Distribución espacial del tamaño de grano en Playa de la Victoria. De marzo a octubre de 2004, la diferencia de volumen encontrada para la sección HV fue de 101,281.46 m3 en un área de 27,988.54 m2, lo cual indica un proceso de depositación y concuerda con el análisis de los perfiles que pone de manifiesto el aumento del ancho de la playa. Esto corrobora el patrón clá- sico de depositación en verano reportado por Komar (1998), debido al oleaje constructivo que remueve sedimento de las barras formadas alrededor de la zona de rompiente. Para la sec- ción FV la diferencia de volumen encontrada fue de –36,556.5 m3 en un área de 33,551.07 m2, la cual indica claramente un proceso de erosión. Como ya se mencionó, éste se debe a la laja rocosa en la que está apoyada la playa, así como al cambio de energía debido a los vientos de Levante que ocurren durante esta temporada (Benavente 2000), y que producen oleaje de mayor altura y energía haciendo que el sedimento sea remo- vido hacia el mar. rate of 170 m3 per linear meter, representing a third of that introduced into section FV (510 m3 per linear meter). This cor- roborates the morphological dynamic differences of the beaches in both sections, and confirms the higher rate of ero- sion at FV. Discussion Evi- dently it was not possible to accurately determine the natural change; however, since sand is deposited at certain intervals, this information will serve as reference for future studies. When La Victoria Beach was regenerated in the spring of 1991, the part of the beach with a rocky platform (section FV) was found to have a higher rate of sediment loss than section HV. In the 1991 regeneration project, 50,000 m3 were added at a 265 Ciencias Marinas, Vol. 35, No. 3, 2009 ol. 35, No. 3, 2009 Figure 8. Distribution of the fluorescent tracers along La Victoria Beach (0, 0 = injection point). Figura 8. Distribución de trazadores a lo largo de Playa de la Victoria. El punto (0,0) indica el punto de inyección. 0 20 40 60 80 100 120 140 160 180 200 -40 -20 0 20 40 0 40 80 120 160 200 240 280 320 360 400 440 480 520 560 Tracers Distance (m) 0 20 40 60 80 100 120 140 160 180 200 -40 -20 0 20 40 0 40 80 120 160 200 240 280 320 360 400 440 480 520 560 Tracers Distance (m) Figure 8. Distribution of the fluorescent tracers along La Victoria Beach (0, 0 = injection point). j ) Figura 8. Distribución de trazadores a lo largo de Playa de la Victoria. El punto (0,0) indica el punto de inyección. 0 20 40 60 80 100 120 140 160 180 200 -40 -20 0 20 40 3.6 4 4.4 4.8 5.2 5.6 6 6.4 6.8 7.2 7.6 8 8.4 8.8 9.2 Distance (m) Figure 9. Spatial distribution of the concentration of carbonates at La Victoria Beach. Figura 9. Distribución espacial de la concentración de carbonatos en Playa de la Victoria. 0 20 40 60 80 100 120 140 160 180 200 -40 -20 0 20 40 3.6 4 4.4 4.8 5.2 5.6 6 6.4 6.8 7.2 7.6 8 8.4 8.8 9.2 Distance (m) Figure 6. Three-dimensional section for October 2004. To the north of section Final Victoria it is flat (left of the arrows) and berm development (arrows) is observed; to the south there is little berm variation. ( ) Figura 6. Sección tridimensional durante octubre de 2004. Discussion Esto se relaciona con el criterio de Sunamura y Horikawa (1978), quienes indican que en condiciones texturales similares existe transporte de sedimento, y en el área estudiada la direc- ción preferentemente resultó ser de norte a sur. Esto también es un indicativo de playas con sedimentos muy bien clasificados, ya que el mayor porcentaje de sedimento es prácticamente del mismo tipo, lo que provoca la disminución de la permeabilidad de la playa incidiendo directamente en la pendiente intermareal (Benavente 2000). The statistical parameters obtained for section HV show the occurrence of two sediment groups, one consisting of fine sands close to the breaker zone and the other of very fine pebbles at the berm. Sunamura and Horikawa (1978) reported that sediment transport occurs under similar textural condi- tions, and in the area surveyed, the preferential direction was from north to south. This is also indicative of beaches with well classified sediments, since most of the sediment is of the same type, a characteristic that results in a reduction in beach permeability and directly affects the intertidal slope (Benavente 2000). La granulometría presentó una predominancia de granos finos hacia el sur (fig. 7), lo que coincide con los resultados obtenidos mediante el uso de trazadores fluorescentes (fig. 8). Esto concuerda con la dirección del transporte litoral de NW a SE reportado para la zona por Meliéres (1974) y Ojeda (1989), quienes analizaron la dinámica sedimentaria en el Golfo de Cádiz y la dinámica litoral reciente en la costa occidental de Andalucía, respectivamente. Asimismo, el sedimento presentó el mismo desplazamiento encontrado por Muñoz-Perez et al. (1999) por medio de trazadores fluores- centes al SW de España, similar al transporte litoral y perpendicular a la costa por el efecto del movimiento de la ola con el conocido relavado que se debe a la incidencia de la ola sobre la cara de la playa y por el retroceso de la misma (Kraus et al. 1982). The grain size analysis indicated a predominance of fine grains towards the south (fig. 7), coinciding with the fluores- cent tracers (fig. 8). This concurs with the NW–SE direction of the littoral transport reported by Melierés (1974) and Ojeda (1989), who analyzed the sedimentary dynamics of the Gulf of Cádiz and the recent coastal dynamics of the west coast of Andalusia, respectively. Moreover, the sediment presented the same displacement as that found by Muñoz-Pérez et al. Discussion From March to October 2004, the difference in volume found for section HV was 101,281.46 m3 in an area of 27,988.54 m2, indicating a deposition process and concurring with the profile analysis that revealed an increase in beach width. This corroborates the classical summer deposition pattern reported by Komar (1998), due to the constructive waves that remove the sediments from the bars formed around the breaker zone. For section FV, the difference in volume found was –36,556.5 m3 in an area of 33,551.07 m2, clearly indicating an erosion process. As already mentioned, this can Los parámetros estadísticos encontrados para la sección HV muestran la presencia de dos grupos de sedimento, el pri- mero de arenas finas que se encuentran cerca de la rompiente y 266 Avila-Serrano et al.: Sediment variations and littoral transport at La Victoria Beach Table 1. Total sediment volume in the surveys conducted. The difference was calculated between the 9 March and 15 October 2004 surveys. Tabla 1. Volumen total del sedimento en las campañas realizadas. La diferencia se realizó entre la campaña del 9 de Marzo y la del 15 de Octubre de 2004. Survey (d/m/yr) Hotel Victoria section Final Victoria section 09/03/2004 20,906.35 m3 112,311.76 m3 03/04/2004 75,520.09 m3 316,350.15 m3 20/04/2004 83,311.35 m3 111,900.44 m3 04/05/2004 73,349.40 m3 100,908.90 m3 18/05/2004 75,566.62 m3 102,695.37 m3 03/06/2004 83,880.91 m3 316,350.15 m3 15/10/2004 122,187.81 m3 75,755.26 m3 Difference 101,281.46 m3 –36,556.50 m3 Surface area 27,988.54 m2 33,551.07 m2 e in the surveys conducted. The difference was calculated between the 9 March and 15 October 2004 surveys. imento en las campañas realizadas. La diferencia se realizó entre la campaña del 9 de Marzo y la del 15 de Octubre able 1. Total sediment volume in the surveys conducted. The difference was calculated between the 9 March and 15 Octob abla 1. Volumen total del sedimento en las campañas realizadas. La diferencia se realizó entre la campaña del 9 de Marzo be attributed to the rocky platform and to the change in energy due to the trade winds that occur during this period (Benavente 2000), increasing the height and energy of the waves with the consequent offshore removal of sediment. el segundo de guijarros muy finos localizados en la berma. Discussion (1999) using fluorescent tracers in SW Spain, similar to the littoral transport and perpendicular to the coast due to wave move- ment, with the known rewashing as a result of the waves encroaching and receding from the beach face (Kraus et al. 1982). La distribución de CaCO3 en Playa de la Victoria registró una mayor concentración en la zona de rompiente (fig. 9), relacionada con la distribución de los granos finos. Benavente (2000) realizó un estudio sobre la morfodinámica litoral en la bahía externa de Cádiz, donde menciona que a mayor tamaño de grano, mayor es la concentración de CaCO3. Esto se debe a que la concentración del CaCO3 es mucho menor que la de cualquier otro componente de los sedimentos, por lo tanto es fácilmente removido por la acción del oleaje. En la sección HV el CaCO3 resultó con máximas concentraciones en zonas donde predominaban los tamaños finos lo que se contrapone a lo The distribution of CaCO3 at La Victoria Beach showed a higher concentration in the breaker zone (fig. 9), related to the distribution of fine grains. In a study on the littoral morphody- namics of the external Cádiz bay, Benavente (2000) found that the concentration of CaCO3 was higher when the grain size was larger. This occurs because the concentration of CaCO3 is much lower than that of any other sediment component and is thus easily removed by wave action. The behaviour of CaCO3 267 Ciencias Marinas, Vol. 35, No. 3, 2009 Table 2. Textural parameters in phi units for section Hotel Victoria of La Victoria Beach. Tabla 2. Parámetros texturales en unidades phi (φ), de la sección Hotel Victoria de Playa de la Victoria. descrito por Benavente (2000) y podría indicar inestabilidad de la playa el día en que se realizó el muestreo. in the Hotel Victoria section showed maximum CaCO3 content in areas dominated by fine grains, differing from that reported by Benavente (2000). This indicates the instability of the beach depending on the sampling date. Acknowledgements El segundo autor agradece a la Universidad Autónoma de Baja California por la beca otorgada para la estancia en Cádiz. Se agradece a la Universidad de Cádiz el uso de sus instala- ciones y materiales necesarios en el procesamiento de las muestras, así como a A Ghetti por su ayuda en la medición de los perfiles topográficos. The second author acknowledges receipt of a scholarship from the Autonomous University of Baja California for the stay at Cádiz. We thank the University of Cádiz for the use of their facilities and providing the material necessary to process the samples, as well as A Ghetti for assistance with the topo- graphic profiles. English translation by Christine Harris. Benavente J. 1997. Introducción al estudio de la dinámica sedimentaria de las playas del norte de la Bahía de Cádiz: Vistahermosa, Santa Catalina y La Puntilla. B.Sc. thesis, Univ. Cádiz, 192 pp. Benavente J. 1997. Introducción al estudio de la dinámica sedimentaria de las playas del norte de la Bahía de Cádiz: Vistahermosa, Santa Catalina y La Puntilla. B.Sc. thesis, Univ. Cádiz, 192 pp. Benavente J. 2000. Morfodinámica litoral de la bahía externa de Cádiz. Ph.D. thesis, Univ. Cádiz, 534 pp. Carter RWG. 1988. Coastal Environments. Academic Press, 617 pp. Discussion Profile Point Mean Deviation Asymmetry Kurtosis V-1 A 2.9 0.5 –0.1 3.4 V-1 B 3.4 0.5 0.6 4.0 V-1 C 2.6 0.5 0.7 2.7 V-1 D 2.5 0.5 0.4 3.4 V-1 E 2.5 0.5 0.6 4.0 V-1 F 2.8 0.5 –0.3 3.8 V-1 G 2.4 0.4 0.5 4.0 V-1 H 2.4 0.5 –0.1 6.0 V-2 A 2.5 0.7 –0.4 4.7 V-2 B 2.4 0.6 0.0 5.0 V-2 C 2.5 0.7 –0.4 4.8 V-2 D 2.9 0.6 –0.4 5.2 V-2 E 3.0 0.7 –0.8 7.1 V-2 F 2.8 0.8 –1.2 7.0 V-2 G 2.7 0.7 –0.8 6.1 V-2 H 2.8 0.6 –0.8 4.7 V-3 A 2.6 0.8 –0.6 4.8 V-3 B 2.8 0.7 –0.6 5.0 V-3 C 2.8 0.7 –0.6 4.7 V-3 D 2.7 0.6 –0.6 6.2 V-3 E 2.7 0.7 –0.8 5.4 V-3 F 3.0 0.7 –0.4 5.0 V-3 G 2.9 0.8 –0.7 5.3 V-3 H 2.9 0.8 –0.9 6.2 Average 2.7 0.6 –0.3 4.9 Table 2. Textural parameters in phi units for section Hotel Victoria of La Victoria Beach. Tabla 2. Parámetros texturales en unidades phi (φ), de la sección Hotel Victoria de Playa de la Victoria. descrito por Benavente (2000) y podría indicar inestabilidad de la playa el día en que se realizó el muestreo. Benavente J. 2000. Morfodinámica litoral de la bahía externa de Cádiz. Ph.D. thesis, Univ. Cádiz, 534 pp. Avila-Serrano et al.: Sediment variations and littoral transport at La Victoria Beach Muñoz-Pérez JJ. 1996. Análisis de la morfología y variabilidad de playas apoyadas en lajas rocosas. Ph.D. thesis, Univ. Cádiz, 150 pp. CERC, Coastal Engineering Research Center. 1984. Shore Protection Manual. US Army Corps of Engineers, Coastal Engineering Research Center, Printing Office, Washington. CMAJA, Consejería de Medio Ambiente de la Junta de Andalucía. 1995. Medio Ambiente en Andalucía. Junta de Andalucía, Sevilla, 406 pp. Muñoz-Pérez JJ, Gutiérrez-Mass JM, Parrado JM, Moreno L. 1999. Sediment transport velocity by tracer experiment at Regla Beach (Spain). J. Waterway Port Coast. Ocean Eng.: 332–335. Folk RL. 1966. A review of grain size parameters. Sedimentology 6: 73–93. Muñoz JJ, López B, Gutiérrez JM, Moreno L, Cuena G. 2001. Cost of beach maintenance in the Gulf of Cadiz (SW Spain). Coast. Eng. 42: 143–153. Folk RL. 1974. Petrology of Sedimentary Rocks. Hemphill Publishing Co., Austin, Texas, 182 pp. Ojeda J. 1989. Dinámica litoral reciente de la costa occidental de Andalucía. In: El Cuaternario en Andalucía Occidental. AEQUA, Monografías 1: 123–132. Horikawa K. 1988. Nearshore Dynamics and Coastal Processes. Univ. Tokyo Press, 522 pp. Short A. 1979. Three dimensional beach stage model. J. Geol. 87: 553–571. Ingle JC. 1966. The Movement of Beach Sand. Elsevier, New York, 221 pp. Sunamura T, Horikawa L. 1978. Predominant direction of littoral transport along Kujyukuri Beach, Japan. Coast. Eng. Japan 14: 107–117. King CAM. 1972. Beaches and Coasts. 2nd ed. Arnold, London, 570 pp. Komar PD. 1969. The longshore transport of sand on beaches. Ph.D thesis, Univ. California, San Diego, 143 pp. Teleki PG. 1966. Fluorescent sand tracers. J. Sediment. Petrol. 36: 469–485. Komar PD. 1998. Beach Processes and Sedimentation. Prentice-Hall, 544 pp. Weisman M, Nehring K. 1951. Agrikulturchemisches Praktikum. Paul Parey, Berlin. Kraus M, Masselink G, Hughes M. 1982. Field experiments on longshore sand transport in the surf zone. Proc. 18th Int. Conf. Coast. Eng. ASCE, pp. 626–644. Wentworth CK. 1922. A scale of grade and class term for clastic sediment. J. Geol. 30: 377–392. Yasso WE. 1966. Formulation and use of fluorescent tracer coatings in sediment transport studies. Sedimentology 6: 287–301. Melierés F. 1974. Reserches sur la dynamique sédimentarire du Golfe du Cadix (Espagne). Ph.D. thesis, Univ. Paris, N° RC CNRS AV 206, 8, 235 pp. Recibido en septiembre de 2008; aceptado en agosto de 2009. Recibido en septiembre de 2008; aceptado en agosto de 2009. MOPU, Ministerio de Obras Públicas y Urbanismo. 1991. Actuaciones en la Costa. Recibido en septiembre de 2008; aceptado en agosto de 2009. References Anfuso G. 2001. Morfología y dinámica sedimentaria del litoral gaditano entre Chipiona y Rota. Ph.D. thesis, Univ. Cádiz, 375 pp. 268 Avila-Serrano et al.: Sediment variations and littoral transport at La Victoria Beach Avila-Serrano et al.: Sediment variations and littoral transport at La Victoria Beach Secretaría General Técnica, Madrid, 307 pp. 269
https://openalex.org/W4361819456	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Methods_Figure_Legends_1-5_Table_Legends_1-3_from_Identification_of_Anaplastic_Lymphoma_Kinase_as_a_Potential_Therapeutic_Target_in_Ovarian_Cancer/22389585/1/files/39835101.pdf	English	null	Supplementary Methods, Figure Legends 1-5, Table Legends 1-3 from Identification of Anaplastic Lymphoma Kinase as a Potential Therapeutic Target in Ovarian Cancer	null	2,023	cc-by	1,040	SUPPLEMENTAL MATERIALS SUPPLEMENTAL MATERIALS Label free quantification (LFQ) analysis of phospho-peptides Label free quantification (LFQ) was performed on 33 tumor samples (OC01, OC02, OC04, OC05, OC07, OC08, OC09, OC10, OC11, OC13, OC14, OC16, OC22, OC24, OC26, OC26a, OC27, OC28, OC29, OC29a, OC31, OC34, OC35) and 10 normal samples (B20, B22, B24, B25, B26, B26a, B27, B28, B29, B30) that were run back to back on the LTQ-Orbitrap mass spectrometer. Peptide m/z values between runs were used in order to define retention time (RT) differences from run to run by simple linear regression. Once RT drift was established, the maximum intensity for an m/z value was extracted from MS1 scans using predicted RT values and a mass tolerance of 10 ppm. An intensity value was considered valid if at least 3 consecutive MS1 scans contained the target m/z. To compare the abundance of phospho-peptides in tumor samples, we calculated the MS1 peak intensity ratios based on the raw intensity in tumor samples and the basal intensity. The basal intensity is represented by the average raw intensities of MS1 peaks in the 10 normal samples. An intensity of 20,000 (estimated noise level) was used as raw intensities for normal samples that had no MS1 intensity value. The MS1 intensity ratio of a phospho-peptide is calculated as the following: Intensity ratio = Raw intensity in a tumor/ Basal Intensity Transfection of 293T cells and down stream signaling analysis Transfection of 293T cells and down stream signaling analysis 293T cells were transfected with MSCV-Neo, MSCV-Neo/ALK and MSCV- Neo/FN1ALK using FuGENE 6 Transfection Reagent (Roche). 24 hours later, cells were serum starved for 24 hours and either left untreated or treated with 2μM Crizotinib or 0.1μM TAE684 for 2 hours. The cells were then lysed in 1X Cell Lysis Buffer with sonication. Cell lysates containing equal amount of protein were analyzed by western blot assay. Immunofluorescent (IF) analysis. Transfected NIH3T3 cells on 8 well culture slide (BD FalconTM) were stained with ALK (D5F3) XP™ Rabbit mAb, a DNA dye DRAQ5® (Cell Signaling Technology), and DY- 554 phalloidin (Dyomics GmbH, Jena, Germany) that binds to actin filament following standard IF protocol (www.cellsignal.com). Immunohistochemical staining. 4 μm ovarian tissue sections or FFPE TMA slides were deparaffinized and rehydrated through xylene and graded ethanol, respectively. Antigen retrieval was performed in a Decloaking Chamber (Biocare Medical, Concord, CA) using 1.0 mM EDTA, pH 8.0. Slides were then quenched in 3% H2O2 for 10 minutes, washed in deionized H2O and blocked with Tris buffered saline /0.5% Tween-20 (TBST)/5% goat serum in a humidified chamber for 60 minutes. Sections were then exposed to ALK (D5F3) XP™ Rabbit mAb overnight at 4°C. Detection was performed with SignalStain® Boost IHC Detection Reagent (Cell Signaling Technology) for 30 minutes. All slides were exposed to NovaRed (Vector Laboratories, Inc., Burlingame, CA) for 1 minute before they were rinsed, dehydrated, cleared and cover-slipped. rinsed, dehydrated, cleared and cover-slipped. Immunofluorescent (IF) analysis. Supplemental Figure 3. A novel fusion: FN1-ALK. A, Schematic diagram of genomic DNA fusion of FN1 and ALK genes. Gene structure diagram (exons, black boxes; introns, lines), location and orientation of FN1 and ALK genes are shown. Exons and joint sequences in FN1 (blue) and ALK (red), positions of the novel breakpoint (Novel BP) and the common breakpoint (Common BP) are indicated. A single PCR product of ~1kb amplified from OC19 gDNA using PCR primers annealing to FN1 Exon23 and ALK Exon 19 is detected by agarose electrophoresis. B, Predicted amino acid sequence of the FN1-ALK fusion protein (198.82 kd). Residues corresponding to FN1 or ALK are indicated in blue and red, respectively. Peptide sequence encoded by ALK Exon 19 is underlined. Amino acid sequence spanning the trans-membrane domain is highlighted in yellow. Supplemental Figure 2. Hyperphosphorylation of signaling molecules in serous carcinomas bearing ALK LFQ analysis was performed as described in Materials and Methods. The MS1 peak intensity ratios of 13 phospho-peptides representing tyrosine phosphorylation of 12 signaling molecules across 23 serous carcinoma samples are shown in the 3D graph. The three serous carcinomas bearing phosphorylated ALK (OC07, OC16 and OC26) are grouped together to facilitate the comparison with other serous carcinomas. Peptide sequences and the values of MS1 peak intensity ratio are listed in Supplemental Table 3. Supplemental Figure 1. Detection of two common ALK peptides containing phospho-Y1507 in 4 patients. Supplemental Figure 1. Detection of two common ALK peptides containing phospho-Y1507 in 4 patients. A, Extracted ion chromatograms (EICs) of two m/z (mass/charge) values corresponding to the two peptides containing ALK phospho-Y1507 (NKPTSLWNPTyGSWFTEKPTK and NKPTSLWNPTyGSWFTEKPTKK) in OC07, OC16, OC19 and OC26. Arrows indicate MS1 peaks (±3ppm) correspondent to the two ALK peptides. The MS1 peak intensities in each sample are indicated. B, Examples of MS2 spectra matched to ALK phospho-Y1507 peptides. The two spectra have normalized intensities of 1-2 x104. Blue lines indicate MS2 peaks matched to theoretical y ion peaks and red lines, b ion peaks. For example, in the left spectrum, the blue line labeled as y19++ is a peak matched to the 1 1 y19 ion with a charge of 2+, the red line labeled as b8+ is a peak matched to the b8 ion with a charge of 1+. Supplemental Figure 4. Activation and ALK inhibitor sensitivity of down stream molecules in 293T cells expressing ALK and FN1-ALK. Whole cell lysates of untreated or Crizotinib treated 293T cells transfected with MSCV- Neo, MSCV-Neo/ALK and MSCV-Neo/FN1-ALK were analyzed by western blot assay using indicated antibodies. The positions of full length ALK/FN1-ALK (220 kd), cleaved ALK (140 kd) and FN1-ALK fragment (~78 kd) are indicated by arrows for ALK and 2 2 phospho-ALK (Y1278/1282/1283) blots on the left. Blot with β-Actin antibody was used as a loading control. treatment. Four to six nude mice carrying 3T3 tumors expressing Src, ALK or FN1-ALK are treated with vehicle or 10mg/kg/day TAE684 by oral gavage when tumors are palpable. The tumors are measured every other day until the mean tumor size of the vehicle treated mice reaches 1500 mm3. Supplemental Table 1. Patient Diagnostic Information Supplemental Table 1. Patient Diagnostic Information Supplemental Table 2. Phospho-tyrosine Peptides of Tyrosine Kinases Identified by LC-MS/MS in Ovarian Tissues Supplemental Table 3. Abundance of phospho-peptides corresponding to specific signaling molecules in selective serous carcinoma patients 3 3
https://openalex.org/W2098649037	http://collections.unu.edu/eserv/UNU:1490/Analysis_of_open_source_biotechnology.pdf	English	null	Analysis of open source biotechnology in developing countries: An emerging framework for sustainable agriculture	Technology in society	2,012	public-domain	14,574	a r t i c l e i n f o Agricultural biotechnology (e.g. genetically modiﬁed (GM) crop technology) is rapidly growing and has immense potential to contribute to sustainable agriculture in developing countries. However, due to the privatization and increased intellectual property rights (IPRs) protection, many people in the developing world ﬁnd it very difﬁcult to access modern biotechnology research tools (e.g. genetic engineering, micro-propagation, mutation breeding etc.) to improve agricultural productivity. This paper reviews the existing open source literature and draws parallels between the open source paradigm and the effect of IPRs on agricultural biotechnology. Using standard qualitative research methodology and examining speciﬁc case studies and initiatives, an innovative Open Source Biotechnology Framework (OSBF) is proposed as part of the solution that could address the challenges with IPR and help bring about sustainable agriculture. This paper further examines the potential impacts, constraints, and adoption of open source for agricultural biotechnology. The paper concludes with a summary of issues arising from adopting the open source paradigm in agricultural biotechnology while proposing a way forward. Article history: Received 7 June 2011 Received in revised form 22 July 2012 Accepted 31 July 2012 Keywords: Agriculture Biotechnology Technology innovation Sustainable development Open source software Intellectual property rights (IPRs) Developing countries 2012 Elsevier Ltd. All rights reserved. Ademola A. Adenle a,b,, Sulayman K. Sowe a,b, Govindan Parayil a, Obijiofor Aginam c a United Nations University, Institute of Advanced Studies (UNU-IAS), 6F International Organisations Centre, Paciﬁco Yokohama, 1-1-1 Minato Mirai, Nishi-k 220-8502 Yokohama, Japan b National Graduate Institute for Policy Studies (GRIPS), 7-22-1 Roppongi, Minato-ku, Tokyo 106-8677, Japan c United Nations University, Institute for Sustainability and Peace (UNU-ISP), 5-53-70 Jingumae, Shibuya-ku, Tokyo 150-8925, Japan Contents lists available at SciVerse ScienceDirect Contents lists available at SciVerse ScienceDirect Corresponding author. United Nations University, Institute of Advanced Studies (UNU-IAS), 6F International Organisations Centre, Paciﬁco Yokohama, 1-1-1 Minato Mirai, Nishi-ku, 220-8502 Yokohama, Japan. Tel.: þ81 45 221 2367; fax: þ81 45 221 2303. Technology in Society 34 (2012) 256–269 Technology in Society 34 (2012) 256–269 0160-791X/$ – see front matter 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.techsoc.2012.07.004 p E-mail address: adenle@ias.unu.edu (A.A. Adenle). * Corresponding author. United Nations University, Institute of Advanced Studies (UNU-IAS), 6F International Organisations Centre, Paciﬁco Yokohama, 1-1-1 Minato Mirai, Nishi-ku, 220-8502 Yokohama, Japan. Tel.: þ81 45 221 2367; fax: þ81 45 221 2303. E-mail address: adenle@ias.unu.edu (A.A. Adenle). 1.1. Research methodology In social and physical sciences research, relying on and integrating data from multiple sources has long been recognized as a standard practice. Triangulation or cross- examination is often used to indicate this research meth- odology [11]. Triangulation increases the reliability of the data by allowing the researcher to make inferences and value judgement by comparing and contrasting informa- tion in one data set against another. In the context of data collection, triangulation serves to corroborate the data gathered from various sources. Furthermore, Denzin (2006) [12] distinguished between four types of triangulation research methods, and pointed out that methodological triangulation (such as the one employed in this research) involves using multiple choices to gather data, such as documents or review of the literatures, observations of initiatives that can add value to what is being studied [11,12]. The application of open source in software development led to the concept of applying open source in agricultural biotechnology [6]. Open source biotechnology is a method of creating genetically modiﬁed crops that does not infringe on patents held by large biotechnology companies. The technique would be made available free to others to use and improve as long as the improvements are also available free. Similar to open source software, the idea is to spur innovation. It is believed that open source will create opportunities through which life science inventions can be made available to the public and broad research commu- nities by effectively opening up the IPRs “logjam” [7,8]. Open source as an alternative to proprietary technologies is gradually becoming popular in developing countries in the area of information and communication technology (ICT) [9]. Free accessibility and low cost are some of the char- acteristics that make open source software technologies an attractive proposition to poorer communities [10]. Devel- oping countries are taking advantage of the inherent beneﬁts of open source (e.g. availability of source code, ability to modify and customize the software, lower total cost of ownership, freedom from vendor lock-in, avail- ability of community support, etc.) to solve practical problems in agriculture, health, environment and educa- tion to improve livelihood in the rural areas. 1. Introduction Given the IPRs “logjam” that constrains these research tools, there is a need for innovative solutions to tackle food security problems. Q3. Are there lessons to be learned from existing initiatives to facilitate access and promote open source approach for biotechnology R&D? The aim of this paper is to provide answers to these questions by investigating the role of open source biotechnology in sustainable agricultural development. 1. Introduction has been recognised as a research tool that can potentially contribute to sustainable agriculture in developing coun- tries. Recent reports have shown that agricultural biotechnology (especially genetically modiﬁed (GM) crop technology) has made a signiﬁcant impact in terms of increased yields, increased income and improved quality of life in developing countries [2,3]. Crop varieties such as drought and herbicide-tolerant, insect and pest resistant traits have been developed using modern biotechnology, particularly genetic engineering. However, there is little or no access to this innovation that has great potential to improve agricultural productivity and sustainable devel- opment in developing countries. Sustainable agriculture is widely acknowledged as a fundamental component of any strategy to ﬁght poverty and food security problems in developing countries. The World Bank estimates more than 86% of poor people living in developing countries rely on agriculture practices as the source of their livelihood [1]. Agricultural biotechnology One notable problem is obvious in the area of biotech- nology research and development (R&D). This problem is A.A. Adenle et al. / Technology in Society 34 (2012) 256–269 257 Q2. What are the relevant examples or case studies of the open source approach that can beneﬁt sustainable agri- cultural development? Are there generalizable solutions than can be drawn from these cases? the increased intellectual property rights (IPRs) protection which can constitute both a problem and an opportunity for the adoption of agricultural biotechnology in devel- oping countries. The GM crop technology is very expensive and investment oriented with a huge capital base market because biotechnology R&D is largely dominated by private-sector ﬁrms [4,5]. For example, in the US, revenue for the biotechnology industry increased from $8 billion 1992 to $ 25 billion in 2000, an increase of over 300% during this time-period alone [4]. Multinationals placing strong IPRs on agricultural inventions including research tools have affected the development, adoption and diffu- sion of new innovation in developing countries. As a result, research tools that are needed for the development of subsistence crops are often not available. Some of the research tools used in modern biotechnology such as micro-propagation, marker-assisted breeding, mutation breeding and genetic engineering have produced different crop varieties in use today. These research tools are needed to overcome the inevitable crop production problems due to low yields, postharvest losses, drought, disease and insects in developing countries. 2. Background The term open source was ﬁrst used in free software development [13]. Free open source software (FOSS) uses free software and open source which is licenced to use, copy, modify, redistribute and gives the opportunity to increase the value of software or technology to a desired taste or for different purposes with the full access to the source code [6,14]. For example, the free software founda- tion (FSF) uses free software licences and the open source initiative (OSI) uses open source licences. Other FOSS movements include the FOSS Bazaar,4 Creative Commons5 and the Debian Linux Community [6,15,16]. The FOSS projects have been remarkably successful with many open source programs available and most recognized among them include Linux, Apache and Mozilla. Open source technology can be made available under a copyleft6 licence that prevents an individual or any organisation modifying and reproducing the technology for proprietary purposes since the initial access to the technology is open and free, and must be modiﬁed or reproduced upon initial agree- ment [17]. The idea of the copyleft licence is to ensure that everyone has free access to the innovations without further restrictions. Agricultural innovations are widely known as the key driving force for rural development in developing coun- tries. And farmers play a vital role in the development process of innovations but are faced with problems of insects and pests, low income, small yield products, lack of communication and other technological problems in developing countries. Some of these problems are partly due to multiple intellectual property claims on key inputs and tools used in agricultural biotechnologies. Because of these factors, and the need for innovation to be more affordable and become more decentralized, the open source development presents an alternative distributive model in technological development for agricultural innovation. However, enhanced value of the technology can still be converted into economic beneﬁt just as in the case of proprietary technology, but at the same time it must be freely offered to the public [18]. The concept of open source is, however, a matter of liberty not necessarily production cost as there are typically no costs evaluated toward labor even though the process is labor intensive. Moreover it is possible that some version of open source software may be directly relevant to agricultural R&D. 4 FOSS Bazaar is an open community of technology and industry leaders that collaborate on how to accelerate the adoption of free and open source software in the enterprise as well as focussing on best practices, education and tools (https://fossbazaar.org/). 5 Creative commons recommends and uses free and open source software licences (General Public Licence–GNU) for software develop- ment (http://creativecommons.org/software). 6 Copyleft is a word used to describe the practice of using copyright law that allows distributing copies and modiﬁed versions of work and granting the same right preserved in modiﬁed version of the work. 1.1. Research methodology Given the paucity of empirical data linking open source and agricultural biotechnology, especially in the context of developing countries, qualitative methodolog- ical approaches are the most appropriate for synthesizing evidence from various sources, increasing conﬁdence in the interpretation of research results, and enabling the results to build or propose frameworks (such as the Open Source Biotechnology Framework-OSBF) which may help others to undertake research in this area. In our ﬁrst data source, the literature and best prac- tices review provided an overview of this ﬁeld of study but also may increase our understanding of the sustain- able aspects of agricultural practices in developing coun- tries. The second data source, the case studies, helped us to ﬁnd out why open source can be a valuable tool in overcoming the IPRs logjam, facilitating access to infor- mation, and mitigating risks. The main reason for using case studies as research instruments is grounded in the argument by Yin (2003, p111) [11], that a case study design should be considered when “the focus of the study is to answer how and why questions” and when the researcher wants to “cover contextual conditions” which are believed to be relevant to the phenomenon (agricul- tural biotechnology) studied. Thus, a case study approach was chosen for this research. In the third data source, we contrast four prominent initiatives that promote open access to agricultural and health biotechnology innovations. In order to use the open source approach, we must understand the role of IPRs with regards to the develop- ment of agricultural biotechnology and how open source can increase (or decrease) access to biotechnology inno- vation in developing countries. Therefore, we ask the following research questions in this investigation: Q1. What role does IPRs play in limiting access to research tools such as modern biotechnology that has great potential to increase agricultural productivity in developing countries? A.A. Adenle et al. / Technology in Society 34 (2012) 256–269 258 Therefore, one should think of “free speech”, not “free beer” when understanding open source software [19]. The increase in the value of investing in the time and labor for open source technology is in downstream cost savings. In science, this can be in the form of a research tool that was reproduced and improved leading to a high quality product through open source development. 1.1. Research methodology This would make the research tool available to the community at no cost, increasing the opportunity for more innovation through broader access. The rest of the paper is structured as follows. Section 2 introduces background and work related to our research, and describes the principles of open source and how the concept of open source software is similar to traditional farming practices of free sharing and exchanging of seed. Section 3 discusses the implications of IPRs and demon- strates relevant examples of tools used by the IPRs system in agricultural biotechnology. Section 4 examines potential beneﬁts and the impact of open source on agricultural development. This section also demonstrates examples through case studies and initiatives promoting agricultural biotechnology innovation. Section 5 examines the constraints associated with the adoption of open source biotechnology and proposes an Open Source Biotechnology Framework (OSBF) with policy implication towards advancing agricultural development. Finally, the paper concludes with a summary of critical issues for open source biotechnology development. Over the past decade, the awareness of open source is increasingly growing worldwide. The open source princi- ples hold substantial promise for developing countries particularly in the area of biotechnology (e.g. agriculture, health). Due to the mode of operation and the beneﬁts associated with open source, different initiatives are being deployed across different ﬁelds, promoting the use of open source for the beneﬁt of the society. This idea has led to numerous innovations in terms of operating systems and products. The free open source operating system (GNU/ Linux Software) as ﬁrst initiated by Linus Torvalds [16,20] has made a signiﬁcant impact in promoting open-source development and many initiatives have followed this example. Some of the initiatives using open-source and ‘copyleft’ approach for their research tools include: The International HapMap project for mapping haplotypes of human genomes [8], computational drug discovery and developments for treating tropical diseases by Tropical Disease Initiative [21], biological R&D by the Open Bio- informatics Foundation [22], advanced genetics for improving agriculture and sharing biological innovations in poor communities by Biological Innovation for Open Society [7] which is founded by Centre for the Application of Molecular Biology to International Agriculture [23] called CAMBIA BiOS Initiative. 2. Background An example was given by some authors in the case of the seed industry relating the event to computer software in the 1970s [24,25]. Douthwaite and Srinivas (2002) [24,25] observed that it was a traditional practice for computer programmers to freely exchange code among themselves which was similar to farmers that shared seeds freely with others for growing, improving, saving and reproducing crops. These days, a very different model has emerged where private companies in software and agriculture lead innovation which limits this older practice of free sharing. A.A. Adenle et al. / Technology in Society 34 (2012) 256–269 259 Organisations (WTOs) works speciﬁcally on strengthening the rights of inventors [30]. As discussed in this section above, an open source mechanism called copyleft was developed so that any innovation resulting from the open source approach would not be protected or stopped from copying or modifying either by the original licensor or his licensees under open source licence [26]. The same copyleft approach will be applied in agriculture where innovations are developed and allowed to be shared freely among the innovators as bound by the rules and agreement of open source licence for further improvements and distributions without any obstacles. Through this method, open source biotechnology can play a signiﬁcant role in future agricultural develop- ment especially in seed industries that have been domi- nated by multinationals restricting access to the distribution of seeds through the intellectual property rights (IPRs) system. Based on these practices and the initiatives described above, we deﬁne open source biotechnology in this investigation as the development and maintenance of agricultural practices and research tools that allow both producers (e.g. companies) and consumers (e.g. farmers) to actively participate in the development process and freely reveal and share their innovations. However, when these organisations are expected to work together towards a common goal, international changes in the IPRs system sometimes cause failure to deliver. For example, UPOV was established by six Euro- pean nations in 1961. And it was subsequently revised in 1972, 1978 and 1991. The emergence of UPOV in 1961 triggered approval and passage of the Plant Variety Protection Act in US in 1970 [31]. The difference in policy and guidelines between North America and Europe which led to the change [32], helped prevent access to informal exchange, as well as farmers saving and replanting of seeds through the enforcement of law. 2. Background This is in sharp contrast to the original UPOV that allowed breeders to exchange or sell their seeds by member countries [33]. At the same time, the current international environ- ment for IPRs adds to the complexity of the IPRs regime because there are multiple entities representing different interests and processes. The lack of coherent international agreements has created loopholes for agricultural IPRs, particularly in the area that relates to agricultural biotechnology, therefore creating more opportunities for multinationals to exploit loopholes in legal agreements with developing countries [29]. For example, CBD was criticised for not recognising the IPRs agreement within the context of TRIPS. In fact the TRIPS agreement undermines the role of CBD in conserving biodiversity and managing genetic resources. Typically, law is being enforced through collaboration between national and international institu- tions on a global scale. Furthermore, the developing countries are under obligation in line with the TRIPS agreement for the protection of plant varieties either by patent or by other means of the IPRs system [29]. This agreement was done without necessarily considering its beneﬁcial effects on consumers or producers and its possible impact on food security in developing countries. However, the UN Food and Agriculture Organisation (FAO) played an important role in reaching a positive agreement for developing countries in the International Treaty on Plant Genetic Resources for Food and Agriculture [34]. 3. The implications of IPRs for agricultural biotechnology Intellectual property rights (IPRs) are a set of laws that confer exclusive rights on inventors or products of inven- tors for a given period of time [27]. The role of IPRs became prominent in the protection of plant varieties in the second half of 20th century [28]. The assignment of IPRs to plant protection ﬁrst took place in United States (US). When the vegetable propagated plant was patented in 1930. Prior to this event, traditional farming was mainly based on freely exchanging, saving, collecting and replanting seeds among the farmers. But the introduction of IPRs, particularly for agricultural research tools and databases through different patenting systems has led to the expression of concerns among different communities such as farmers, universities, plant scientists, industries, and governments particularly in developing countries. The concerns are based on agricul- tural innovations being hindered through introduction of IPRs that interferes with traditional farming practices. This section will focus on the implication of IPRs for agricultural biotechnology with relevant examples, while examining the role of patents, terminator technology and freedom to operate innovative technology. The issue of IPRs became even more challenging when two independent events took place in the US the same year. These events were pivotal for innovation in life sciences: the legislative approval and implementation of the Bayh- Dole established Act of 1980, and Supreme Court decision in favour of patent protection on genetically modiﬁed organisms in the landmark Diamond v. Chakrabarty in 1980 [35]. After this remarkable development, a series of events followed, in the US and across Europe where large invest- ments were made in agricultural technology in the private sector in collaboration with the universities. Modern IPRs regimes (patent regimes) have been heavily criticised by some due to non-disclosure of many innovations that can beneﬁt the public. The US is arguably the largest world patent portfolio. The total number of patents analysed between 2002 and 2009 was 7469 [5]. Of these ﬁgures, the US accounts for 5, 690 ﬁled under the US Patent and Trademark Ofﬁce (USPTO) while 1779 were ﬁlled under the European Patent Ofﬁce (EPO). The annual trend in crop biotechnology patents is shown in Fig. 1. The patents 3.1. The role of the patent in agricultural biotechnology The existing intellectual property rights (IPRs) relevant to agricultural biotechnology are complex [29]. Most of the international agreements focus on different aspects of agricultural IPRs that are governed by different sets of rules, guidelines and policies. For example, the International Union for the Protection of New varieties of Plants (UPOV) focuses on protecting the right of seed producers (e.g. commercial plant breeders). The Convention on Biological Diversity (CBD) focusses on protecting the rights of farmers using landraces. The Traded-Related Aspects of Intellectual Property Rights (TRIPS Agreement) of World Trade A.A. Adenle et al. / Technology in Society 34 (2012) 256–269 260 Fig. 1. Annual trends in patents of crop biotechnology between 2002 and 2009. Source: (Frisio et al., 2010) [5]. resulting transaction cost limits innovations. According to Isaac and Walter (2004) [41], economists refer to the need for such negotiations under the general rubric of “trans- actions costs”. The most famous example is “Golden Rice” which has experienced a lot of delays due to a patent thicket of about 40 contractual obligations that initially caused the Golden Rice project setback [40]. Golden Rice was a biotech innovation that could potentially solve vitamin-A deﬁciency problems in developing countries where millions of young children were dying of malnutri- tion. It became a “bone of contention” among more than two dozen biotech companies claiming the patents. Golden Rice saga demonstrated that multiple owners holding overlapping and fragmented IPRs to different components of a large innovation can be a problem for developing and disseminating innovation. Academic researchers are affected when there is high transaction cost on a wide range of innovations. Their research becomes more difﬁcult when there is uncertainty as to whether they might infringe company patents, sometimes regarded as “research exemptions” without seeking licences [42]. Fig. 1. Annual trends in patents of crop biotechnology between 2002 and 2009. Source: (Frisio et al., 2010) [5]. This has led to questions of whether enforcement of patent rights stand in the way of basic research in universi- ties. Genetech argued that open science was being encour- aged without enforcing patent rights on research tools [43]. This is in contrast to arguments presented by Eisenberg which was based on high transaction cost for acquiring proprietary research tools that involved many different institutions [44]. 3.1. The role of the patent in agricultural biotechnology Two cases cited by Eisenberg include patent research tools such as rDNA and polymerase chain reaction (PCR) of Cohen Boyer and Hoffman La Roche respectively. Eisenberg (2002) [44] further argues that strong IPRs on research tools lead to reduced innovations and underused knowledge that creates rights of exclusion (anti-common) by IPRs holders. An example of the expressed sequence tag (EST) was given, a situation where some groups attempted to patent EST but abandoned this due to resistance from other competitors. This view suggests that once there is patent lock on innovative tools it creates a logjam in health and agricul- ture, development worldwide [37]. related to the cultivars were only patentable at the USPTO which suggests that the US controls agro-biotech markets due to the large R&D investments [5]. The private sector is mostly involved in biotech indus- tries and makes extensive use of IPRs in form of patents [5,36–39]. Multinationals such as Monsanto, DuPont, Syn- genta and Bayer are famous for acquiring large number of patents with more than 70% of the patents in agricultural biotechnology in between 2002 and 2009 [5]. About 80% of biotech patents under the cultivars category can be attrib- uted to three of six integrated major multinational companies (Monsanto, DuPont and Syngenta). According to Jefferson [37], Monsanto alone has a patent application for over 460,000 genes which is more than 10 times the number in any plant species. Some of these biotech companies have large markets in developed countries with a strong IPRs on cash crops (corn, cotton, and soybean) through patent. The lack of free access to innovative tools was largely responsible for the slow development of genetically modiﬁed (GM) crops with herbicide tolerance traits according to American Cyanamid [38]. In addition, multinationals mainly concentrate on crops of primary interest which will result in the largest ﬁnancial beneﬁt derived from cultivating these crops, therefore neglecting the innovation needed to develop “orphan crops”7 that are simple and cost effective for poorer countries. This is consistent with the argument raised by Parayil (2003) [39] that certain crops are developed to increase shareholder value for private companies as opposed to solving the problems of hunger and deprivation in developing countries. 7 Orphan crops are a diverse set of minor crops such as millet, yam, cassava, cowpea, sorghum grown by poor farmers. These crops are not traded around the world and also receive little or no attention from research networks but play an important role in regional food security. 3.1. The role of the patent in agricultural biotechnology While Eisenberg and others presented some evidence with regards to the argument that patents create less innovation and adversely affect R&D [35,44], some litera- ture has argued that patents enable collaboration and facilitate negotiations between research tool users and producers leading to development [45–47]. 3.2. What role can ‘terminator’ technology play? biggest company supplier of cotton) for the two types: trait-speciﬁc (T-GURT) and variety-level (V- GURT) [50]. While T-GURT is designed for GM trait-speciﬁc (e.g. disease resistance), V-GURT is designed for GM crop varieties through seed sterility [51,52]. g y [ ] The terminator technology is mostly targeted at devel- oping countries where IPRs are non-existent and termi- nator technology may not have much effect on developed nations as other technologies can be used to unravel and relocate innovative characteristics for plant breeding [53]. The idea of introducing terminator technology is not only to protect IPRs and stimulate private R&D but to prevent the ﬂow of unwanted genes from genetically modiﬁed (GM) crops, and to help solve plant-back problems in GM crops [52,54]. According to Eaton et al., (2002) [52], the imple- mentation of terminator technology can potentially lead to a reduced atmosphere for sharing genetic resources through increased IPRs protection, particularly among competing companies and institutions. In developing countries, poor farmers may not be able to afford GURT seeds from suppliers every year and also may limit their ability of saving and exchanging seed practices. And Mon- santo may sue farmers that save and reuse their seeds. For example, an infringement suit was brought against a farmer that saved 1500 bushels of Bt soybean seeds from his ﬁeld enough to grow 1500 acres another year [55]. This approach may discourage poor farmers from buying patented seeds. Moreover, traditional crops may suffer reduction in viable seed production from sterile GURT seed technology due to pollen transfer [56,57]. The researchers at the universities or academic insti- tutions ﬁnd it difﬁcult to use research tools that could lead to more innovations due to associated risks, particularly in the light of patented technologies, while advancing their research work. Some literature evidence suggests that researchers are experiencing difﬁculty, delay and re- directing of research due to the high cost and problem of accessing permission to patented technology that can beneﬁt research programs [66,67]. Even though, this kind of problem often happens in developed countries, without doubt, it becomes a spill-over problem when the tech- nologies are transferred to developing countries [68]. According to Hoekman et al., (2004) [68], the improve- ment that occurs at a rapid rate may not be located in another industry without the policies for the productivity improvement that depend on a country’s own R&D. 3.2. What role can ‘terminator’ technology play? One of the primary purposes of intellectual property rights (IPRs) in biotech industries is to gain proﬁts through the application of patenting, temporarily allowing compa- nies to have a monopoly on certain innovative technologies, particularly in agricultural biotechnology. This is demon- strated by the revenue generation trajectory of biotech ﬁrms where plant patents that are held by private ﬁrms situated in developed countries generate revenue from the poorest for multinationals in the wealthiest countries [39,48]. With the development of new plant varieties by these companies, a different strategy is devised where IPRs cannot effectively serve their intended purpose. The use of The issue of ‘‘patent thicket’’ arises when there are numerous negotiations involving high transaction cost and uncertainty with different patent holders [40]. The A.A. Adenle et al. / Technology in Society 34 (2012) 256–269 261 particularly in the areas critical for the public goods (such as genetic resources for agriculture). The problem is that government funding for R&D is far less than private sector funding meaning that the proﬁt motive becomes a priority. While this drives technological innovation and economic development in many instances, in some instances it can inhibit the diffusion of innovation. For example, when a company or institution invents a technology, a patent is usually ﬁled so that such invented technology is protected from free use. In most cases, the company prevents exclu- sive use of technology to regain any investment made on the invention for about 20 years when the patent would have run out [63]. In contrast, open source technology is free and company owned technology is protected. However, while some successes have been recorded in open source projects, particularly in software development, many open source projects have experienced little or no development due to a lack of momentum behind them [64,65]. Given this challenge, efforts should be increased for the involvement of country government, private sector and international organizations to facilitate and encourage freedom to innovate where access is limited to biotech- nology R&D, particularly in developing countries. terminator technology has the potential to circumvent this problem. The word ‘terminator’ was coined by activist groups in an attempt to ban the use of sterile seed tech- nology [49]. The original name is called Genetic Use Restriction Technologies (GURTs). GURTs were granted US patent (5,723,765) in 1998 to joint partnership between U.S. Department of Agriculture (USDA) and Delta & Pine Land Company (the U.S. 3.2. What role can ‘terminator’ technology play? When this problem occurs, freedom to operate becomes a difﬁ- culty due to a lack of easy and quick access to material held by others. Again, the cost of freedom to operate may be too high for public institutions to afford [69]. Additionally, the use of IPRs as constraining innovation is seen as a problem in universities and in developing countries. Most signiﬁ- cantly it can be a serious threat to the supply of food due to the problem of access to IPRs as expressed by the inter- national research and donor communities [70]. Although, it may be possible that the so called T-GURT form of what is referred to as terminator technology would serve a good purpose in terms of disease resistance [58], saving and replanting the seeds (excluding transgenic traits) but farmers will have to pay for activating chemicals each year [59]. Given little or no detailed application of V-GURT in the literature [60], more evidence-based scientiﬁc research will need to be done, but the opportunity may not exist in the future as the terminator technology kind of approach in agricultural biotechnology is widely opposed. For example, as a result of wide-spread criticism of terminator technology (V-GURT, in particular), the application of V-GURTs for different varieties was disqualiﬁed in India [51] and was rejected by the Rockefeller Foundation [61] and by the Consultative Group on International Agricultural Research (CGIAR) [62]. Given this opposition, it may be difﬁcult for GURT technology to replace IPR in developing countries. 4. Potential beneﬁts and impacts of open source for agriculture and biotechnology The role of information and communication technology (ICT) in improving the quality of life in rural areas of developing countries is fast gaining recognition [71]. The beneﬁts of open source innovations are common in many areas of ICT for educational, social and economic activities around the world. Now, open source has started to beneﬁt agricultural practices in developing countries for improving agricultural productivity and providing better 3.3. Freedom to operate innovative bio-technology Open source has become an important mechanism to deliver free access in a global market for public goods such as innovative bio-technology. When monopoly rights are conferred on innovation, it narrows the ﬁeld of innovation, A.A. Adenle et al. / Technology in Society 34 (2012) 256–269 262 communications within farming communities that connects farmers to the rest of the world. India, Malawi, Pakistan and Kenya. For example, in Kenya, Frontline SMS facilitates and increases access to sales and deliveries as well as providing more accurate price, quality, and quantity information among small farmers and medium-size retailers. The ICT sector unit of World Bank group has described several mobile applications for agri- culture and rural development (m-ARD) that can be made available via open source in developing countries [75]. For example, m-ARD applications are under detailed case studies for agriculture markets and extension services and these applications are active in many developing countries including India, China, Philippine, Colombia, Argentina, Uruguay, Tanzania, Uganda and Kenya. Although many of these mobile applications are pilot studies they still hold promise for advancing future agricultural production and enhancing food security in developing countries. Table 1 demonstrates speciﬁc cases of open source for agricultural development in developing countries. Exam- ples of case studies such as Agribazzar, OSCAR (Simple Computer for Agriculture in Rural), SOPAC (South Paciﬁc Applied Geoscience) and e-Transform Agriculture are established by the country governments in partnership with developed countries and international organizations to enhance sustainable agricultural development in developing countries. This kind of open source innovation will provide opportunities for farmers to increase their knowledge on best farming methods, provide relevant information on agricultural inputs (e.g. fertilizer and seed), weather, storage and other farming activities, and widen their markets and gain new customers without internet connection, particularly in the rural areas [10,72,73]. While the impact of Frontline SMS is growing in health and agricultural development [74,76], effort is required in the area of ICT education which is the key to successful adoption. Qiang et al., (2011) [75] emphasised that the private sector, the public sector and donors must increase their efforts by improving the infrastructure, training young professionals and opening up access to publicly available data to facilitate development of mobile application. 3.3. Freedom to operate innovative bio-technology Apart from the examples demonstrated in the Table 1, there are several other initiatives being implemented through Open Mobile Consortium in developing countries particularly in Africa to facilitate access to information between non-governmental organisations (NGOs) and small farmers in the rural communities. For example, the United Nations Children’s Fund (UNICEF) Innovation has created an open source platform called Rapid SMS to monitor food distribution in Ethiopia and to assist in improving process of tracking the nutritional trends of Malawian children [73]. Frontline SMS is another open source platform that provides free access to software that is being used mostly in developing world in the area of agricultural development [74]. The biggest impact of Frontline SMS has been recorded in Philippines, Nigeria, Biotechnology is another ﬁeld where open source has great potential to contribute to agricultural development. Open source biotechnology may be key to knowledge and innovation for agricultural development, and part of the solution to the increasing population problem in devel- oping countries. Given the patent impediments on new agricultural innovations, different initiatives are emerging Asia-Europe collaboration. French institute of Pondicherry (IFP) lead project. Agriculture Department of Malaysia and the Malaysian Institute of Microelectronic Systems Table 1 Table 1 Open source for agricultural development case studies. Case study Summary of activities Consortium/partners Open source rational and beneﬁts References AgriBazaar AgriBazaar is an internet based agric- related trading for producers and suppliers. It provides real-time market information and prices of farm produce. Agriculture Department of Malaysia and the Malaysian Institute of Microelectronic Systems Free accessibility, ability to customize the and translate the software interface (GUI) into local languages, low maintenance and cost effectiveness [10] OSCAR – simple computer for agriculture in rural areas For farming community of the Indo- Gangetic Plains (IGP) using open source software applications for identifying/controlling weed in rice and wheat crop system that suit local languages and cultural practices in different regions. Asia-Europe collaboration. French institute of Pondicherry (IFP) lead project. To assist decision making on farm-level concerns in agriculture with relevance among farmers, extension ofﬁcers, scientists and students [77] SOPAC–South Paciﬁc applied geoscience Aimed to reduce vulnerability using GeoCMS application software to help collect and publish geographic data for access and sharing over the Internet. European funded project in collaboration with 14 African, Caribbean and Paciﬁc Group of States (ACP) To facilitate sharing of information and awareness promotion on hazard mitigation and risk assessment, water resources supply and agricultural practices [10] e-Transform Africa mAgri programme is an example of eAgriculture to read, capture and store data through mobile technology being promoted in African countries A partnership between the World Bank and the African Development Bank (AfDB), supported by the African Union (AU) To facilitate multi-stakeholder partnerships and services among farmers, extension ofﬁcers, scientists and other organisations for free agriculture knowledge sharing. [78] Consortium/partners Open source rational and beneﬁts References Agriculture Department of Malaysia and the Malaysian Institute of Microelectronic Systems Free accessibility, ability to customize the and translate the software interface (GUI) into local languages, low maintenance and cost effectiveness [10] Asia-Europe collaboration. French institute of Pondicherry (IFP) lead project. Agriculture Department of Malaysia and the Malaysian Institute of Microelectronic Systems Table 2 Initiatives promoting open access to agricultural and health biotechnology innovation. Source: [8,79–82]. The Centre for the Application of Molecular Biology to International Agriculture (CAMBIA) was ﬁrst formulated in mid-1980s by Richard Jefferson but developed into an integrated, full-text database of patents in the agricultural sciences that was funded by the Rockefeller Foundation in 1999. CAMBIA makes biotechnological research tools widely available through BiOS (Biological open source) initiative, Patent Lens database and BioForge. The BiOS Framework creates, validates and promulgates licencing tools, along with the norms and new business models to make use of strategies for “open source” creation, improvement, and sharing of enabling technology without impediment by patents and licences for the greater good of the public. BiOS initiative has led to emergence of biotechnological innovation such as b-glucuronidase (GUS) reporter gene system and Rhizobium strains (TransBacter system). GUS reporter gene system has been a mainstay of plant biotech research that led to the ﬁrst release of transgenic potatoes in 1987. The Patent Lens is a platform to focus, understand, and investigate the patent rights and to inform practitioners and policy-makers. It is a free full text searchable database for intellectual property informatics and analysis containing over 1.6 million patents in the life sciences. BioForge is an online interface where enabling technologies is made available via creating and distributing key “pump-priming”. Creating an innovation where IPR or patent restricts free access to diagnostic technology can be a useful tool to solve farming problems under BioForge open-source initiative. Since its establishment, efforts have been geared towards a strong campaign to provide communicative systems that allow dispersed individuals to participate and beneﬁt from decentralised innovative research. The Public Intellectual Property Resource for Agriculture (PIPRA) involves a group of non-proﬁt institutions from more than 15 countries around the world with focus on intellectual property issue (e.g. patent) by providing free access to patented technology, particularly agricultural biotechnology under a set of shared principles. PIPRA membership is open to any university, public agency, or non-proﬁt research institution actively engaged in research and development, but subject to supporting PIPRA’s mission and agreeing to the terms laid out in the PIPRA Memorandum of Understanding (MOU). A concerted effort has been made to develop a database of 6600 agricultural patents involving 45 different countries. Table 1 To assist decision making on farm-level concerns in agriculture with relevance among farmers, extension ofﬁcers, scientists and students [77] European funded project in collaboration with 14 African, Caribbean and Paciﬁc Group of States (ACP) To facilitate sharing of information and awareness promotion on hazard mitigation and risk assessment, water resources supply and agricultural practices [10] A partnership between the World Bank and the African Development Bank (AfDB), supported by the African Union (AU) To facilitate multi-stakeholder partnerships and services among farmers, extension ofﬁcers, scientists and other organisations for free agriculture knowledge sharing. [78] AgriBazaar is an internet based agric- related trading for producers and suppliers. It provides real-time market information and prices of farm produce. Asia-Europe collaboration. French institute of Pondicherry (IFP) lead project. For farming community of the Indo- Gangetic Plains (IGP) using open source software applications for identifying/controlling weed in rice and wheat crop system that suit local languages and cultural practices in different regions. mAgri programme is an example of eAgriculture to read, capture and store data through mobile technology being promoted in African countries A.A. Adenle et al. / Technology in Society 34 (2012) 256–269 263 is arguably the most useful for developing a framework for agriculture and has been at the forefront of promoting open source for sharing biological innovation [83]. For example, the GUS reporter system arguably remains the most widely used staining technique in plant science with over 4000 literature citations. Two different academic institutions have beneﬁted from using open source research tools for carrying out experimental research in agriculture [84]. The scientists at Cornell University in collaboration with a Hawaiian Papaya Growers Co-operative (made up of a small group of farmers) used CAMBIA open source research tools to ﬁnd a solution to a virus problem in papayas. The second example of use took place at Huaz- hong University where more than 20,000 unique lines were created by Zhang Qifa, a leading Chinese scientist in plant biotechnology. through open source development to facilitate access to modern agricultural biotechnology. Table 2 shows example of initiatives that are promoting open access to agricultural biotechnology with the exception of the International HapMap project promoting open access to health biotechnology. The fundamental rationale for the open source movement is to ensure the development, distribution and adoption of agricultural biotechnology that will beneﬁt researchers and ultimately small-holder farmers in developing countries. International HapMap project Table 1 International HapMap project The international HapMap project was origin common human sequence variation from pop project is to store and share data, and make t composed of scientists, funding agencies, pub Canada, China, Nigeria, and the United Kingd disease and responses to medications that wi Table 2 Initiatives promoting open access to agricultural and health biotechnology innovation. Source: [8,79–82]. Initiative Major areas of activity CAMBIA The Centre for the Application of Molecular Biology to International Agriculture (CAMBIA) was ﬁrst formulated in mid-1980s by Richard Jefferson but developed into an integrated, full-text database of patents in the agricultural sciences that was funded by the Rockefeller Foundation in 1999. CAMBIA makes biotechnological research tools widely available through BiOS (Biological open source) initiative, Patent Lens database and BioForge. The BiOS Framework creates, validates and promulgates licencing tools, along with the norms and new business models to make use of strategies for “open source” creation, improvement, and sharing of enabling technology without impediment by patents and licences for the greater good of the public. BiOS initiative has led to emergence of biotechnological innovation such as b-glucuronidase (GUS) reporter gene system and Rhizobium strains (TransBacter system). GUS reporter gene system has been a mainstay of plant biotech research that led to the ﬁrst release of transgenic potatoes in 1987. The Patent Lens is a platform to focus, understand, and investigate the patent rights and to inform practitioners and policy-makers. It is a free full text searchable database for intellectual property informatics and analysis containing over 1.6 million patents in the life sciences. BioForge is an online interface where enabling technologies is made available via creating and distributing key “pump-priming”. Creating an innovation where IPR or patent restricts free access to diagnostic technology can be a useful tool to solve farming problems under BioForge open-source initiative. Since its establishment, efforts have been geared towards a strong campaign to provide communicative systems that allow dispersed individuals to participate and beneﬁt from decentralised innovative research. PIPRA The Public Intellectual Property Resource for Agriculture (PIPRA) involves a group of non-proﬁt institutions from more than 15 countries around the world with focus on intellectual property issue (e.g. patent) by providing free access to patented technology, particularly agricultural biotechnology under a set of shared principles. Table 1 PIPRA membership is open to any university, public agency, or non-proﬁt research institution actively engaged in research and development, but subject to supporting PIPRA’s mission and agreeing to the terms laid out in the PIPRA Memorandum of Understanding (MOU). A concerted effort has been made to develop a database of 6600 agricultural patents involving 45 different countries. PIPRA’s goal is to ensure farmers, researchers and other organizations are provided with right resource for clarity and analysis of patented technology through effective implementations. PIPRA acts as a resource for technology transfer programs, and for scientists that work in the public sector, to facilitate the transfer and adoption of their technologies. Part of their goals is to mobilize and encourage innovative technologies among various institutions for the development and distribution of subsistence crops based on humanitarian purposes in developing countries. AATF The African Agricultural Technology Foundation (AATF) is an Africa-based non proﬁt organisation that negotiates and facilitates for the delivery and access of appropriate proprietary agricultural technologies to smallholder farmers in Sub-Saharan Africa. AATF engages in public/private partnerships to facilitate sharing and transfer of technology and research products that will beneﬁt African resource-poor farmers without access constraint. For example AATF obtained a royalty-free Monsanto GM technology, a Bacillus thuringiensis (Bt) gene (cry-1Ab) and sublicensed it to the Australian Commonwealth Scientiﬁc and Industrial Research Organisation (CSIRO) and Nigerian-based International Institute for Tropical for Agriculture (IITA) for the development of pod borer resistant cowpea crop. When the GM cowpea is ready for use in future, the biotech product will be made available through the commercialization and public institution for humanitarian use. International HapMap project The international HapMap project was originally launched in October 2002 to create genome wide database of common human sequence variation from population with ancestry parts of Africa, Asia and Europe. The goal of this project is to store and share data, and make the information freely available in the public domain. The consortium is composed of scientists, funding agencies, public and private organisations from six countries; the United States, Japan Canada, China, Nigeria, and the United Kingdom. The aim is to enable researchers to ﬁnd genes that affect health, disease and responses to medications that will lead to development of diagnostic tools. Table 1 The same concept is adopted by the International HapMap project for health biotechnology to facilitate open access to the develop- ment of diagnostic tools between developed and devel- oping countries [79], as described in the Table 2. Efforts by the CAMBIA BiOS initiative (Table 2) are not just about the campaign or creating awareness, but they demonstrate practical examples of donating free technol- ogies through open source licencing. Of all the initiatives, the BiOS initiative under the leadership of Richard Jefferson However, the Public Intellectual Property Resource for Agriculture (PIPRA) can be distinguished from open- source as they have different approaches for sharing Table 2 Initiatives promoting open access to agricultural and health biotechnology inn Initiative Major areas of activity CAMBIA The Centre for the Application of Molecular B mid-1980s by Richard Jefferson but develope sciences that was funded by the Rockefeller F widely available through BiOS (Biological ope Framework creates, validates and promulgate make use of strategies for “open source” crea impediment by patents and licences for the g of biotechnological innovation such as b-gluc (TransBacter system). GUS reporter gene syst release of transgenic potatoes in 1987. The Pa patent rights and to inform practitioners and property informatics and analysis containing interface where enabling technologies is mad Creating an innovation where IPR or patent r farming problems under BioForge open-sourc a strong campaign to provide communicative from decentralised innovative research. PIPRA The Public Intellectual Property Resource for more than 15 countries around the world wit access to patented technology, particularly ag membership is open to any university, public research and development, but subject to sup PIPRA Memorandum of Understanding (MOU agricultural patents involving 45 different cou organizations are provided with right resourc implementations. PIPRA acts as a resource for public sector, to facilitate the transfer and ad encourage innovative technologies among va crops based on humanitarian purposes in dev AATF The African Agricultural Technology Foundati and facilitates for the delivery and access of a in Sub-Saharan Africa. AATF engages in publi and research products that will beneﬁt Africa obtained a royalty-free Monsanto GM techno Australian Commonwealth Scientiﬁc and Indu Institute for Tropical for Agriculture (IITA) for cowpea is ready for use in future, the biotech public institution for humanitarian use. 5.1. Constraints in adopting open source biotechnology As demonstrated above, open source biotechnology has potential to beneﬁt human development, but there are fundamental challenges that must be properly addressed to ensure that open source delivers on its potential. Even though challenges will vary from country to country, most challenges will more likely be faced in developing coun- tries largely due to the high level of poverty, political instability, economic instability and limited resources. Moreover, the challenge of open source adoption will remain a global problem as long as the majority of the world population has little or no access to it. The challenge in using open source biotechnology will not only be in the area of agriculture practices but in every aspect of bio- logical innovation. q y gy Patent misuse through grant back mechanisms is another challenge in open source biotechnology [89]. As the technology advances, it may be difﬁcult to keep the research tools and inventions within the conﬁnes of open source arena as technology becomes available to the wider research community. For example, in an attempt to seek rewards or compensation for any kind of improvement made to the technology, it may result in legal proceedings if the innovator involved feels that improvement made to the technology should attract greater rewards [89]. Under normal principles of patent law, inventors making novel improvements to the core technology are entitled to apply for patents as long as the requirements of patentability are met and the right improvements are followed. Therefore, the open source licencing agreement may be ignored by the inventor if there is greater reward. This can sometimes lead to shrouded or overlapping rights on research tools among multiple parties. The issue of the grant back mechanism as related to the open source licence may discourage collab- oration that would facilitate ideas among young genera- tions of scientists towards increasing creativity and better innovations. For example the grant back mechanism and BiOS viral licence have led to the rejection of BiOS licencing terms for the PIPRA project [90]. Moreover, in the context of the grant back approach, researchers who are interested in commercializing their research work could decide not to Introducing open source biotechnology in developing countries will require a considerable amount of training. Where there is a fair amount of knowledge of ICT, some amount of retraining may be required. Table 2 In open source, service is focused on cumulative improvement which requires downstream transfer of the open-sourced materials under a copyleft style “grant back mechanism” (licensees must agree to share back and allow the right to re-use improvement made on research tools under open-source licence) [86]. While both initiatives differ in a way, they serve the research community well and achieve their purpose of encouraging and sharing innovation by creating more free access to research tools that can beneﬁt various institu- tions across different countries. delay the open source development. Each of these cases requires enormous ﬁnances for training and retraining processes, and constructing new infrastructure for IT purposes. p p The challenge of IPRs is arguably the most complex due to the high cost involved. In the case of resource-poor farmers, an open source consortium that requires IPRs to protect farmer’s resources can be very expensive to obtain and maintain, and this may be a big burden to farmers that have limited ﬁnancial resources [87]. In spite of the fact that the general open source licence policy is based on the promise to keep source code free and allow free access to copyright-protected aspect of the code, challenges still remain in open source biotechnology. There are challenges of the translation of open source software model to biotechnology due to different characteristics governing the patent versus the copyright laws. To achieve protected common access (allow users to access) in open source, copyright as the dominant IPRs licence is the key legal right access in open source software, whereas patent is the dominant IPR form of protection open source biotechnology [20]. In terms of standards, ”patentability” is a much higher standard than the “copyrightability” [88]. The cost of getting patents for the innovations in biotechnology can be exorbitant and time-consuming, compared to open source software that costs little and requires less time. Thus, patents can cause a setback in research and limit the experimentation by scientists or individual farmers. In addition, the research culture in open source software is different from open source biotechnology [26]. For example, the equipment used in biotechnology research is very expensive, when compared to software technology that requires a computer and a desk. Considering these factors, the open source approach would be more difﬁcult to diffuse or move quickly in biology than in software. Table 2 PIPRA’s goal is to ensure farmers, researchers and other organizations are provided with right resource for clarity and analysis of patented technology through effective implementations. PIPRA acts as a resource for technology transfer programs, and for scientists that work in the public sector, to facilitate the transfer and adoption of their technologies. Part of their goals is to mobilize and encourage innovative technologies among various institutions for the development and distribution of subsistence crops based on humanitarian purposes in developing countries. p p p p g The African Agricultural Technology Foundation (AATF) is an Africa-based non proﬁt organisation that negotiates and facilitates for the delivery and access of appropriate proprietary agricultural technologies to smallholder farmers in Sub-Saharan Africa. AATF engages in public/private partnerships to facilitate sharing and transfer of technology and research products that will beneﬁt African resource-poor farmers without access constraint. For example AATF obtained a royalty-free Monsanto GM technology, a Bacillus thuringiensis (Bt) gene (cry-1Ab) and sublicensed it to the Australian Commonwealth Scientiﬁc and Industrial Research Organisation (CSIRO) and Nigerian-based International Institute for Tropical for Agriculture (IITA) for the development of pod borer resistant cowpea crop. When the GM cowpea is ready for use in future, the biotech product will be made available through the commercialization and public institution for humanitarian use. The international HapMap project was originally launched in October 2002 to create genome wide database of common human sequence variation from population with ancestry parts of Africa, Asia and Europe. The goal of this project is to store and share data, and make the information freely available in the public domain. The consortium is composed of scientists, funding agencies, public and private organisations from six countries; the United States, Japan, Canada, China, Nigeria, and the United Kingdom. The aim is to enable researchers to ﬁnd genes that affect health, disease and responses to medications that will lead to development of diagnostic tools. A.A. Adenle et al. / Technology in Society 34 (2012) 256–269 264 innovations. PIPRA offers services in terms of collaboration with institutional members on intellectual property policy analysis, biotechnology resources, and commercialization strategy to improve and develop shared technology packages [81,85]. Table 2 While the African Agricultural Technology Foundation (AATF) and the International HapMap project are under different pilot phases, there is a need to encourage a wide range of stakeholders in private and public sector to increase their commitment so as to make it a reliable and effective open source biotechnology operating system. 5.2.1. Proposed open source agricultural biotechnology framework f Based on the analysis of the case studies and initiatives presented, an Open Source Biotechnology Framework (OSBF) shown in Fig. 2 is proposed as a possible solution for the adoption and promotion of agricultural biotechnology R&D for sustainable development in developing countries. Using a similar principle to the open source software development process [6], the OSBF identiﬁes agricultural biotechnology research and cooperation priorities which are highly dependent on technology for their effective implementation. As shown in Fig. 2, two developing countries (A and B) working on the same or similar biotech products (e.g. maize or sorghum) can leverage the principle of open source software development and openly collabo- rate in their R&D activities. According to this framework, agricultural extension workers, research institutions, farmers, governments and other stakeholders can freely and collectively leverage the expertise of biotechnology developer and user bases. They can also beneﬁt or learn from other biotechnology companies and vendors of the software who are willing to reveal and share their inno- vation, share their experiences and risks, associated with a particular agricultural product. This kind of intra/inter research and collaboration can even be extended to other countries or forms of international alliances where open source biotechnology research and innovation networks are already formed. 5.2.2. Policy implications for open source biotechnology 5.1. Constraints in adopting open source biotechnology For example, Agri- Bazaar (Table 1), using FOSS tools in Malaysia were difﬁcult due to the lack of familiarity among software developers. Some of the engineers are used to proprietary software products, and similarly it will take time to adjust to a new product in agricultural biotechnology. Also, the interoper- ability problems may cause reluctance among the users where organisations need to change to open source [10]. In many places in developing countries, particularly in Africa, the lack of basic infrastructure such as communications and electricity may delay the application or adoption of open source biotechnology. For example, high-bandwidth Internet access is lacking in universities and research institutions coupled with an erratic supply of power. This can limit the ability to participate in open source projects. Furthermore, factors such as a shortage of IT professionals and the absence of IT industries in developing countries can A.A. Adenle et al. / Technology in Society 34 (2012) 256–269 265 participate when confronted with the policy and process for commercialization. participate when confronted with the policy and process for commercialization. Beneﬁts associated with this research and innovation network may include reducing or sharing costs and risks on: (1) using open source technologies and services, (2) exchanging staff and sharing of skills, (3) outsourcing essential services. However, as highlighted in Section 5.1, considerable training, re-training and education in the area of open source software development is essential for implementing this kind of framework. Cooperation and collaboration in areas such as open source awareness, policy harmonization across agricultural zones, farming communities and countries is fundamental for ensuring successful implementation and sustainability of this open source agricultural biotechnology framework. 5.2. Agricultural biotechnology framework and policy implications for open source development 5.2. Agricultural biotechnology framework and policy implications for open source development 5.2.1. Proposed open source agricultural biotechnology framework 5.2.2. Policy implications for open source biotechnology 5.2.2.1. Provision of adequate training. Training is the hall- mark of effective and good management. Open source development requires adequate training in various aspects of FOSS in order to facilitate the introduction and growth of open source biotechnology in developing countries. It is necessary to establish a technical support and training policy for open source development, particularly in infor- mation and communication technology (ICT). ICT policies should be a part of the process and part of what is needed in the developing countries to promote and support open source. Policies adopted should support education and training at different levels. Due to the low level of ICT literacy in developing countries, strategies or policies designed for training should reﬂect basic skills (e.g. basic understanding in IT) and user-friendly methodology in any language of communication for the rural poor. For example, FOSS software used for AgriBazaar in connecting farmers and buyers in Malaysia was designed to serve the Fig. 2. Open source agricultural biotechnology R&D framework. Fig. 2. Open source agricultural biotechnology R&D framework. Fig. 2. Open source agricultural biotechnology R&D framework. A.A. Adenle et al. / Technology in Society 34 (2012) 256–269 266 Providing local professionals with adequate training and acquiring skills in relevant software development to remain viable in a competitive market; communities in various local languages [10]. This can facilitate the acceptance and participation among the tar- geted group at grass-roots levels. Moreover, training should be provided regularly in the course of the evaluation and adaptation process. Where necessary, retraining should be provided for sustainability and continuity. Identifying and prioritization areas of need such as local software development and IT industries; Adopting open standards for storage and preservation of data. 5.2.2.2. Provision of adequate resources and facilities. Given the importance of open source biotechnology for the beneﬁt of society, adequate resources would need to be committed to support open source development in devel- oping countries. Apart from education and training, basic facilities such as telecommunication and electricity that will facilitate the application of open source in developing countries should be provided. In all of these, open source will require a big ﬁnancial investment from the govern- ments. Nonetheless, the cost of providing open source can be cheaper relative to proprietary software. For example, the migration of Ugandan University to open source resulted in signiﬁcant cost reduction [91]. 5.2.2. Policy implications for open source biotechnology For example, it should be practiced under the “four freedoms of choice”: 1) Access without restriction; 2) Availability of source code; 3) Improve and add to the source code; 4) Redistribute the source code and software. The availability of genomic data- bases through bioinformatics provides ﬂexibility for the terms chosen by the users. By contrast, biotechnology research does not usually provide an avenue for freedom of choice. Also, the current licencing policy of BiOS does not encourage freedom of choice due to the grant back mecha- nism [89]. BiOs allows users to refrain from the grant back improvement if kept as trade secrets where it prevents their improvement from being disclosed to another users. A recent report emphasises the fact that an enhanced open source model through a trade secret could offer a ﬂexible policy agreement as proposed for the HapMap project [96]. In addition, a business-friendly licencing policy that will maxi- mize the growth potential of open source biotechnology should be encouraged among the users. Encouraging ﬂexible licencing policies under the open source approach could lead to the rapid development of research tools and increased economic beneﬁts for users in developing countries. 5.2.2.3. Collaboration and network expansion. Collaborative data sharing should be encouraged among the academic communities particularly in developed countries, as this will provide opportunities for researchers in developing countries to beneﬁt. Most researchers in developing countries depend on scientiﬁc data and publication from developed countries to advance research in their areas. For example, developing countries have created successful science policies through a two-way contribution of international scientiﬁc exchange [95]. Therefore, collaborative data sharing, through open source is one approach to encourage scientiﬁc networking between scientists in developed and developing countries. Moreover, collaboration based on common tools where individual sharing of discoveries in biological innovation could be encouraged between universities and multina- tionals, paving the way for open source biotechnology. 5.2.2. Policy implications for open source biotechnology Other examples [92,93] similarly show that open source is relatively inex- pensive and easily adapted to local needs for various purposes. This is in sharp contrast to proprietary vendors that are globally proﬁt-oriented with little connection to local needs [94]. Therefore, the government’s policy should recognise the importance of FOSS and address the issues concerning the provision of right resources and allocation of budget for the development of open source project. In addition, governments should work in conjunction with international agencies such as CBD, TRIPS and UPOV to create a friendly IPRs system that will encourage open source development for innovative technologies in devel- oping countries. This can decentralize patented technologies and make them freely accessible to researchers in devel- oping countries, particularly in agriculture biotechnology. 5.2.2.5. Flexible licencing policies. Open source licencing policy should be ﬂexible enough to allow interested party to use innovation under the freedom of choice. For example, it should be practiced under the “four freedoms of choice”: 1) Access without restriction; 2) Availability of source code; 3) Improve and add to the source code; 4) Redistribute the source code and software. The availability of genomic data- bases through bioinformatics provides ﬂexibility for the terms chosen by the users. By contrast, biotechnology research does not usually provide an avenue for freedom of choice. Also, the current licencing policy of BiOS does not encourage freedom of choice due to the grant back mecha- nism [89]. BiOs allows users to refrain from the grant back improvement if kept as trade secrets where it prevents their improvement from being disclosed to another users. A recent report emphasises the fact that an enhanced open source model through a trade secret could offer a ﬂexible policy agreement as proposed for the HapMap project [96]. In addition, a business-friendly licencing policy that will maxi- mize the growth potential of open source biotechnology should be encouraged among the users. Encouraging ﬂexible licencing policies under the open source approach could lead to the rapid development of research tools and increased economic beneﬁts for users in developing countries. 5.2.2.5. Flexible licencing policies. Open source licencing policy should be ﬂexible enough to allow interested party to use innovation under the freedom of choice. 6. Conclusion The open source paradigm as applied to agricultural practices has the potential to compensate farmers for contributing to the growth of plant resources, and may serve as information resources for farming communities [87]. While this article discusses the complexity of IPRs in agricultural biotechnology, it also mentions the potential beneﬁt and impact of open source in agricultural and biotechnology development. Addressing the three research questions posed in a systematic way provided insight into the development of open source biotechnology in devel- oping countries. Most importantly, the analysis of speciﬁc case studies and initiatives led to the proposal of an Open Source Biotechnology Framework (OSBF) that can facilitate development of biotechnology R&D, exchange of skills and ideas and collaboration among agricultural research insti- tutes between different countries as well as policy 5.2.2.4. Effective policy and legislation. The success of open source development requires that effective policy and legislation be developed and implemented in developing countries. Given the importance of political support, it can facilitate processes and increase the adoption rate if there is a high level of commitment. Therefore, government should formulate policies that encourage open source development and the policy should focus on the following areas: Support of ICT in education and the government sector at all levels; Enabling an environment for the accessibility of ICT by citizens, business and the government; A.A. Adenle et al. / Technology in Society 34 (2012) 256–269 267 recommendations to foster open source development in agriculture. authors are grateful to the three anonymous reviewers for their comments on the article. Comments and suggestions from the editor, Charla Griffy-Brown, in improving the quality of the manuscript are gratefully acknowledged. The adequate provision of basic infrastructure and ﬁnancial resources will play a vital role in the adoption of open source biotechnology. Education forms the basis through which open source development can be promoted among the citizens, therefore quality education on ICT programs should be provided to encourage wide participa- tion from the grass-root levels. The lack of political support due to a low level of awareness among the government ofﬁcials on open source in developing countries can slow down open source development. Therefore, a concerted effort should be made to educate and encourage govern- ment ofﬁcials in all relevant institutions. References [1] WorldBanK. World development report, agriculture for development. Washington, DC: Ofﬁce of the Publisher, the World Bank; 2008. [2] James C. Global status of commercialized biotech/GM crops. The International Service for the Acquisition of Agri-biotech Applica- tions (ISAAA); 2012. Brief 43. [3] Adenle AA. Global capture of crop biotechnology in developing world over a decade. Journal of Genetic Engineering and Biotech- nology 2011;9(2):83–95. [4] Hemphill TA. Preemptive patenting, human genomics and the US biotechnology sector: balancing intellectual property rights with societal welfare. Technology in Society 2003;25:337–49. [5] Frisio DG, Ferrazzi G, Ventura V, Vigani M. Public vs. private agbiotech research in the United States and European Union, 2002– 2009. AgBioForum 2010;13(4):333–42. [6] Gay J, Lessig L. Free software, free society: selected essays of Richard M. Stallman. Boston, MA: GNU Press, http://www.gnu.org/philosophy/ fsfs/rms-essays.pdf; 2002 [accessed April 2012]. [7] BiOS. Biological innovation for open society, http://www.bios.net/ daisy/bios/2518.html; 2005. While this article has reviewed the existing literature with relevant information about the adoption of open source biotechnology in agricultural practices, not many initiatives or organisations are advocating for open source development for innovations that can enhance sustainable agricultural development. More effort is required from a variety of actors including the private sector, individuals, national governments and international agencies to support and promote open source biotechnology for sustainable agricultural development in developing coun- tries. Moreover, a lot of work needs to be done in terms of further case study analysis to more fully assess the major areas where open source is being adopted, as well as the beneﬁts and the constraints that are associated in open source adoption in developing countries. [8] Thorisson GA, Smith AV, Krishnan L, Stein LD. The international HapMap project web site. Genome Research 2005. [9] Sowe SK. Free and open source software sustainability and innovation: lessons learnt from Sub-Saharan African. In: Interna- tional conference on ICT for Africa, Nigeria, March 23–26, 2011; 2011. p. 199–203. p [10] Hoe NS. Breaking barriers: the potential of free and open source software for sustainable human development; a compilation of case studies from across the world. UNDP-APDIP; 2006. [11] Yin RK. Case study research: design and methods. In: Applied social research methods series. 3rd ed., vol. 5. Sage Publications; 2003. [12] Denzin N. Sociological methods: a sourcebook. Aldine Transaction; 2006. [13] Stallman R. Free software, free society. Boston, MA: GNU Press; 2002. [14] Voas J, Miller KW, Costello T. References Free and open source software. IT Professional 2010;12(6):14–6. [15] Raymond E. The cathedral and the bazaar: musings on linux and open source by an accidental revolutionary. Sebastopol, CA: O’Reilly Media; 1999. Finally, if the introduction of open source biotech- nology is to contribute to sustainable agriculture in developing countries, enabling environments must be put in place that include policy formulation and imple- mentation for establishing, supporting and providing the capacity building and resources required to develop open source. Moreover, the attention and focus of open source development should not be restricted to one area but it should address other needs such as health and the envi- ronment. The opportunities offered by open source biotechnology can make a signiﬁcant impact on sustain- able agricultural development through free access to modern biotechnology techniques. Open source biotech- nology is making slow but steady progress in agriculture, but many issues will have to be addressed to enjoy the beneﬁts of this new innovation in developing countries. This article has raised important concerns that will lead to further debates among stakeholders including scientists and policymakers. [16] Weber S. The success of open source. Cambridge MA: Harvard University Press; 2004. [17] Cassier M. New “enclosures” and the creation of new “common rights” in the genome and in software. Contemporary European History 2006;15(2):255–71. y ( ) [18] Hope J. Open source licensing. In: Krattiger A, Mahoney RT, Nelson L, et al., editors. Intellectual property management in health and agricultural innovation. A handbook of best practices. Oxford, U.K./Davis, U.S.A.: MIHR/PIPRA; 2007. [19] FSF. Free Software Foundation. Free software deﬁnition, http:// www.gnu.org/philosophy/free-sw.html; 2008. [20] Hope J. The open source revolution and biotechnology. Cambridge, MA: Harvard University Press; 2008. [21] Wake S, Ridley RG. Virtual drug discovery and development for neglected diseases through public-private partnerships. Nature Review 2003:919. [22] OBF. Open Bioinformatics Foundation. (www.open-bio.org). [23] CAMBIA. Center for the Application of Molecular Biology to Inter- national Agriculture, http://www.cambia.org/daisy/cambia/about/ 3692/2518.html; 1991. / [24] Douthwaite B. Enabling innovation: a practical guide to understanding and fostering technical change. Boston, MA: Zed Books; 2002. [25] Srinivas K. The case for biolinuxes: and other pro-commons innovations. In: Vasudevan R, Sundaram R, Bagchi J, Narula M, Lovink G, Sengupta S, editors. Sarai reader 2002: the cities of everyday life. New Delhi: Center for the Study of Developing Socities; 2002. p. 321–8. 6. Conclusion Given the impact of IPRs in creating a research environment for biotechnology, the IPRs system must be designed in a research-friendly way, particularly with regards to protection under law that will facilitate commercialization and technology transfer to developing countries. Added to this, institutional and legal frameworks must be established and encouraged to protect IPRs in developing countries so as to enhance the economic beneﬁt of open source biotechnology products. Acknowledgement [59] Thies JE, Devare M. An ecological assessment of transgenic crops. Journal of Developmental Studies 2007;43:97–129. [34] Cooper HD. The international treaty on plant genetic resources for food and agriculture. Review of European Community & Interna- tional Environmental Law 2002;11:116. [60] Steinbrecher RA. V-GURTs (terminator): can it be effective as a bio- logical containment tool? EcoNexus. CoP-MoP2, http://www. econexus.info/sites/econexus/ﬁles/ENx_V-GURTs_brief_2005.pdf; 2005 [accessed 30.05.2012]. [35] Rai AK, Eisenberg RS. Bayh-Dole reform and the progress of biomedicine. Law & Contemporary Problems 2003;66:289–300. [36] de Janvry A, Graff G, Sadoulet E, Zilberman D. Technological change in agriculture and poverty reduction. Concept paper for WDR on Poverty and Development 2000/2001. Berkeley: University of Cal- ifornia; 2000. p. 6–7. [ ] [61] RF Rockefeller Foundation. “Food gains for the world’s poor are being threatened by furor over genetically modiﬁed (GM) foods”. Press release, Rockefeller Foundation Washington DC. 1999. p [37] Jefferson R. Freely sharing innovation is the only way to face the future. Australian Leadership Retreat Special Report. The Australian August 31. 2007. [62] CGIAR. Shaping the CGIAR’s future: summary of proceedings and decisions. Consultative Group on International Agricultural Research (CGIAR) International Center Week, October 26-30. Washington, DC: CGIAR Secretariat; 1998. [38] Pray CE, Naseem A. Intellectual property rights on research tools: incentive or barriers to innovation? Case studies of rice genomics and plant transformation technologies. AgBioForum 2005;8:108–17. [63] OECD. Organisation for Economic Co-operation and Development (OECD). Intellectual property and competition policy in the biotechnology industry. June, Policy Brief; 2005. [64] Thomas D, Hunt A. Open source ecosystems. IEEE Software 2004; 21(4):89–91. [39] Parayil G. Mapping technological trajectories of the green revolu- tion and the gene revolution from modernization to globalization. Research Policy 2003;32:971–90. [65] Fitzgerald B. Open source software adoption: anatomy of success and failure. International Journal of Open Source Software & Processes 2009:1–23. [40] Kryder DR, Kowalski SP, Krattiger AF. The intellectual and technical property components of pro-vitamin A rice (Golden Rice): a preliminary freedom-to-operate review. In. International service for the acquisition of agri-biotech applications (ISAAA) brief no. 20. ISAAA, Ithaca, NY; 2002. [66] Wright BD. Public germplasm development at a crossroad: biotech- nology and intellectual property. California Agriculture 1998;56:8–13. [67] Erbisch FH, Maredia KM. Intellectual property rights in agricultural biotechnology. CAB International; 2004. [41] Isaac AG, Walter GP. On intellectual property rights: patents vs. free and open development, chapter 18 of Colombatto, Enrico, the Elgar companion to the economics of property rights. Acknowledgement Aldershot, UK: Edward Elgar; 2004. [68] Hoekman BM, Maskus KE, Saggi K. Transfer of technology to developing countries: unilateral and multilateral policy options. Institute of Behavioral Science (IBS). University of Colorado; 2004. Working Paper. [42] Heller MA, Eisenberg RS. Can patents deter innovation? The anti- commons in biomedical research. Science 1998;280:698–701. g p [69] Fenton GM, Chi-Ham C, Boettiger S. Freedom to operate: the law ﬁrms approach and role. In: Krattiger A, Mahoney RT, Nelsen L, Thomson JA, Bennett AB, Satyanarayana K, et al., editors. Intellectual property management in health and agricultural innovation: a handbook of best practices. Oxford, U.K./Davis, U.S.A: MIHR/PIPRA, http://www. iphandbook.org/handbook/chPDFs/ch14/ipHandbook-Ch%2014% 2004%20Fenton-Chi-Ham-Boettiger%20FTO%20and%20Law%20Firm %20Roles.pdf; 2007 [accessed April 2012]. [43] Rimmer M. Genentech and the stolen gene: patent law and pioneer inventions. Bio-Science Law Review 2003;5(6):198–211. [44] Eisenberg RS. Why the gene patenting controversy persists. Academic Medicine 2002;77(12):1381–7. [45] Arora A, Merges P. Specialised supply ﬁrms, property rights and ﬁrm boundaries. Industrial and Corporate Change 2004;13: 451–75. [46] Bureth A, Penin J, Wolff S. Entrepreneurship in biotechnology: the case of four start-ups in the upper-Rhine biovalley. Working paper BETA, no.2006-21; 2006. [70] Pardey PG, Wright BD, Nottenburg C, Binenbaum E, Zambrano P. Intellectual property and developing countries: freedom to operate in agricultural biotechnology. Brief 3. Biotechnology and Genetics Resource Policies; 2003. [47] David PA. Can ‘open science’ be protected from the evolving regime of intellectual property rights protections. Journal of Theoretical and Institutional Economics 2004;160:1–26. [71] USAID. The U.S Agency for International Development (USAID). Selecting mobile ICT Devices for agriculture services and application in Sub-Saharan Africa. Brieﬁng Paper; 2011. [48] Fisher WM. The impact of “terminator gene” technologies on developing countries. Report to the United Kingdom department for international development. December. http://www.law.harvard. edu/faculty/tﬁsher/terminator.html [accessed April 1999]. [72] Pickernell DG, Christie MJ, Rowe PA, Thomas BC, Putterill LG, Grifﬁth JL. Farmers in Wales: marketing the network? British Food Journal 2004;106:194–210. [49] ETCGroup. Ban terminator before it’s too late. ETC News Release 5 April. 2002. [73] Schechter M. Innovation of the week: open source software for agriculture and Nutrition. State of the World 2011: innovations that nourish the planet. http://blogs.worldwatch.org/ nourishingtheplanet/innovation-of-the-week-open-source- software-for-agriculture-and-nutrition/[accessed April 2011]. [50] Jefferson R. Transcending transgenics – are there ‘babies’ in the bathwater, or is that a dorsal ﬁn? In: Pardey PG, editor. The future of food; biotechnology markets and policies in an international setting. Washington, DC: International Food Policy Research Institute; 2001. Ch.5. [74] FSU. Acknowledgement [26] Hope J. Open source biotechnology. A thesis submitted for the degree of Doctor of Philosophy at The Australian National University. Available at: http://rsss.anu.edu.au/wjaneth/ OpenSourceBiotechnology27July2005.pdf. 2004. The ﬁrst two authors would like to acknowledge the support of Japan Society for the Promotion of Science. The A.A. Adenle et al. / Technology in Society 34 (2012) 256–269 268 [27] Blakeney M. Recent development in intellectual property and power in the private sector related to food and agriculture. Food Policy 2011;36(Suppl. 1):S109–13. Elsevier Ltd. the third green revolution. Biotechnology Law Report 2004;23: 1–29. [52] Eaton D, Van Tongeren F, Louwaars N, Visser B, Van der Meer I. Economic and policy aspects of ‘terminator’ technology. Biotech- nology and Development Monitor 2002;49:19–22. [28] Wright BD, Pardey PG, Koo B. Agricultural innovation: investments and incentives. In: Evenson R, Pingali P, editors. Handbook of agri- cultural economics, vol. 3; 2007. p. 2537–8. [53] Swason T, Goschl T. Genetic use restriction technologies (GURTs): impacts on developing countries. International Journal of Biotech- nology 2000;2(1/2/3). [29] Safrin S. Treaties in collision? The biosafety protocol and the world trade organization agreements. The American Journal of Interna- tional Law 2002;96:22. [54] Goeschl T, Swanson T. The development impact of genetic use restriction technologies: a forecast based on the hybrid crop experi- ence. Environment and Development Economics 2003;8:149–65. [30] Moschini GC. Intellectual property rights and the world trade organization: retrospect and prospects, http://www.card.iastate. edu/faculty/proﬁles/giancarlo_moschini/moschini-trips-preprint- oct-04.pdf; 2012 [accessed 28.05.2012]. [55] Martin MA. Biotechnology, gene ﬂow, and intellectual property rights: an agricultural summit. In: Proceedings of a conference held the anticommons in biomedical research September 13, in Indian- apolis, Indiana; 2002. [31] Van Overwalle G. Patent protection for plants. A comparison of American and European approaches. The Journal of Law and Tech- nology 1999;39:143–94. p [56] Giovannetti M. The ecological risks of transgenic plants. Rivista Di Biologia 2003;96:207–23. [32] Bocci R. Seed legislation and agrobiodiversity: conservation varie- ties. Journal of Agriculture and Environment for International Development 2009;103:31–49. [57] Daniell H. Molecular strategies for gene containments in transgenic crops. Nature Biotechnology 2002;20:581–6. [33] Pardey PG, Alston JM, Chan-Kang C, Castello MagaIhaes E, Vosti SA. Assessing and attributing the beneﬁts from varietal improvement research in Brazil. IFPRI Research Report. No. 136. Washington, DC: International Food Policy Research Insitutute; 2004. [58] Shoemaker R. Economics issues in agricultural biotechnology. Agricultural Information Bulletin No.762. Washington DC: Economic Research Service, United States Department of Agricul- ture; 2001. Acknowledgement Sowe holds a PhD (summa cum laude) in Computer Science from Aristotle University, Greece (2007), MSc and Advanced Diploma in Computer Science from Sichuan University, China (1997), BEd in Science Education from University of Bristol, UK (1991), and a Higher Teachers Certiﬁcate (HTC) from Gambia College, the Gambia (1988). Dr. Sowe previously worked as a senior researcher at United Nations University of Maastricht Economic and social Research and training centre on Innovation and Technology (UNU-MERIT), Netherlands, applying qualitative and quantitative data analysis tech- niques to identify patterns of Open Source Software innovation and technological sustainability in ﬁrms and communities. He is currently pursuing research funded by the Japan Society for the Promotion of Science (JSPS), at the United Nations University Institute of Advanced Studies (UNU-IAS) in Yokohama, Japan. He is also a visiting scholar at the National Graduate Institute for Policy Studies (GRIPS), Tokyo, Japan. [81] PIPRA. PIPRA (Public Intellectual Property Resource for Agriculture). http://www.pipra.org/about/[accessed April 2012]. http://www.pipra.org/about/[accessed April 2012]. [82] Boadi RY, Bokanga M. The African agricultural technology founda- tion approach to IP management. In: Krattiger A, Mahoney RT, Nelson L, et al., editors. Intellectual property management in health and agricultural innovation: a handbook of best practices. Oxford, U.K: MIHR; 2007. [83] Boetigger S, Wright BD. Open source in biotechnology: open ques- tions. Innovations Case Discussion: CAMBIA-BiOS; 2006. [84] Thomas Z. Open source agricultural biotechnology. Current Science 2005;88:1212–3. [85] Atkinson RC, Beachy RN, Conway G, Cordova FA, Fox MA, Holbrook KA, et al. Intellectual property rights. Public sector collaboration for agricultural IP management. Science 2003;301: 174–5. [86] Penin J, Wack J. Research tool patents and free-libre biotechnology: a suggested uniﬁed framework. Research Policy 2008;37:1909–21. Professor Govindan Parayil, an Indian national, joined the United Nations University (UNU) as Vice-Rector in August 2008, and as Director of United Nations University Institute of Advanced Studies in January 2009. He currently serves as both Director of UNU-IAS and Vice-Rector of UNU. He holds a Bachelor of Science degree (Electrical Engineering) from the University of Calicut (India), a Master of Science degree (Science, Tech- nology and Values) from Rensselaer Polytechnic Institute (USA), a Master of Arts degree (Development Economics) from American University (USA), and a Ph.D. in Science and Technology Studies from Virginia Polytechnic Institute and State University (USA). Acknowledgement FrontlineSMS Users (FSU). Learning more about FrontlineSMS users: results from our ﬁrst ever survey, http://www.frontlinesms. com/2011/04/13/learning-more-about-frontlinesms-users-results- from-our-ﬁrst-ever-survey/; 2012 [accessed 28.05.2012]. [51] Pedleton CN. The peculiar case of terminator technology: agri- cultural and intellectual property protection at the crossroads of A.A. Adenle et al. / Technology in Society 34 (2012) 256–269 269 [75] Qiang CZ, Kuek SC, Dymond A, Esselaar S. Mobile application for agriculture and rural development. ICT sector unit. Washinghton DC: The World Bank; 2011. Dr. Ademola A. Adenle holds a M.Sc in Genetic Manipulation from University of Sussex, UK and a Ph.D. from University of Nottingham, UK. Dr. Adenle has published in several peer-reviewed international journals including book chapters. He has won prestigious awards including the best student prize for his PhD research presentation at the British Toxi- cology Society’s Annual Congress in Surrey, UK. He ﬁrst authored a reviewed paper on spaceﬂight which attracted interest from UK and US media respectively. His current research at the United Nations University- Institute of Advanced Studies (UNU-IAS) is based on the role of biotech- nology in sustainable agriculture and climate change mitigation/adaption in developing countries, particularly in Africa. He has visited and looked into the procedures leading to the development of biosafety regulatory frameworks across different African countries including Ghana, Nigeria, South Africa, Kenya, Egypt and Tunisia. He is also a visiting scholar at the National Graduate Institute for Policy Studies (GRIPS), Tokyo, Japan. [76] Rannu R, Saksing S, Mahlakõiv T. The mobile government: 2010 and beyond. The white paper. European Union Regional Development Fund; 2010. [77] Balasubramanian D, Lie R, Grard P. ICT and agriculture in India: a perspective from the case study based on the oscar project (open source simple computer for agriculture in rural area). NETCOM 2009;23(3/4):281–92. ( / ) [78] ETA. eTransform Africa. Agriculture sector study sector assessment and opportunities for ICT, http://etransformafrica.org/sites/default/ ﬁles/Readers-Digest-Agriculture.pdf; 2012 [accessed April 2012]. / g g p [ p ] [79] Lander ES, Lai EH, Nickerson DA, Abecasis GR, Altshuler D, Bentley DR, et al. The international HapMap project. Nature 2003; 426:789–96. [80] BiOS. BiOS (Biological Innovation for Open Society). http://www. bios.net/daisy/bios/2518.html. [accessed April 2012]. Dr. Sulayman K. Acknowledgement He authored Conceptualizing Tech- nological Change (1999) and edited Kerala: The Development Experience (2000) and Political Economy and Information Capitalism in India (2006), and has written numerous book chapters and articles in international journals. His newest book (co-edited with A.P. D’Costa) on The New Asian Innovation Dynamics: China and India in Perspective was published in January 2009. He is active in research and advocacy work in science, technology and innovation for sustainable societies [87] Beck RH. Farmers’ rights and open source licencing. Express; 2010. [88] Lemley MA, O’Brien DW. Encouraging software reuse. Stanford Law Review 1997;49:225. [89] Feldman R. The open source biotechnology movements: is it patent misuse? Minnesota Journal of Law Science & Technology 2004;6: 118–67. [90] PIPRA. PIPRA’s evaluation of the BIOS license. PIPRA’s summer 2006 quarter newsletter, issue 5, http://www.pipra.org/en/documents/ PIPRA-Newsletter-Issue5.pdf; 2006. [91] Bruggink M. Open source in Africa: towards informed decision- making. IICD Research Brief-No 7; 2003. [92] Weber S. Open source software in developing economies. Berkeley: University of California, http://webarchive.ssrc.org/programs/itic/ publications/ITST_materials/webernote2.pdf; 2012 [accessed 30.05. 2012]. ] [93] Ariyabandu R, Zengpei X. Free open source software for disaster management: a case study of Sahana disaster management system of Sri Lanka. ESCAP Technical Paper; 2009. Dr. Obijiofor Aginam was educated in Nigeria and Canada. He holds a Bachelor of Laws (magna cum laude) from University of Nigeria; Master of Laws from Queen’s University at Kingston, Canada, and a Ph.D. from the University of British Columbia, Canada. Before joining United Nations University (UNU), he held a tenured academic position as Associate Professor of Law at Carleton University, Ottawa, Canada where he taught and researched emerging global issues that cut across globalization, global governance of health and environmental issues, South–North relations, international organizations, and Third World Approaches to International Law (TWAIL). [94] Ghosh RA, Schmidt P. Open source and open standards: a new Frontier for economic development?; 2006. Policy Brief. No 1. [95] Forero-Pineda C. Convergence of research processes, big and small scientiﬁc communities. In. IIASA workshop, Laxenburg, Austria; May 1997. [96] Gitter DM. Resolving the open source paradox in biotechnology: a proposal for a revised open source policy for publicly funded genomic databases. Computer Law & Security Report 2008;24: 529–39.
https://openalex.org/W2042243208	https://www.scielo.br/j/cagro/a/Nr5Wcvr7fbdfdFcwtW7QmRf/?lang=pt&format=pdf	Portuguese	null	Germinação de sementes de nogueira-macadâmia submetidas à incisão e imersão em ácido giberélico	Ciência e Agrotecnologia	2,010	cc-by	3,708	1Universidade Estadual do Oeste do Paraná/Unioeste – Marechal Cândido Rondon, PR 2Universidade Federal de Lavras/UFLA – Departamento de Agricultura/DAG – Cx. P. 3037 – 37200-000 – Lavras, MG – rafaelpio@hotmail.com 3Universidade de São Paulo, Escola Superior de Agricultura “Luiz de Queiroz” – USP/ESALQ – Departamento de Produção Vegetal – Piracicaba, SP RESUMO Neste trabalho, objetivou-se avaliar a emergência de plântulas de nogueira-macadâmia, por meio do uso de incisão nas sementes e imersão em ácido giberélico. Sementes do cultivar IAC-Campinas B, extraídas de frutos maduros foram padronizadas quanto ao tamanho e sanidade. Foram imergidas em recipiente preenchido por água. Em 80 sementes, realizou-se incisão de 0,5 cm na casca, no sentido longitudinal ao embrião, deixando as amêndoas expostas. Outras 80 sementes foram mantidas intactas. Em seguida, todas as sementes foram submersas em diferentes concentrações de ácido giberélico: 0, 150, 300 e 450 mg L-1, por 90 horas, com sistema de oxigenação. Após esse período, as sementes foram colocadas em bandejas plásticas, entre camadas de areia grossa autoclavada, preservadas em laboratório com temperatura controlada (em torno de 25ºC). As bandejas foram umedecidas diariamente, com auxílio de regador manual. Após 180 dias, mensurou-se a porcentagem de emergência, os comprimentos médios das raízes, das partes aéreas e a massas secas médias totais das plântulas. Concluiu-se que a incisão e imersão de nogueira-macadâmia em ácido giberélico foram prejudiciais à emergência e desenvolvimento das plântulas. Termos para indexação: Macadamia integrifolia, superação de dormência, fitorregulador, propagação, produção de m Germinação de sementes de nogueira-macadâmia... 641 GERMINAÇÃO DE SEMENTES DE NOGUEIRA-MACADÂMIA SUBMETIDAS À INCISÃO E IMERSÃO EM ÁCIDO GIBERÉLICO Germination of macadamia nut seeds submitted to incision and immsertion in gibberelic acid Idiana Marina Dalastra1, Rafael Pio2, Fábio Albuquerque Entelmann 3, Tatiana Werle1, Marcelo Bortoli Uliana1, João Alexio Scarpare Filho3 ABSTRACT The objective of the present work was to evaluate the emergency of macadamia nut seedlings through incision and immersion of the seeds in the gibberellic acid. Seeds of IAC-Campinas B cultivar, extracted from mature fruits, were standardized according to size and health. They were immerged in recipient filled with water. In 80 seeds a 0.5 cm incision was done in the peel, longitudinally in relation the embryo, leaving the almonds exposed. The other 80 seeds were kept intact. Soon after, the seeds were submerged in different concentrations of gibberellic acid: 0, 150, 300 and 450 mg L-1, for 90 hours, with oxygen system. After that period, the seeds were placed in plastic trays, among layers of autoclaved sand, and preserved in laboratory with controlled temperature (around 25ºC). The trays were humidified daily. After 180 days, the emergency percentage, average length of the roots, average length of the aerial part and average total dry mass of the seedlings were evaluated. It was concluded that the incision and immersion of the seeds of macadamia nut gibberellic acid were harmful to the emergence and development of the seedlings. Index terms: Macadamia integrifolia, dormancy, phytoregulator, propagation, seedlings production. MATERIAL E MÉTODOS empregados são, ainda, adaptações de pesquisas desenvolvidas em outros países, como Austrália e Estados Unidos (Sacramento & Pereira, 2003). Frutos de nogueira-macadâmia ‘IAC-Campinas B’, provenientes de plantas matrizes situadas no município de Palotina-PR, foram coletados em março de 2007 e transportados para o Laboratório de Tecnologia de Sementes e Mudas do Centro de Ciências Agrárias, da Universidade Estadual do Oeste do Paraná (Unioeste), Marechal Cândido Rondon-PR, para a extração de suas sementes. Os carpelos foram removidos manualmente, com auxílio de faca e martelo de metal. Procedeu-se a classificação das sementes por tamanho, considerando-se, apenas, as de dimensões de dois centímetros de diâmetro e as que não sobrenadaram após imersão em recipientes com água. A amostra foi, então, dividida em duas porções de sementes. Apenas nas sementes da primeira porção foram realizadas incisões de 0,5 cm de comprimento na casca, com auxilio de uma serra pequena (escarificação), no sentido longitudinal ao embrião, expondo as amêndoas. A outra porção ficou intacta. Em seguida, as sementes de ambas as porções foram colocadas para embeber em Beckers, com capacidade de um litro, que continham soluções de ácido giberélico (GA3), nas concentrações de 150, 300, 450 mg L-1, além do controle composto somente com água destilada. O produto utilizado foi o ProGibb, que possui em sua fórmula 10% de ácido giberélico e 90% de material inerte. Durante o período de imersão de 90 horas, as soluções foram oxigenadas mediante o emprego de bombas de aquário, segundo as recomendações de Ono et al. (1993). O processo de propagação da nogueira-macadâmia é realizado por sementes, tanto para a formação de mudas de pé-franco como de porta-enxerto. Todavia, o tempo demandado para a germinação das sementes é superior a 120 dias após a semeadura, dependendo do tratamento aplicado às sementes (Simão, 1998). Um dos grandes entraves observados na propagação da nogueira-macadâmia refere-se à reduzida porcentagem de germinação das sementes e irregularidade na emergência das plântulas, decorrentes, possivelmente, de influências do fenômeno da dormência (Hamilton, 1954). A ocorrência de embriões imaturos e de tegumentos impermeáveis à água ou ao oxigênio, de restrições mecânicas ou de substâncias inibidoras da germinação, estão relacionadas à dormência (Bewley & Black, 1994; Campo Dall’Orto et al., 2007; Entelmann et al., 2009). INTRODUÇÃO 199 propriedades rurais. O principal município paulista produtor é Dois Córregos, região de Jaú, seguido dos municípios de Bauru, Avaré e São Sebastião da Grama, cujas áreas de produção correspondem a 45% da área cultivada com a espécie no Estado (Barbosa et al., 2003). A nogueira-macadâmia (Macadamia integrifolia Maiden & Betche), originária das florestas subtropicais australianas é, atualmente, cultivada em diversos países, a exemplo do Brasil e Estados Unidos, pela qualidade e importância nutricional dos seus frutos. Por ser de procedência subtropical, a nogueira-macadâmia desenvolve-se, adequadamente, nos Estados de São Paulo, Espírito Santo, Bahia e Rio de Janeiro, que produzem 92% da safra nacional estimada em 3.200 toneladas de “noz em casca” (Sobierajski et al., 2005). Como a cultura é relativamente recente no País, estudos e dados técnicos são escassos. Poucas instituições têm investido em pesquisas com essa noz; uma das pioneiras é o Instituto Agronômico (IAC) que, desde 1948, realiza seleção de material adaptado e desenvolvimento de tecnologias para sua produção (Barbosa et al., 1991). Embora tenha sido introduzida no Brasil na década de 40, muito dos tratos culturais A nogueira-macadâmia é a principal noz cultivada no Estado de São Paulo e ocupa uma área de 2.166 ha com Ciênc. agrotec., Lavras, v. 34, n. 3, p. 641-645, maio/jun., 2010 DALASTRA, I. M. et al. 642 MATERIAL E MÉTODOS A dureza dos tecidos de cobertura as sementes, como é o caso da nogueira-macadâmia, frequentemente causam consideráveis problemas para os viveiristas, em razão da restrição à entrada de água e do oxigênio, e da resistência física à retomada do embrião (Moussa, 1998). Entre os métodos utilizados para superação da dormência tegumentar, a escarificação mecânica é frequentemente utilizada e constitui a opção mais prática e segura para pequenos agricultores. Além de simples e de baixo custo, é eficaz para promover germinação rápida e uniforme das sementes (Hermansen, 2000). No entanto, deve ser empregada com cuidado para evitar danos a tecidos vitais das sementes e consequente redução da germinação. Após o período de imersão, as sementes foram colocadas em bandejas plásticas (35 x 25 cm), entre camadas de areia grossa autoclavada, mantidas em condições de ambiente de laboratório com controle da temperatura em torno de 25ºC. O umedecimento do substrato foi realizado diariamente, com auxílio de borrifador manual, sempre se aplicando 50 mL de água. Além da escarificação mecânica, a imersão em ácido giberélico das sementes de nogueira-macadâmia pode elevar os índices germinativos e uniformizar a emergência das plântulas. A giberelina, importante regulador endógeno de crescimento, produz, também, outros efeitos como indução da germinação de sementes, promoção do alongamento do hipocótilo e do caule (Peng & Harberd, 2000; Richards et al., 2001). A ação da giberelina está relacionada à síntese de enzimas envolvidas no enfraquecimento dos tegumentos, como endo-b-manases, de expansinas e de enzimas hidrolíticas de reserva nutritiva contida no endosperma, como á-amilase. Esses eventos estão relacionados ao alongamento embrionário e à protrusão da radícula (Bewley & Black, 1994; Bewley, 1997). O experimento foi conduzido em esquema fatorial 2 x 4 (sementes com e sem incisão x concentrações de ácido giberélico), com quatro repetições e parcelas compostas por 20 sementes. Passados 180 dias, foram mensurados a porcentagem de emergência, os comprimentos médios das raízes e das partes aéreas, com auxílio de escalímetro, e as massas secas médias totais das plântulas, por meio da secagem do material vegetal em estufa de circulação de ar forçado à 65ºC, durante 48 horas, e posterior pesagem em balança analítica. Ciênc. agrotec., Lavras, v. 34, n. 3, p. 641-645, maio/jun., 2010 Germinação de sementes de nogueira-macadâmia... 643 Germinação de sementes de nogueira-macadâmia... Gomes (2000), utilizando-se do Sistema para Análise de Variância - SISVAR (Ferreira, 2000). A porcentagem de emergência das plântulas de nogueira-macadâmia sem qualquer tratamento (42,13%), apresentou padrões idênticos ao relatado por Ojima et al. (1989), que obtiveram 34% de germinação para ‘IAC 5-10’ e 51% de germinação para a seleção ‘336 HAES’. RESULTADOS E DISCUSSÃO Conforme a Tabela 1, apenas para a porcentagem de emergência de plântulas foi verificada interação entre os fatores estudado. Efeito isolado ao fator incisão das sementes foi constatado para as demais características mensuradas. Os resultados não foram os esperados quanto ao emprego do ácido giberélico. O ácido giberélico, considerado ativador enzimático endógeno, promove a germinação das sementes. A aplicação exógena desse promotor influencia o metabolismo protéico e pode duplicar a taxa de síntese de proteínas das sementes (McDonald & Khan, 1983). Segundo Khan et al. (1978), o uso de compostos químicos biologicamente ativos, como o ácido giberélico, pode sobrepujar efeitos de fatores adversos na qualidade e no desempenho das sementes. De acordo com a Figura 1, foi verificada em sementes não incisas, redução linear da emergência das plântulas de nogueira-macadâmia com a elevação da concentração da solução de imersão de ácido giberélico, da ordem de 25,51%, quando comparada a maior concentração (450 mg L-1) em relação ao tratamento controle. Tabela 1 – Resumo da análise de variância para porcentagem de emergência (PE), comprimento médio da parte aérea (CMPA), comprimento médio da raiz (CMR) e massa seca média total de plântulas de nogueira-macadâmia ‘IAC- Campinas B’, em função da incisão de suas sementes e imersão em diferentes concentrações de ácido giberélico. Marechal Cândido Rondon-PR, Unioeste, 2008. Tabela 1 – Resumo da análise de variância para porcentagem de emergência (PE), comprimento médio da parte aérea (CMPA), comprimento médio da raiz (CMR) e massa seca média total de plântulas de nogueira-macadâmia ‘IAC- Campinas B’, em função da incisão de suas sementes e imersão em diferentes concentrações de ácido giberélico. Marechal Cândido Rondon-PR, Unioeste, 2008. Quadrados Médios FV GL PE CMPA CMR MSMT Incisão (I) 1 5216,78* 197,34* 585,31* 406,19* Concetrações de ácido giberélico (A) 3 360,54* 100,91 87,36 22,32 I x A 3 299,31* 34,32 27,89 28,25 Erro 23 47,28 51,73 54,75 15,12 * Significativo a 5% de probabilidade. y = -0,0567x + 42,13 R 2 = 0,97 - sem incisão y = -0,000083x 2 + 0,0292x + 4,37 R 2 = 0,74 - com incisão 0 5 10 15 20 25 30 35 40 45 0 150 300 450 Concentrações de ácido giberélico (mg L -1) % de emergência sem incisão com incisão Figura 1 – Porcentagem de emergência de plântulas de nogueira-macadâmia ‘IAC-Campinas B’, em função da incisão de suas sementes e imersão em diferentes concentrações de ácido giberélico. MATERIAL E MÉTODOS Os dados foram submetidos à análise de variância pelo teste F e as médias foram comparadas pelo teste Tukey (fator incisão), ao nível de 5% de probabilidade e à regressão linear ou quadrática (concentrações de ácido giberélico), de acordo com as indicações de Pimentel Neste trabalho, objetivou-se avaliar a emergência e desenvolvimento de plântulas de nogueira-macadâmia, em função da escarificação mecânica e da concentração de ácido giberélico aplicada às sementes. Ciênc. agrotec., Lavras, v. 34, n. 3, p. 641-645, maio/jun., 2010 RESULTADOS E DISCUSSÃO Marechal Cândido Rondon-PR, Unioeste, 2008. Quadrados Médios FV GL PE CMPA CMR MSMT Incisão (I) 1 5216,78* 197,34* 585,31* 406,19* Concetrações de ácido giberélico (A) 3 360,54* 100,91 87,36 22,32 I x A 3 299,31* 34,32 27,89 28,25 Erro 23 47,28 51,73 54,75 15,12 * Significativo a 5% de probabilidade. Quadrados Médios FV GL PE CMPA CMR MSMT Incisão (I) 1 5216,78* 197,34* 585,31* 406,19* Concetrações de ácido giberélico (A) 3 360,54* 100,91 87,36 22,32 I x A 3 299,31* 34,32 27,89 28,25 Erro 23 47,28 51,73 54,75 15,12 * Significativo a 5% de probabilidade. y = -0,0567x + 42,13 R 2 = 0,97 - sem incisão y = -0,000083x 2 + 0,0292x + 4,37 R 2 = 0,74 - com incisão 0 5 10 15 20 25 30 35 40 45 0 150 300 450 Concentrações de ácido giberélico (mg L -1) % de emergência sem incisão com incisão Figura 1 – Porcentagem de emergência de plântulas de nogueira-macadâmia ‘IAC-Campinas B’, em função da incisão de suas sementes e imersão em diferentes concentrações de ácido giberélico. Marechal Cândido Rondon-PR, Unioeste, 2008. Ciênc. agrotec., Lavras, v. 34, n. 3, p. 641-645, maio/jun., 2010 644 DALASTRA, I. M. et al. DALASTRA, I. M. et al. Tabela 2 – Comprimento médio da parte aérea (CMPA), comprimento médio da raiz (CMR) e massa seca média total de plântulas (MSMT) de nogueira-macadâmia ‘IAC- Campinas B’, em função da incisão ou não de suas sementes. Marechal Cândido Rondon-PR, Unioeste, 2008. O efeito do ácido giberélico sobre o alongamento celular e crescimento, destacado na literatura consultada, não foi confirmado neste trabalho. Os resultados podem ser atribuídos também às concentrações do ácido giberélico empregadas, ou mesmo ao tempo de submersão das sementes. No entanto, Ono et al. (1993), recomendam que as sementes de nogueira-macadâmia devem ser submersas em água por 90 horas, para proporcionar alta porcentagem de germinação, de acordo com os índices característicos das espécies (ao redor de 50%). Variáveis analisadas* Incisão na semente CMPA (cm) CMR (cm) MSMT (mg) Sem incisão 12,58 a 17,72 a 12,48 a Com incisão 7,53 b 9,02 b 5,24 b C.V. (%) 22,18 23,41 24,74 *Médias seguidas pela mesma letra na coluna não diferem significamente entre si, pelo teste Tukey, ao nível de 5% de probabilidade. Quanto às sementes incisas, a aplicação do ácido giberélico também mostrou-se ineficaz para incrementar a porcentagem de emergência. RESULTADOS E DISCUSSÃO As porcentagens de emergência de plântulas foram bem inferiores em relação às sementes que não sofreram incisão (Figura 1). Novos estudos devem ser realizados com a nogueira- macadâmia, não só no que tange o processo germinativo de suas sementes, mas também técnicas auxiliares que venham a facilitar o enraizamento de suas estacas (Chagas et al., 2008; Bastos et al., 2009; Ohland et al., 2009). A incisão das sementes também não foi benéfica ao desenvolvimento das plântulas de nogueira- macadâmia, pois comprimentos médios da parte aérea e das raízes e as massas secas médias totais das plântulas foram significamente inferiores nas mesmas, quando comparadas àquelas que não sofreram incisão (Tabela 2). CONCLUSÃO A imersão de sementes de nogueira-macadâmia em ácido giberélico por 90 horas prejudica a emergência das plântulas. A incisão, como forma de escarificação, diminui significamente a porcentagem de emergência e desenvolvimento das plântulas. Os resultados obtidos no presente trabalho concordam com Rêgo et al. (1991), que compararam sementes sem e com incisão de nogueira-macadâmia e observaram que a ausência de tratamento proporcionou 52,5% de sementes germinadas após 90 dias da semeadura, 40% a mais que as sementes com incisão. Hamilton (1954) atribui a rachadura mecânica como causa de redução da germinação. Ciênc. agrotec., Lavras, v. 34, n. 3, p. 641-645, maio/jun., 2010 arid of Niger, West Africa. Forest Ecology and Management, Amsterdam, v.104, n.1, p.27-34, 1998. How gibberellin regulates plant growth and development: a molecular genetic analysis of gibberellin signaling Annual Review of Plant Physiology and Plant HERMANSEN, L.A. Pretreatments to overcome seed coat dormancy in Dimorphandra mollis. Seed Science & Technology, Zurich, v.28, n.1, p.581-595, 2000. KHAN, A.A. Incorporation of bioactive chemicals into seeds to alleviate environmental stress. Acta Horticulturae, The Hague, v.83, n.2, p.2255-2264, 1978. HERMANSEN, L.A. Pretreatments to overcome seed coat dormancy in Dimorphandra mollis. Seed Science & Technology, Zurich, v.28, n.1, p.581-595, 2000. KHAN, A.A. Incorporation of bioactive chemicals into seeds to alleviate environmental stress. Acta Horticulturae, The Hague, v.83, n.2, p.2255-2264, 1978. SACRAMENTO, C.K.; PEREIRA, F.M. Fenologia da floração da nogueira-macadâmia (Macadamia integrifolia Maiden & Betche) nas condições climáticas de Jaboticabal, São Paulo, Brasil. Revista Brasileira de Fruticultura, Cruz das Almas, v.25, n.1, p.19-22, 2003. MALAVASI, M.M. Germinação de sementes. In: ______. Manual de análise de sementes florestais. Piracicaba: Fundação Cargill, 1988. 100p. arid of Niger, West Africa. Forest Ecology and Management, Amsterdam, v.104, n.1, p.27-34, 1998. arid of Niger, West Africa. Forest Ecology and Management, Amsterdam, v.104, n.1, p.27-34, 1998. arid of Niger, West Africa. Forest Ecology and Management, Amsterdam, v.104, n.1, p.27-34, 1998. OHLAND, T.; PIO, R.; CHAGAS, E. A.; BARBOSA, W.; KOTZ, T. E.; DANELUZ, S. Enraizamento de estacas apicais de figueira ‘Roxo de Valinhos’ em função de época de coleta e AIB. Ciência e Agrotecnologia, Lavras, v. 33, n.1, p. 74-78, jan./fev., 2009. CHAGAS, E. A.; PIO, R.; BETTIOL NETO, J. E.; SOBIERAJSKI, G. da R.; CAMPO DALL´ORTO, F. A.; SIGNORINI, G. Enraizamento de estacas lenhosas de pessegueiro e clones de umezeiros submetidos à aplicação de AIB. Ciência e Agrotecnologia, Lavras, v. 32, n.3, p. 986-991, maio/jun., 2008. OJIMA, M.; CAMPO DALL’ORTO, F.A.; RIGITANO, O. Germinação de sementes de nogueira-macadâmia. 2.ed. Campinas: Instituto Agronômico, 1989. 17p. (Boletim técnico, 33). DALL’ORTO, F. A. C.; OJIMA, M.; PIO, R.; CHAGAS, E. A. Avaliação da capacidade reprodutiva de algumas cultivares de marmeleiros visando a obtenção de porta- enxertos. Ciência e Agrotecnologia, Lavras, v.31, n.2, p.274-278, mar./abr., 2007. ONO, E.O.; RODRIGUES, J.D.; SABINO, J.C.; PINHO, S.Z. Estudos da imersão e da viabilidade de sementes de macadâmica (Macadamia integrifolia Maiden & Betche). Scientia Agrícola, Piracicaba, v.50, n.1, p.40-44, 1993. ENTELMANN, F. A.; PIO, R.; CHAGAS, E. A., SCARPARE FILHO, J. A.; ALVARENGA, A. A.; ABRAHÃO, E. Estratificação à frio de sementes de ‘Japonês’, porta- enxerto para marmeleiros. Ciência e Agrotecnologia, Lavras, v.33, Edição Especial, p.1877-1882, 2009. PENG, J.; HARBERD, N.P. The role of GA-mediated signalling in the control of seed germination. Current Opinion in Plant Biology, London, v.5, p.376-381, 2000. FERREIRA, D.F. Análise estatística por meio do SISVAR (Sistema para Análise de Variância) para Windows versão 4.0. In: REUNIÃO ANUAL DA REGIÃO BRASILEIRA DA SOCIEDADE INTERNACIONAL DE BIOMETRIA, 45., 2000, São Carlos. Anais... São Carlos: UFSCar, 2000. p.255-258. PIMENTEL GOMES, F. Curso de estatística experimental. 14.ed. Piracicaba: USP/ESALQ, 2000. 477p. RÊGO, F.A.O.; COSTA, M.M.M.N.; ABREU, S.M.; SILVA, A.Q.; SILVA, H. Influência do tamanho da semente e escarificação na germinação da macadâmia (Macadamia integrifolia). Informativo ABRATES, Londrina, v.1, n.4, p.85, 1991. HAMILTON, R.A. Make your own nutcracker. Hawaii Farm Science, v.3, n.2, p.5-6, 1954. RICHARDS, D.E.; KING, K.E.; AIT-ALI, T.; HARBERD, N.P. How gibberellin regulates plant growth and development: a molecular genetic analysis of gibberellin signaling. Annual Review of Plant Physiology and Plant Molecular Biology, Dordrecht, v.52, p.67-88, 2001. RICHARDS, D.E.; KING, K.E.; AIT-ALI, T.; HARBERD, N.P. REFERÊNCIAS BIBLIOGRÁFICAS BASTOS, D. C.; SCARPARE FILHO, J. A.; LIBARDI, M. N.; PIO, R. Estiolamento, incisão na base da estaca e uso de ácido indolbutírico na propagação da caramboleira por estacas lenhosas. Ciência e Agrotecnologia, Lavras, v. 33, n.1, p. 313-318, jan./fev., 2009. BASTOS, D. C.; SCARPARE FILHO, J. A.; LIBARDI, M. N.; PIO, R. Estiolamento, incisão na base da estaca e uso de ácido indolbutírico na propagação da caramboleira por estacas lenhosas. Ciência e Agrotecnologia, Lavras, v. 33, n.1, p. 313-318, jan./fev., 2009. De acordo com os resultados obtidos no presente trabalho e na literatura consultada, percebe que a incisão como forma de escarificação, para a quebra de dormência das sementes de nogueira-macadâmia, não traz benefícios a emergência e desenvolvimento das plântulas. Era esperado que a incisão nas sementes, ao facilitar o contato do embrião com o ácido giberélico, promovesse melhorias significativas aos processos germinativos da nogueira-macadâmia, o que não se confirmou. Possivelmente, o contato do embrião com umidade antes de se iniciar o desencadeamento do processo germinativo possa ter prejudicado a emergência das plântulas. Segundo Malavasi (1988), a embebição demasiada das sementes de algumas espécies, reduz o período disponível para que as membranas celulares se reorganizem e, como consequência, há uma expressiva liberação de solutos, diminuindo acentuadamente a germinação. BARBOSA, W.; CAMPO-DALL’ORTO, F.A.; OJIMA, M.; SANTOS, R.R.; FRANCO, J.A.M. Seleções de nogueira-macadâmia do Instituto Agronômico. O Agronômico, Campinas, n.43, p.94-99, 1991. BARBOSA, W.; POMMER, C.V.; RIBEIRO, M.D.; VEIGA, R.F.A.; COSTA, A.A. Distribuição geográfica e diversidade varietal de frutíferas e nozes de clima temperado no Estado de São Paulo. Revista Brasileira de Fruticultura, Cruz das Almas, v.25, n.2, p.341-344, 2003. BEWLEY, J.D. Breaking dow the walls: a role for endo betamananase in release from seed dormancy. Plant Science, Shannon, v.2, p.139-144, 1997. BARBOSA, W.; POMMER, C.V.; RIBEIRO, M.D.; VEIGA, R.F.A.; COSTA, A.A. Distribuição geográfica e diversidade varietal de frutíferas e nozes de clima temperado no Estado de São Paulo. Revista Brasileira de Fruticultura, Cruz das Almas, v.25, n.2, p.341-344, 2003. BEWLEY, J.D. Breaking dow the walls: a role for endo betamananase in release from seed dormancy. Plant Science, Shannon, v.2, p.139-144, 1997. Ciênc. agrotec., Lavras, v. 34, n. 3, p. 641-645, maio/jun., 2010 645 Germinação de sementes de nogueira-macadâmia... BEWLEY, J.D.; BLACK, M. Seeds: physiology of development and germination. New York: Plenum, 1994. 445p. BEWLEY, J.D.; BLACK, M. Seeds: physiology of development and germination. New York: Plenum, 1994. 445p. SIMÃO, S. Tratado de fruticultura. Piracicaba: FEALQ, 1998. 760p. SIMÃO, S. Tratado de fruticultura. Piracicaba: FEALQ, 1998. 760p. McDONALD, M.D.; KHAN, A.A. Acid scarification and protein synthesis during seed germination. Agronomy Journal, Madison, v.2, n.75, p.111-114, 1983. MOUSSA, H. Factors affecting the germination of doum palm (Hyphaene thebaica Mart.) seeds from the semi- McDONALD, M.D.; KHAN, A.A. Acid scarification and protein synthesis during seed germination. Agronomy Journal, Madison, v.2, n.75, p.111-114, 1983. SOBIERAJSKI, G.R.; FRANCISCO, V.L.F.S.; ROCHA, P.; GHILARDI, A.A.; MAIA, M.L. Noz-macadâmia: produção, mercado e situação no Estado de São Paulo. Informações Econômicas, São Paulo, v.36, n.5, p.25-36, 2005. MOUSSA, H. Factors affecting the germination of doum palm (Hyphaene thebaica Mart.) seeds from the semi- Ciênc. agrotec., Lavras, v. 34, n. 3, p. 641-645, maio/jun., 2010
https://openalex.org/W4327791019	https://cyberleninka.ru/article/n/patterns-in-applied-art-of-the-uzbek-folk/pdf	English	null	PATTERNS IN APPLIED ART OF THE UZBEK FOLK	European journal of arts	2,023	cc-by	3,180	I. Introduction the shape of crown of the rooster), tabadoniy, iris nusha, tumor gul (similar to the shape of a amulet), shabaqa and others not only in the 19th century, but also in present days are used by all handicraftsmen. If the patterns on the objects related to traditional applied art are studied seriously, the specific ideological content of each pattern can realize. The culture, ancient history and natural landscape of Uzbekistan are very rich and varied. It consists of mixtures of cultural traditions of various nations. They are represented in music, art, handicrafts, dances and national clothes. The rich and traditional culture of the Uzbek people has developed over many centuries and differs from the oriental cultures with specific and unique features. On these days, the development of the types of applied arts, the changes in the system of patterns based on tradition and innovation in the decoration of objects occupy a special place as the main field of artistic culture that maintains and develops the succession. For instance, the pattern of “kordi osh” expresses the meaning of a kitchen knife, and the image of “feathers, birds” means wisdom, intelligence; “almond” is a sym bol of life and fertility. “Palak” is derived from the word “falak” and it expresses the meaning of the mutuality of the universe and the earth. The image of the moon sewn around the sky shows the meaning of the bright beauty of the universe. They are polished in the colours of green, blue, pistachio as a symbol of the life and vitality. The image of snakes on the borders of the embroidered item represents as a symbol of savior who protects people, families, and homes. Love for life and thirst for beauty are reflected in the colors of embroidered item and flow er of universe, which give grace and refreshment to the households. In addition, in the folk applied art, in par ticular, there are also basic compositions in embroidery, To basis of the theme and its actuality. The seman tics of patterns used in the applied folk art of Uzbek na tion, in particular, the semantics patterns used in pottery, carving, wood carving, embroidery, weaving a carpet, and jewelry, are extremely complex. UDC: 75.021.4 UDC: 75.021.4 DOI: 10.29013/EJA-23-1-11-14 DOI: 10.29013/EJA-23-1-11-14 DOI: 10.29013/EJA-23-1-11-14 R. R. JABBAROV 1 1 Tashkent State Pedagogical University named after Nizami, Tashkent, Uzbekistan Abstracth Abstract The purpose of the research. It is to contribute to the preservation of the history and traditions of Uzbek folk applied art, and to pass it on to the future generation. Research methods. In this scientific work, the semantics of patterns in the applied decorative arts of Uzbekistan, the feelings of deep connection of the Uzbek people with the past world of values, the process of restoring the original layers and traditions of applied arts, technological methods, pattern compositions, decoration types were analyzed. Research results. This study provides information about the types of patterns in Uzbek folk applied decorative h i f hi f i i d di i Research methods. In this scientific work, the semantics of patterns in the applied decorative arts of Uzbekistan, the feelings of deep connection of the Uzbek people with the past world of values, the process of restoring the original layers and traditions of applied arts, technological methods, pattern compositions, decoration types were analyzed. g p p p p , p g g layers and traditions of applied arts, technological methods, pattern compositions, decoration types were analyzed. Research results. This study provides information about the types of patterns in Uzbek folk applied decorative art, their forms, history of origin, and traditions. t Research results. This study provides information about the types of patterns in Uzbek folk applied decorative art, their forms, history of origin, and traditions. y g Practical application. All scientific intellectuals working in the field of art studies can take full advantage of this research work. Keywords: authentic, semantics, composition, stylization, epigraphic, panno, friezes, engr European Journal of Arts 1 (2023) Section 1. Visual, decorative and applied art ISSN 2310-5666 ISSN 2310-5666 II. To object and subject of the theme The historical origin of applied art dates back to the period of childhood of mankind. As humanity grew up, applied art also developed, as long as there was a struggle for living, and in the process of increasing the needs for a good life, mental labor started to separate from manual labor. The demand for hunting weapons and household items increased. First of all, stone carving, bone carving, and later wood carving slowly found its development. In the centuries-old history of the Uzbek people, the types of folk applied decorative arts include the most amazing and mass part of our rich and colorful cultural inheritance. The types of art that flourished and appeared in the area of Uzbekistan are famous in the world for their uniqueness and originality. If we think about the stages of such devel opment, we will witness that the roots of Uzbek applied decorative arts dates back to the childhood of mankind which to primitive society. As evidenced by the historical monuments found as a result of excavations of soil layers on the land of our country, the activity of creating items in the way of artistic handling to the human body began in the Stone Age and continues till present days for centu ries. As a result of such a deep philosophical approach to applied art, the creation of applied artistic decorative art works based on conditionality, stylization has increased. This historical factor motivated the rapid development of the Uzbek national decorative art, as a result today our famous architectural monuments, their ganch carving, the art of decorating with tiles, the art of design, the art of calligraphy, stone carving and other types of art be came famous in the world. Folk applied decorative art enriches the spiritual world of people, forms artistic taste, and brings up their spirit.h Pottery. Since ancient times, pottery has been the most common occupation for humans. Even today, several famous pottery workshops are working in some cities, towns and villages of Uzbekistan. Uzbekistan is a place that has preserved the richest heritage of pottery art, because the representatives of modern, traditional and non–traditional pottery art (historically formed in the 19th century) are interested in the historical layers of indigenous culture.t Since ancient times, the craftsmanship centers of all oases have been formed in Uzbekistan. ISSN 2310-5666 ISSN 2310-5666 tribes who were very rich in wool products (mainly sheep and camels) living in a semi-nomadic (herding) lifestyle. The weaving carpet by hand is extremely painstaking and complicated task, it demands diligence, taste and crafts manship from the weaver. In the periods before the Mon gols, the woven carpet items of the Oguz, which are part of the Turkic tribes, became famous in the world. In the period of the Timurids, carpet weaving combined with Iranian and Turkish traditions, and from the 16 th centu ry, the carpet weaving items of the Dashtikipchak Uzbek tribes spread widely around the world in Movarounnahr. The historical relevance of the carpet-weaving traditions of these Turkic peoples forms the basis of the ethno-cul ture of the national carpet-making of Uzbekistan. which serve to become the item richer. For example, the medallion composition consists of 1 central figure and 4 bushes or bouquets of flowers located in 4 corners. The name of this pattern is “yak mohu – chor shah” – “four horns and one moon” (mainly Bukhara, Nurota). I. Introduction Many types of patterns such as bodomgul (similar to the shape of an almond), anorgul (similar to the shape of an a pomegranate), qo‘chqorak (similar to the shape of a horn of ram), zomucha, kilichak, kordi osh (similar to the shape of a knife), kadj bayt, toji xo‘roz gul (similar to R. R. JABBAROV PATTERNS IN APPLIED ART OF THE UZBEK FOLK 11 Section 1. Visual, decorative and applied art European Journal of Arts 1 (2023) R. R. JABBAROV PATTERNS IN APPLIED ART OF THE UZBEK FOLK ISSN 2310-5666 This style was especially fully reflected in the works of the master Mul ladost Muhammad, who decorated “kashkul” (a deep cop per vessel with a lid and a handle, in which ingredients are usually stored) items in the style of “Kufic” with engraved calligraphy. At that time, masters such as Hakim Bukhari and Dostmuhammad Rizo, representatives of the Bukhara school of engraving, many times used as ornament elegant verses of copper items. III. Conclusiontt Wood engraving. The fame of wood engraving has always been high in Uzbekistan. The most famous type of traditional wood engraving is the decoration of any surface through patterns and decorations related to plants, as well as the ornamental technique called “Abso lute Infinity”. The maple, mulberry, walnut, larch, cherry or apricot were mainly used for wood engraving. In the applied art of Uzbekistan, wood engraving differs from other types of art by its uniqueness, the main reason that masters related to this branch of art type are not only household items, but also architectural parts (pillars, doors, gates and others) is also very active in decorating home furnishings. Wide range of use of wooden columns, doors, gates and other architectural parts can be seen in the architecture of Khiva, Bukhara, Kokand, Samarkand and Tashkent, the capitals of the Uzbek khanates. One of the mature centers of wood engraving and the art of patterning is Khiva, and the art of Khiva wood craftsmen can be seen on the columns and doors of the buildings of the Ichan-Kala complex (in cluding the carved pillars in the interior of the Juma Mosque monument). One of the popular representatives of the Khi va wood engraving school is the dynasty of Polvonov, many carved columns, doors and gates were established by them in Khiva at the beginning of 19 th and 20 th centuries. K. Hay darov, one of the famous masters of Kokand, preserved the traditions of the Kokand school of wood engraving in his creation and created unique items. His creative works of art include architectural parts such as doors, panels (a piece of patterned wall), friezes, columns, carved tables, chairs, rehal (book rest), pencil cases, and other household items. Tash kent was one of the main centers of wood engraving in the 19 th and 20 th centuries. ISSN 2310-5666 ISSN 2310-5666 others). The lead glaze and yellow–green, brown paints play an important role in manufacturing the Bukhara–Sa markand ceramic items with sonorous and elegant (rep resentatives Alisher and Abdulla Narzullaev (Gijduvan), Namaz and Noman Oblokulov (Urgut), H. Hakberdiev (Samarkand). In the pottery of Kashkadarya (Kasbi dis trict) currently manufactures only unglazed items. since 1990. In the area of Uzbekistan, there are mainly four schools of wood engraving (Kokand, Samarkard, Tashkent, Khiva), which differ from each other with their styles and directions. Art of coppersmithing. In the period of the reign of the Timurids in the 14th century, the production of Uzbek national handicraft items was more developed. Amir Temur brought foreign handicraftsmen to Samarkand during his military campaigns to other countries. Thereby, the names of foreign masters can be seen on the metal objects related to that period. For instance, there is a legendary cauldron made of seven different metals in the Hermitage Museum, it was decorated with a plant-like pattern and high artistic writing, and it was made by Abdulaziz ibn Sharofiddin, a metal caster from Tabriz. The Spanish ambassador Clavijo wrote in his memoirs that the wonderful dishes saw at the reception and party of Amir Temur were made to a high level. Young Bukhara is one of the oldest centers of cop per engraving, and the patterns and inscriptions on dishes made by native masters there are amazing with elegance of the ornaments. At the beginning of the 19 th and early 20 th centuries, the art of calligraphy, together with the patterns and decorations that were given into the objects in a cross– sectional manner, took a special place, and the inscriptions were embossed in the form of plant–like patterns. Espe cially, the copper items of Gijduvan found their own re flection as the major ornament the elegant inscriptions on items such as kashkul (a deep copper vessel with a lid and a handle, in which ingredients are usually stored), buckets, pail, juice bowls, copper bowls, dolly–tub, weapons, and candlesticks, as well as it fully demonstrated the unique style of epigraphy characteristic of the Bukhara school of copper engraving. It is known from historical sources that during the period of the Bukhara Khanate, engravers al ways used the style of calligraphy, they decorated the belly, neck or handle of the jugs, and the wide surface side of the tray with the inscriptions of calligraphy. ISSN 2310-5666 Artistic decoration of large architec tural buildings and household carvings have an important place in the work of Tashkent masters. The artistic decora tion of huge architectural buildings and household carvings play an important role in the creation of Tashkent masters. The traditions of famous masters from Tashkent – S. Kho jaev, M. Kasimov, A. Fayzullaev, H. Kasimov, N. Ibrohimov have been continued by A. Azlarov, master of the dynasty M. Ibrohimova, S. Rakhmatullaev, H. Hasanov and others II. To object and subject of the theme According to the method of production, pottery is divided into two main types – glazed and unglazed pottery. Unglazed pottery has a long history. The glazed pottery was widely spread out in the cities of Movarounnahr at the end of the 8th century – the beginning of the 9th century. In the 9th – 18th centuries, this style gained artistic excellence and high technological quality. Since the 20th century, the main schools and centers have been founded in the re gions of present–day Uzbekistan: a) Samarkand-Bukhara school, Tashkent, Samarkand, Urgut, Bukhara, Gijdu von, Shahrisabz, Kitab, Kattakorgan, Denov centers; b) Fergana school, Rishton and Gurumsaray centers; c) Khorezm school, Khanka, Modir village, Kattabog, Chimboy centers. Each center has its own, unique local features. Nowadays, the household items and other pot tery products with flat (bowls, plates), long, upwardly directed (jugs, long pitchers) are being manufactured in the centers which above–mentioned. In the pottery of Fergana and Khorezm, the preparation of traditional blue alkaline glaze was started, but they are distinguished by their unique patterns, delicate ornaments and variety of the items (representatives M. Turopov (Gurumsaray), I. Komilov (Rishton), R. Matchonov (Khorezm) and Carpet weaving. The carpet making traditions of an cient folk developed as a result of the inventive attempts of many descendants, and has extremely long and wide roots. Home-made carpets mainly by women in rural areas are ordinary, but it means that they are produced in perfect technique, thus they are exhibiting the perfect brightness of colors, shapes, combinations of delicate pat tern and creative ornaments. In Uzbekistan, carpet weav ing and felt weaving are one of the most ancient type of folk handicraft and were the main occupation of native R. R. JABBAROV PATTERNS IN APPLIED ART OF THE UZBEK FOLK 12 European Journal of Arts 1 (2023) Section 1. Visual, decorative and applied art References 1. Xurshida Xayitboboeva. Mustaqillik davri O‘zbekiston amaliy san’atida an’ana va innovatsiya. Dissertasiya ishi, – Toshkent. 2022.t 1. Xurshida Xayitboboeva. Mustaqillik davri O‘zbekiston amaliy san’atida an’ana va innovatsiya. Dissertasiya ishi, – Toshkent. 2022. 2. Xamidova Z. O‘zbekistonda san’at turlari va ularning rivojlanishi. URL: https://www. bukharamuseums.uz/uz/ maqolalar/item/ozbekistonda-sanat-turlari-va-ularning-rivo jlanishi. 3. “O‘zbekiston nomoddiy madaniy merosi”. URL: http://ich.uz/uz/ich-of-uzbekistan/nat ional-list/domain‑5/ 460-yoghoch-oymakorligi. 4. Юsupov Ismat, Abrarova Gulchexra, Usanova Umida. Ma’danga bitilgan naqshlar. Oriental Art and Culture, (V), 2020. – P. 63–67. 5. Sayfullaeva G. I., Rashidova N. N., Эgamova F. I. O‘zbek xalqining amaliy san’ti tarixi va xalq hunarmandchiligi fanini o‘qitishning asosiy vazifalari. Scientific progress, – 2 (7). 2021. – P. 138–141. fanini o‘qitishning asosiy vazifalari. Scientific progress, – 2 (7). 2021. – P. 138–141. 6. Bulatov S. O‘zbek xalq amaliy bezak san’ati. – Toshkent: “Mehnat, 1991. gi g tov S. O‘zbek xalq amaliy bezak san’ati. – Toshkent: “Mehnat, 1991. i 6. Bulatov S. O‘zbek xalq amaliy bezak san’ati. – Toshkent: “Mehnat, 1991. 7. Bulatov S. S., Gulyamov K. M. Amaliy sanat. – T.: Iqtisod-moliya, 2014. 7. Bulatov S. S., Gulyamov K. M. Amaliy sanat. – T.: Iqtisod-moliya, 2014. 8. Bulatov S. S. Naqqoshlik. – T: “Iqtisod-moliya, 2014. – P. 22–23. 9. Bulatov S. S., Gulyamov K. M. Формирование чувства патриотизма средствами общечеловеческиx ценностей. Устойчивое развитие науки и образования, – (10). 2017. – С. 184–189. Bulatov S. S., Gulyamov K. M. Формирование чувства патриотизма средствами общечеловеч 9. Bulatov S. S., Gulyamov K. M. Формирование чувства патриотизма средствами общечеловеческиx ценностей. Устойчивое развитие науки и образования, – (10). 2017. – С. 184–189. 10. Xolmuratovich M.X., Ravshanovich J.R. Amaliy va badiiy bezak san’ati, 2020. 9. Bulatov S. S., Gulyamov K. M. Формирование чувства патриотизма средствами общечеловеческиx ценностей. Устойчивое развитие науки и образования, – (10). 2017. – С. 184–189. Устойчивое развитие науки и образования, – (10). 2017. – С. 184–189. 10. Xolmuratovich M. X., Ravshanovich J. R. Amaliy va badiiy bezak san’ati, 2020. р у р , ( ) 9 10. Xolmuratovich M. X., Ravshanovich J. R. Amaliy va badiiy bezak san’ati, 2020. р у р , ( ) 10. Xolmuratovich M. X., Ravshanovich J. R. Amaliy va badiiy bezak san’ati, 2020. р р 10. Xolmuratovich M. X., Ravshanovich J. R. Amaliy va badiiy bezak san’ati, 2020. ISSN 2310-5666 orative arts, which have been created as a result of the creative work of the people over the centuries, teaching the young future generation to increase their esthetic taste and educating them as highly cultured person are one of the important tasks of our time. In the process of fulfilling this important task, the role and significance of innovative technologies is important, because this is the requirement of today. Handicraftsmen such as patterning art, ganch carving art, coppersmithing, knife making, mat making, carpet mak ing, and wood carving have their own special masters. Every handicraftsman has his own disciple. If any master has no disciples, he can be compared to a tree without fruit. Because our masters have traditionally passed their crafts from generation to generation. Therefore, preserv ing, appreciating and using the Uzbek folk applied dec III. Conclusiont No matter what kind of craft there is in the world, it will definitely have its own masters and disciples. R. R. JABBAROV PATTERNS IN APPLIED ART OF THE UZBEK FOLK 13 Section 1. Visual, decorative and applied art European Journal of Arts 1 (2023) R. R. JABBAROV PATTERNS IN APPLIED ART OF THE UZBEK FOLK Information about the author Rustam Ravshanovich Jabbarov, Acting Associate Professor, Tashkent State Pedagogical University named after Nizami, Tashkent, Uzbekistan Rustam Ravshanovich Jabbarov, Acting Associate Professor, Tashkent State Pedagogical University named after Nizami, Tashkent, Uzbekistan Address: prospekt. Bunyodkor, 27, Toshkent, Uzbekistan E-mail: jabbarovrustam7@gmail.com; Tel.: +998 71 276-80-86, +998 71 276-75-93 ORCID: 0000-0002-1485-4563 Rustam Ravshanovich Jabbarov, Acting Associate Professor, Tashkent State Pedagogical University named after Nizami, Tashkent, Uzbekistan Address: prospekt. Bunyodkor, 27, Toshkent, Uzbekistan E-mail: jabbarovrustam7@gmail.com; Tel.: +998 71 276-80-86, +998 71 276-75-93 ORCID: 0000-0002-1485-4563 Address: prospekt. Bunyodkor, 27, Toshkent, Uzbekistan R. R. JABBAROV PATTERNS IN APPLIED ART OF THE UZBEK FOLK 14
https://openalex.org/W2956341737	https://boris.unibe.ch/142721/1/pone.0219146.pdf	English	null	Response of peripheral arterial pulse wave velocity to acute exercise in patients after recent myocardial infarction and healthy controls	PloS one	2,019	cc-by	7,001	RESEARCH ARTICLE Editor: Lena Jonasson, Linkoping University, SWEDEN In 21 patients after recent myocardial infarction (CAD), 11 HAM and 10 HY pPWV was mea- sured by applanation tonometry at the proximal femoral artery and the posterior tibial artery at rest and from 5 to 15 min after cessation of exhaustive exercise. Heart rate (HR) and blood pressure (BP) were monitored continuously. Resting central PWV (cPWV) was measured between the carotid and femoral arteries. Resting values and reponses to acute exercise were compared between the three groups and predictors for pPWV response were sought. Copyright: © 2019 Trachsel 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. Funding: This study was funded by a grant from the Swiss Heart Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. OPEN ACCESS Many studies found increased central arterial stiffness and poor endothelial function in patients with coronary artery disease (CAD). Acute exercise has been shown to decrease peripheral pulse wave velocity (pPWV) in young healthy volunteers. We hypothesized the response to acute exercise to be diminished in CAD patients compared to healthy young (HY) and age-matched (HAM) controls. Citation: Trachsel Y, Herzig D, Marcin T, Zenger N, Dysli M, Trachsel LD, et al. (2019) Response of peripheral arterial pulse wave velocity to acute exercise in patients after recent myocardial infarction and healthy controls. PLoS ONE 14(7): e0219146. https://doi.org/10.1371/journal. pone.0219146 Response of peripheral arterial pulse wave velocity to acute exercise in patients after recent myocardial infarction and healthy controls Y. Trachsel1, D. Herzig1, T. Marcin1, N. Zenger1,2, M. Dysli1, L. D. Trachsel1, M. Wilhelm1, P. Eser1* Y. Trachsel1, D. Herzig1, T. Marcin1, N. Zenger1,2, M. Dysli1, L. D. Trachsel1, M. Wilhelm1, P. Eser1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Preventive Cardiology & Sports Medicine, University Clinic for Cardiology, Inselspital, Bern University Hospital, University of Bern, Switzerland, 2 Institute for Biomechanics, ETH Zu¨rich, Zu¨rich, Switzerland a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * prisca.eser@insel.ch Methods Editor: Lena Jonasson, Linkoping University, SWEDEN Results The response in pPWV to acute exercise seen in HY (lowering in pPWV by 17%) was absent in both CAD and HAM. Resting pPWV was not statistically different between the three groups, while cPWV was comparable in CAD and HAM but 17% lower in HY. Predic- tors for response in pPWV to exercise were age (Spearman r = 0.48), cPWV (r = 0.34) and response in diastolic BP (r = 0.32). Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Introduction Exercise training in patients with coronary artery disease (CAD) has been shown to increase exercise capacity [1] and decrease cardiovascular and all-cause mortality [2]. Beneficial cardio- vascular effects of exercise are manifold and include improved cardiac and pulmonary func- tion, as well as a lowering of resting heart rate and systolic and diastolic blood pressure, the latter being caused by a reduced peripheral resistance [3]. Improved peripheral circulation after chronic exercise has been shown to be due to an increase in endothelial function, vasculo- genesis and capillary density [3]. Due to the dominant effect of vasomotor tone in resting condition [4], dilation after occlu- sion or exercise offers a more reproducible state for measurements of vessel properties and the reaction to this challenge test provides valuable information of vascular reactivity. Dilation of peripheral large arteries as a response to acute exercise has been quantified by measuring artery diameter changes, flow changes or changes in pulse wave velocity (PWV). Several studies in heathy young cohorts have shown that there is a significant decrease in peripheral arterial PWV (pPVW) in the lower limbs immediately post-exercise which persists into the recovery period for approximately 30 min [5–13], while central PWV is not altered [14]. Previous stud- ies in young healthy subjects and elderly cardiac patients have suggested that pPVW measured during hyperemia after occlusion may be a surrogate for flow-mediated dilatation (FMD, rela- tive change in arterial diameter) following occlusion [15–18]. However, two studies that have directly compared post-occlusion pPWV measurements to FMD measured by ultrasound have found only partial agreement [17,19]. This implies that endothelial function is likely to play a role in post-exercise decrease of pPWV, but may not be the only important variable. Other important factors may be blood pressure [20] and vessel wall properties [21]. In CAD patients it has been shown that arterial stiffness is heightened [22] and endothelial function impaired compared to healthy populations [23]. Conclusion The response in pPWV to acute exercise observed in HY was absent in CAD and HAM. In dilated peripheral arteries, PWV may reflect stiffness of passive vessel structures, which are likely to increase with age in healthy persons and CAD alike. Competing interests: The authors have declared that no competing interests exist. 1 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0219146 July 9, 2019 Post-exercise peripheral PWV after myocardial infarction Introduction Ankle-brachial PWV in combination with flow-mediated dilation has been shown to be useful for risk stratification of chronic CAD patients,[24] and exercise therapy has been shown to improve peripheral vascular function in these patients.[25] However, the responsiveness of peripheral arteries to peak exercise has not been measured in CAD patients before, and the need for studies assessing acute and chronic adaptations in populations with cardiovascular diseases has recently been pointed out.[26] We hypothesized that the response of peripheral arteries to acute exercise seen in young healthy people with pPWV decreasing acutely and transiently would be impeded in CAD patients. We further hypothesized that the response in pPWV to acute exercise would also be lower in CAD patients compared to healthy age-matched controls. Therefore, we measured pPWV at rest and acutely after exhaustive exercise in CAD patients with recent myocardial infarction, healthy aged-matched physically active men and healthy young men. Pulse wave velocity Aortic (central) PWV (cPWV) was measured between the pulse sites of the carotid and femo- ral arteries by applanation tonometry (Sphygmocor, AtCor Medical, West Ryde, NSW, Austra- lia). Distance between the location of the two pulse sites was measured using a caliper referenced against a measuring tape (100% of path length was used). Measurements were repeated if heart rate between both measurements differed by more the 5 bpm or if the stan- dard deviation of the mean travelling time of the pulse waves (over the 10 s of the recording) exceeded 10% of the mean value. Peripheral pulse wave velocity (pPWV) was measured between the right femoral artery and the right posterior tibial artery pre-exercise and continuously from 5 to 15 min post-exercise. Simultaneous recordings of the pressure waves of the femoral and the posterior tibial artery were obtained using an automated pressure transducer system (Complior SP, Artech Medical, Pantin, France). The distances between the measurement sites was again measured by caliper. The measurements taken post-exercise were obtained continuously and recorded by the median PWV of every 10 s. Methods Consecutive male CAD patients after recent myocardial infarction who were willing to enroll in a cardiac rehabilitation programme were recruited for the purpose of a related study (NCT02627586) at the University Clinic of Cardiology, University Hospital Bern, Switzerland between 1.9.2016 and 30.4.2018. Inclusion criteria were a first ST-segment elevation myocar- dial infarction or equivalent, with onset of symptoms of ischemia and treated by primary per- cutaneous coronary intervention within four weeks prior to study inclusion. Exclusion criteria were the inability to perform a maximal exercise test, a known chronic heart failure with left ventricular ejection fraction 45% before the acute index event, any medical intervention PLOS ONE \| https://doi.org/10.1371/journal.pone.0219146 July 9, 2019 2 / 12 Post-exercise peripheral PWV after myocardial infarction preventing a subject from performing exercise, or subjects with alcohol or drug abuse, or dementia. Healthy young men (HY) between 20–35 years of age and healthy middle-aged men (HAM, matching the age distribution of the CAD population, between 40–75 years of age) were recruited from hospital staff and their friends or relatives. These subjects were recreation- ally moderately to very active (with a minimum of 60 min on at least 3 days/week). Exclusion criteria were smoking, hypertension (> 140/90 mmHg), a history of cardiovascular disease, diabetes and intake of regular medication. The study was approved by the Ethics Committee of the Canton Berne and all participants gave their written informed consent. Testing procedure Subjects reported to the laboratory and changed into light sports gear. Height was measured using a wall-mounted stadiometer (KIRCHNER & WILHELM GmbH + Co. KG, Asperg, Ger- many), and weight was measured with a digital scale (InBody 720, Biospace Co., Seoul, Korea). Then they rested supine in a quiet, dimly lit room for 5 min. First, they had resting central and peripheral pulse wave velocity (PWV) measurements done. Then, still in supine position, they had resting heart rate (HR) and blood pressure (BP) recorded for 5 min while subjects were breathing at a paced rate of 0.25 Hz. After this, they mounted a stationary cycle ergometer and performed a cardiopulmonary exercise test with an individually determined ramp protocol (increasing by 10, 15, or 20 Watts/min) in order to reach exhaustion within 8–12 min. Oxygen consumption (VO2) was recorded continuously breath-by-breath in an open spirometric sys- tem (Jaeger Oxycon pro, ViaSys Healthcare GmbH) and peak VO2 was determined as the highest average of 8 breaths. After exhaustion, they performed a 2-min cool-down with light pedaling, after which they laid supine again for the 15 min post-exercise pPWV, BP and HR monitoring. Heart rate and blood pressure BP and HR were also measured pre-exercise and continuously from 5 to 15 min post-exercise. Baseline BP and HR mean values were calculated from a one-min period after a 3-min rest in supine position. BP and HR were measured by the Task Force Monitor (CNSystems Medizin- technik AG, Graz, Austria), which consists of a finger cuff (CNAP sensor) with pressure cham- bers and an infrared light sensor, an oscillometric upper arm blood pressure cuff, a four 3 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0219146 July 9, 2019 Post-exercise peripheral PWV after myocardial infarction channel ECG and a corresponding software that enables beat-to-beat BP measurements on the finger artery.[27]. Statistical analysis Sample size calculation was based on previous studies who found statistically highly significant decreases in pPWV acutely after exercise in 12 and 25 healthy volunteers.[5,6] According to their results, 6 subjects are needed to find a decrease in pPWV at a one-sided alpha of 0.05 and a power of 80%. Therefore, we planned for 10 subjects in each of our healthy groups and 20 in our CAD group to accommodate for potentially missing data. Statistical analysis was performed using the software R (Version 3.4.0, R Core Team, 2017). Baseline characteristics were compared between the three groups by ANOVA and between CAD and HAM/HY by t-test. Values at baseline and at 5 min post-exercise were compared between time points and between groups by mixed linear models (function lme of package nlme) with time point and group as fixed effects and subject as random intercept. Likewise, the same mixed linear models were also performed for data at each minute from 5 min to 14 min post-exercise. The difference in peripheral PWV (delta pPWV) was calculated as peripheral PWV at 5 min post-exercise (or 6 or 7 min in 4 subjects who had no available PWV data at 5 min) minus peripheral PWV at baseline. Accordingly, delta systolic BP and delta diastolic BP were also cal- culated. A correlation matrix with Spearman correlation coefficients was composed with the following variables: Delta pPWV, age, VO2 peak, central PWV at baseline, peripheral PWV at baseline, mean BP, and difference in systolic BP from before to after exercise. Linear models were performed with delta PWV as dependent variable and as independent variables group, and parameters that had a correlation coefficient 0.3 with delta pPWV. Normal distribution of data was assessed by visual inspection of QQ-plots. A p-value of less than 0.05 was consid- ered statistically significant. PLOS ONE \| https://doi.org/10.1371/journal.pone.0219146 July 9, 2019 CAD, coronay artery disease group; HAM, healthy age-matched group; HY, healthy young group; BMI, body mass index; VO2 peak, peak oxygen uptake; HR, heart rate; BP, blood pressure; PWV, pulse wave velocity. Results Twenty-one CAD subjects, 11 HAM and 10 HY were recruited for the present study. Three CAD patients were excluded due to pPWV post-exercise measurements that only had a valid signal after more than 7 min post-exercise. One HAM participant was excluded because he turned out to have hypertension as assessed at the pre-exercise measurement. Baseline charac- teristics of the analysed 18 CAD, 10 HAM and 10 HY subjects are presented in Table 1. Disease characteristics and medication of the CAD group are given in Table 2. Mean beta- blocker dose was 27.8% of maximal dose. None of the CAD patients took vasodilators or Ca- antagonists. PPWV was not different at 5 min post-exercise from baseline in the CAD and HAM group, while it decreased significantly by 1 m/s in the HY (p = 0.022, Table 3 and Fig 1). Between min 5 and min 14, pPWV decreased slightly and similarly in all groups (CAD group: 0.04 m/s per min, p = 0.022). Systolic BP increased in all groups similarly from baseline to 5 min post-exercise (CAD group: 3.26 mmHg/min, p<0.001) without a linear trend thereafter in any group (Fig 1). Dia- stolic BP increased by 1.39 mmHg/min in the CAD group (p = 0.011) from baseline to 5 min post-exercise, with insignificantly smaller increases in the other groups (Table 2 and Fig 1). Between min 5 and min 14, diastolic BP remained stable in the CAD and HAM group, but increased significantly in the HY group (by 0.64 mmHg per min, p = 0.037, Table 3 and Fig 1). Resting HR was comparable between the three groups. Peak HR was significantly lower in the CAD group compared both to HAM (-29.2 bpm, p<0.0001) and HY (-45.0 bpm, PLOS ONE \| https://doi.org/10.1371/journal.pone.0219146 July 9, 2019 4 / 12 Shown are means ± standard deviation or number of patients (percentage of all patients). Shown are means (standard deviations). P-value are from ANOVAs with fixed factor group. CAD, coronay artery disease group; HAM, healthy age-matched group; HY, healthy young group; BMI, body mass index; V rate; BP, blood pressure; PWV, pulse wave velocity. M, healthy age-matched group; HY, healthy young group; BMI, body mass index; VO2 peak, peak oxygen uptake; HR, heart ve velocity. ACE, angiotensin-converting-enzyme. PLOS ONE \| https://doi.org/10.1371/journal.pone.0219146 July 9, 2019 https://doi.org/10.1371/journal.pone.0219146.t002 , p0.05 p0 01 P-value are from ANOVAs with fixed factor group. For pPWV at 5 min data was available for n = 14 in CAD, n = 8 in HAM, and n = 10 in HY. pPWV, peripheral pulse wave velocity; BP, blood pressure; CAD, coronary artery disease group; HAM, healthy age-matched group; HY, healthy young group. https://doi.org/10.1371/journal.pone.0219146.t003 p<0.0001). At 5 min post-exercise the CAD group had lower HR compared to HAM (-10.0 bpm, p = 0.057) and HY (-21.4 bpm, p = 0.0001), with a comparable decrease until 14 min post-exercise thereafter (Fig 1). While normal distribution was acceptable for all parameters in all groups at most time points, box plots are shown because of the small group sizes of HAM and HY. The following variables had Spearman correlation coefficients 0.3 with delta pPWV: Age (0.48), cPWV at baseline (0.34), and delta diastolic BP (0.32). The mixed linear model that explained the greatest adjusted percentage of variance in delta pPWV was when only age and delta diastolic BP were included (r2 = 0.24, p = 0.008). Post-exercise peripheral PWV after myocardial infarction Table 1. Comparison of baseline characteristics of the three groups. Parameter CAD HAM HY p-value Number of subjects 18 10 10 Age [yrs] 55.7 (7.3) 55.9 (9.5) 24.9 (3.0) 0.000 Height [cm] 175.7 (8.5) 178.9 (6.9) 182.5 (6.0) 0.085 BMI [kg/m2] 26.6 (3.7) 23.6 (1.8) 24.2 (2.7) 0.029 Resting parameters Resting HR [bpm] 58.6 (8.3) 59.4 (7.7) 55.8 (6.5) 0.540 Resting systolic BP [mmHg] 111 (10) 120 (8) 122 (12) 0.013 Resting diastolic BP [mmHg] 73 (7) 80 (7) 75 (5) 0.042 Resting mean BP [mmHg] 89 (7) 95 (10) 92 (6) 0.216 Resting pulse pressure [mmHg] 38 (6) 40 (7) 48 (11) 0.014 Resting central PWV [m/s] 10.1 (1.3) 10.4 (2.0) 8.3 (0.7) 0.003 Resting peripheral PWV [m/s] 9.5 (1.3) 9.6 (1.1) 9.1 (1.4) 0.577 Peak parameters Exercise test duration [min] 13.2 (2.4) 14.8 (2.3) 13.7 (2.8) 0.310 VO2 peak [ml/kg/min] 29.5 (5.6) 41.9 (6.5) 54.1 (11.0) 0.000 Peak HR [bpm] 143.2 (18.8) 173.2 (14.9) 185.4 (10.4) 0.000 HR reserve [bpm] 84.6 (19.9) 113.8 (15.8) 129.6 (6.6) 0.000 Peak systolic BP [mmHg] 124 (18) 135 (11) 141 (12) 0.041 Table 1. Comparison of baseline characteristics of the three groups. Shown are means (standard deviations). P-value are from ANOVAs with fixed factor group. p , p0.001, all asterices refer to difference to reference group CAD by t-test Table 2. Baseline Characteristics and medication of the CAD group. Parameter 18 CAD patients Disease severity Ejection Fraction [%] 46.3 ± 12.8 3-vessel disease 9 (50%) 2-vessel disease 1 (4.5%) 1-vessel disease 8 (44.5%) Number of stents 1.76 ± 1.30 Cardiovascular Medication Beta-blockers 18 (100) Aspirin 18 (100) Antithrombotics 18 (100) Anticoagulants 2 (11) ACE inhibitors/Sartans 17 (94.5) Statins 18 (100) Non-statins 2 (11) Diuretics 4 (22) Shown are means ± standard deviation or number of patients (percentage of all patients). ACE, angiotensin-converting-enzyme. https://doi.org/10.1371/journal.pone.0219146.t002 Table 2. Baseline Characteristics and medication of the CAD group. 5 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0219146 July 9, 2019 PLOS ONE \| https://doi.org/10.1371/journal.pone.0219146 July 9, 2019 PLOS ONE \| https://doi.org/10.1371/journal.pone.0219146 July 9, 2019 https://doi.org/10.1371/journal.pone.0219146.t003 Post-exercise peripheral PWV after myocardial infarction Table 3. Mixed linear models for pPWV and systolic as well as diastolic BP. Baseline to 5 min post-exercise 5 min to 14 min post exercise Fixed effects Estimate Std. Error p-value Estimate Std. Error p-value pPWV CAD 9.52 0.30 0.000 9.66 0.32 0.000 HAM 0.12 0.50 0.810 0.46 0.52 0.388 HY -0.44 0.50 0.387 -1.67 0.52 0.003 CAD x time interaction -0.02 0.05 0.751 -0.04 0.02 0.022 HAM x time interaction 0.02 0.08 0.807 -0.02 0.03 0.382 HY x time interaction -0.19 0.08 0.022 0.04 0.03 0.136 Systolic BP CAD 111.0 2.52 0.000 126.01 4.26 0.000 HAM 8.88 4.21 0.042 12.33 7.88 0.129 HY 11.44 4.21 0.010 16.86 7.73 0.037 CAD x time interaction 3.26 0.64 0.000 -0.48 0.32 0.143 HAM x time interaction 0.31 1.07 0.776 -0.24 0.59 0.683 HY x time interaction 0.36 0.98 0.716 -0.02 0.57 0.978 Diastolic BP CAD 72.70 1.57 0.000 77.65 2.38 0.000 HAM 6.89 2.63 0.013 4.76 4.41 0.289 HY 1.88 2.63 0.480 -6.30 4.34 0.157 CAD x time interaction 1.39 0.49 0.011 0.11 0.15 0.441 HAM x time interaction -0.57 0.82 0.492 0.07 0.26 0.783 HY x time interaction -1.36 0.83 0.119 0.53 0.25 0.037 Mixed linear models were performed separately for time periods between baseline and 5 min as well as between 5 min and 14 min after exercise cessation. Shown are treatment contrasts with CAD as reference group. Number of available data for pPWV is n = 18 for CAD, n = 10 for HAM, n = 10 for HY, and for systolic and diastolic BP n = 17 for CAD, n = 7 for HAM, and n = 7 for HY. For pPWV at 5 min data was available for n = 14 in CAD, n = 8 in HAM, and n = 10 in HY. Table 3. Mixed linear models for pPWV and systolic as well as diastolic BP. Mixed linear models were performed separately for time periods between baseline and 5 min as well as between 5 min and 14 min after exercise cessation. Shown are treatment contrasts with CAD as reference group. Number of available data for pPWV is n = 18 for CAD, n = 10 for HAM, n = 10 for HY, and for systolic and diastolic BP n = 17 for CAD, n = 7 for HAM, and n = 7 for HY. Mixed linear models were performed separately for time periods between baseline and 5 min as well as between 5 min and 14 min after exercise cessation. Shown are treatment contrasts with CAD as reference group. Number of available data for pPWV is n = 18 for CAD, n = 10 for HAM, n = 10 for HY, and for systolic and diastolic BP n = 17 for CAD, n = 7 for HAM, and n = 7 for HY. For pPWV at 5 min data was available for n = 14 in CAD, n = 8 in HAM, and n = 10 in HY. pPWV, peripheral pulse wave velocity; BP, blood pressure; CAD, coronary artery disease group; HAM, healthy age-matched group; HY, healthy young group. PLOS ONE \| https://doi.org/10.1371/journal.pone.0219146 July 9, 2019 Discussion This is the first study to show that the drop in pPWV found in the legs of YH following a bout of exhaustive exercise was absent in CAD patients and HAM. Similar to previous studies [5,6,26], our YH group showed a decrease in pPWV of 17% at 5 min after peak exercise com- pared to pre-exercise. Confirming our hypothesis, this drop was absent in the CAD patients, but contrary to our hypothesis, this drop was also absent in HAM, leading to the interpretation that the absence of the drop was not due to an altered physiology with CAD but more likely due to age. Namely, change in pPWV from pre- to post-exercise was related to age, cPWV at rest, and change in diastolic BP, but not to pPWV pre-exercise. Brachial systolic BP was 6 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0219146 July 9, 2019 Post-exercise peripheral PWV after myocardial infarction Fig 1. Box plots of data measured before (PRE) and from 5 to 14 min after the acute maximal exercise bout. Shown are peripheral pulse wave velocity (upper left panel), heart rate (upper right panel), systolic blood pressure (lower left panel) and diastolic blood pressure (lower right panel) of the CAD, HAM and HY groups. Boxes include the IQR, whiskers the outer quartiles without outliers, and outliers are defined as distance to median >1.5+IQR. CAD, coronary artery disease group; HAM, healthy age-matched group; HY, healthy young group; IQR, inter-quartile range. Fig 1. Box plots of data measured before (PRE) and from 5 to 14 min after the acute maximal exercise bout. Shown are peripheral pulse wave velocity (upper left panel), heart rate (upper right panel), systolic blood pressure (lower left panel) and diastolic blood pressure (lower right panel) of the CAD, HAM and HY groups. Boxes include the IQR, whiskers the outer quartiles without outliers, and outliers are defined as distance to median >1.5+IQR. CAD, coronary artery disease group; HAM, healthy age-matched group; HY, healthy young group; IQR, inter-quartile range. https://doi.org/10.1371/journal.pone.0219146.g001 https://doi.org/10.1371/journal.pone.0219146.g001 consistently 10 mmHg and diastolic BP 5–8 mmHg lower in CAD patients compared to age- matched healthy controls (significance reached not at all time points). Most existing studies on post-exercise pPWV have been performed with healthy young vol- unteers. They have generally found a decrease in pPWV measured at the leg immediately post- exercise (<5 min) persisting further into the recovery period (> 5 min) [26]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0219146 July 9, 2019 Discussion To our knowl- edge, there are no studies who have compared the effect of acute exercise on leg pPWV in older healthy subjects to CAD patients. Information on pPWV acutely after exercise in middle-aged to elderly persons is sparse, with only few studies who have assessed other parameters of arterial compliance than PWV. Only one study compared middle-aged CAD patients with a control group of patients with chest pain but no CAD [28]. They found post-exercise decreases in ankle-brachial PWV in both groups. However, their measure of PWV was a composite measure of central and periph- eral arteries, therefore, comparison with our results of post-exercise pPWV is difficult. Post-exercise pPWV is likely to reflect endothelial function dependent flow-mediated vaso- dilation due to exercise-induced hyperemia, resulting in a greater arterial radius, a lesser wall thickness and probably reduced elastic modulus due to the absence of vasoconstriction. A sim- ilar vasodilatory response to what is found after exercise can be achieved by occlusion. There is an immediate vasodilation post-exercise lasting up to 20 min probably due to central sympa- thetic inhibition and local dilatory mechanisms and a sustained post-exercise vasodilation PLOS ONE \| https://doi.org/10.1371/journal.pone.0219146 July 9, 2019 7 / 12 Post-exercise peripheral PWV after myocardial infarction lasting up to several hours due to mainly histamine H1 and H2 receptor activation [29]. We can therefore assume that in our study participants, at 5–15 min post-exercise, peripheral ves- sels to exercising muscles were dilated, leg blood flow elevated, and vascular smooth muscle tone largely abolished [30]. It is likely that post-exercise pPWV reflects two concomitant vessel characteristics: vessel dilation due to increased shear stress, which theoretically lowers pPWV via an increased vessel diameter, a decrease in vasomotor tone, and a reduced vessel wall thickness. However, in a fully dilated vessel, the passive structures of the vessel wall may be stiffer due to the elastic modulus of the wall being dominated by the collagen fibers rather than the more elastic elastin fibers [31]. This would explain why in older individuals (our CAD and HAM group), despite the most likely dilated arteries, post-exercise pPWV was not reduced. It is likely that, similar to the central arteries, the passive wall structure of their peripheral arteries were stiffer than those of young individuals, but that this higher stiffness was not detected at rest, when the effect of vasomotor tone had an overriding effect on pPWV. Discussion Age-associated changes of the brachial, radial and popliteal artery have been found as an increase in lumen diameter and intima media thickness, but not for compliance.[32–34] However, these measurements have been performed at rest and not in the dilated state after acute exercise. Post-exercise, pPWV in the HY may have decreased due to higher elasticity of the passive vessel wall structures of the dilated femoral artery compared to the older subjects. Stiffer arterial intrinsic wall properties due to accumulation of collagens and proteoglycans[35] are likely to be detectable only in the vasodilated state when not masked by the amount of smooth muscle tone, as suggested previ- ously by Naka and colleagues [5]. In fact, we found a significant linear correlation between the change in pPWV from baseline to 5 min post-exercise and age, explaining 25% of the total var- iance in delta pPWV (p = 0.001). The only other variables that were associated with delta pPWV was resting cPWV (r2 = 0.13, p = 0.015) and delta diastolic BP (r2 = 0.11, p = 0.064), however, changes in diastolic BP from pre- to post-exercise were similar in all groups. Surprisingly, in our study cPWV was comparable in CAD patients and age-matched healthy controls. This is in contrast to a study by Hofmann and colleagues [36], who found higher cPWV in CAD patients compared to normal reference values and who found cPWV to increase with age and increasing severity of coronary vessel disease. We did not find an associ- ation of cPWV with severity of vessel disease. However, heart rate, systolic and diastolic BP were all reduced in CAD patients compared to our healthy age-matched controls, probably as a consequence of medication, namely all CAD patients were on beta-blockers and 95% on ACE-inhibitors or Sartans. In contrast, in the study cited above [36], systolic BP was higher in CAD patients than controls and was considerably higher than in our CAD patients. Conse- quently, in our study we can not automatically conclude that central arterial stiffness was com- parable in the two groups, since cPWV may have been lowered secondary to the BP-lowering effect of beta-blockers [37]. CPWV has been shown to directly depend on systolic and diastolic BP by approximately 1 m/s for every 10 mmHg change in diastolic BP [20,38–40]. Discussion Similarly, pPWV at baseline and after cessation of exercise was comparable between CAD and HAM, however, both systolic and diastolic BP were significantly lower in the CAD compared to the HAM group pre-exercise with a continuing trend to remain lower after exercise (Fig 1), sug- gesting that peripheral arteries in CAD were either stiffer or their diameter smaller. Acknowledgments This study was funded by a grant from the Swiss Heart Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Supporting information S1 File. S1_File. (XLSX) S1 File. S1_File. (XLSX) Limitations PWV is only a surrogate for arterial stiffness and directly dependent on other factors like vessel diameter, intrinsic wall stiffness (including wall thickness and elastic modulus), and blood vis- cosity, and indirectly dependent on blood pressure [20] and to a lesser extent heart rate [41]. 8 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0219146 July 9, 2019 Post-exercise peripheral PWV after myocardial infarction As we did not measure sympathetic nerve activity, we cannot exclude that vasodilation was incomplete post-exercise in our CAD patients and middle-aged healthy subjects. PWV measurements have known limitations with regard to precision, with reproducibili- ties of between 5–10% [42,43]. Reproducibility of post-exercise lower limb PVW was per- formed in five subjects and shown visually but not quantified [5]. We could not measure central and peripheral PWV simultaneously, which prevented us from also measuring cPWV following acute exercise. However, a recent meta-analysis con- cluded that effects of aerobic exercise on cPWV were equivocal,[14] while they have been well established for pPWV. Further, we did not measure blood viscosity and can therefore not exclude the group differ- ence to be due to different responses in blood viscosity. Namely, exercise has been shown to increase blood viscosity immediately post-exercise by approximately 12%-20% in young healthy volunteers [5,44,45]. Last but not least, the sample sizes of the healthy groups was small, suggesting that the dis- tinction between the CAD and HAM group may have lacked statistical power. However, the differences in SBP and DBP between CAD and HAM group pre-exercise were significant, sup- porting that the absence of a difference between these two groups in pPWV and cPWV pre- exercise was likely due to a lower BP of the CAD group and did not imply comparable arterial stiffness. Conclusions We conclude that post-exercise dilatation of the leg arteries did not lead to a concomitant decrease in pPWV in CAD patients and age-matched healthy controls, as was found in our young healthy population. We suggest that the reason for this may be a stiffer vessel wall of the dilated arteries in older subjects, as the change in pPWV was related to age and cPWV at rest. Post-exercise measurements may offer a maneuver to assess intrinsic stiffness of peripheral arteries. It remains to be studied in longitudinal intervention studies whether this parameter can be influenced by chronic exercise training, however, the absence of a difference between our CAD and HAM group does not make this probable. References 1. Chen YC, Tsai JC, Liou YM, Chan P (2017) Effectiveness of endurance exercise training in patients with coronary artery disease: A meta-analysis of randomised controlled trials. Eur J Cardiovasc Nurs 16: 397–408. https://doi.org/10.1177/1474515116684407 PMID: 28565969 2. Anderson L, Oldridge N, Thompson DR, Zwisler AD, Rees K, Martin N, et al. (2016) Exercise-Based Cardiac Rehabilitation for Coronary Heart Disease: Cochrane Systematic Review and Meta-Analysis. J Am Coll Cardiol 67: 1–12. https://doi.org/10.1016/j.jacc.2015.10.044 PMID: 26764059 3. Gielen S, Schuler G, Adams V (2010) Cardiovascular effects of exercise training: molecular mecha- nisms. Circulation 122: 1221–1238. https://doi.org/10.1161/CIRCULATIONAHA.110.939959 PMID: 20855669 4. Bank AJ, Kaiser DR, Rajala S, Cheng A (1999) In vivo human brachial artery elastic mechanics: effects of smooth muscle relaxation. Circulation 100: 41–47. https://doi.org/10.1161/01.cir.100.1.41 PMID: 10393679 5. Naka KK, Tweddel AC, Parthimos D, Henderson A, Goodfellow J, Frenneaux MP (2003) Arterial disten- sibility: acute changes following dynamic exercise in normal subjects. Am J Physiol Heart Circ Physiol 284: H970–978. https://doi.org/10.1152/ajpheart.00529.2002 PMID: 12433655 6. Kingwell BA, Berry KL, Cameron JD, Jennings GL, Dart AM (1997) Arterial compliance increases after moderate-intensity cycling. Am J Physiol 273: H2186–2191. https://doi.org/10.1152/ajpheart.1997.273. 5.H2186 PMID: 9374752 7. Campbell R, Fisher JP, Sharman JE, McDonnell BJ, Frenneaux MP (2011) Contribution of nitric oxide to the blood pressure and arterial responses to exercise in humans. J Hum Hypertens 25: 262–270. https://doi.org/10.1038/jhh.2010.53 PMID: 20505750 8. Rakobowchuk M, Stuckey MI, Millar PJ, Gurr L, Macdonald MJ (2009) Effect of acute sprint interval exercise on central and peripheral artery distensibility in young healthy males. Eur J Appl Physiol 105: 787–795. https://doi.org/10.1007/s00421-008-0964-7 PMID: 19125283 9. Tordi N, Mourot L, Colin E, Regnard J (2010) Intermittent versus constant aerobic exercise: effects on arterial stiffness. Eur J Appl Physiol 108: 801–809. https://doi.org/10.1007/s00421-009-1285-1 PMID: 20187285 10. Heffernan KS, Rossow L, Jae SY, Shokunbi HG, Gibson EM, Fernhall B (2006) Effect of single-leg resistance exercise on regional arterial stiffness. Eur J Appl Physiol 98: 185–190. https://doi.org/10. 1007/s00421-006-0259-9 PMID: 16896730 11. Lane AD, Ranadive SM, Yan H, Kappus RM, Cook MD, Sun P, et al. (2013) Effect of sex on wasted left ventricular effort following maximal exercise. Int J Sports Med 34: 770–776. https://doi.org/10.1055/s- 0032-1329990 PMID: 23526590 12. Yan H, Ranadive SM, Heffernan KS, Lane AD, Kappus RM, Cook MD, et al. (2014) Hemodynamic and arterial stiffness differences between African-Americans and Caucasians after maximal exercise. Am J Physiol Heart Circ Physiol 306: H60–68. https://doi.org/10.1152/ajpheart.00710.2013 PMID: 24186094 13. Author Contributions Conceptualization: Y. Trachsel, D. Herzig, T. Marcin, L. D. Trachsel, M. Wilhelm, P. Eser. p , g, , , , Data curation: Y. Trachsel, D. Herzig, T. Marcin, N. Zenger, M. Dysli, P. Eser. Formal analysis: N. Zenger, P. Eser. Data curation: Y. Trachsel, D. Herzig, T. Marcin, N. Zenger, M. Dysli, P. Eser. Investigation: Y. Trachsel, D. Herzig, T. Marcin, N. Zenger, M. Dysli, L. D. Trachsel, M. Wil- helm, P. Eser. PLOS ONE \| https://doi.org/10.1371/journal.pone.0219146 July 9, 2019 9 / 12 Post-exercise peripheral PWV after myocardial infarction Methodology: Y. Trachsel, D. Herzig, T. Marcin, M. Wilhelm, P. Eser. Project administration: Y. Trachsel, T. Marcin, N. Zenger, M. Dysli, L. D. Trachsel. Supervision: D. Herzig, M. Wilhelm, P. Eser. Validation: T. Marcin, N. Zenger. Writing – original draft: P. Eser. Writing review & editing: Y Trachsel D Herzig T Marcin N Zenger M Dysli L D Methodology: Y. Trachsel, D. Herzig, T. Marcin, M. Wilhelm, P. Eser. Project administration: Y. Trachsel, T. Marcin, N. Zenger, M. Dysli, L. D. Trachsel. Writing – review & editing: Y. Trachsel, D. Herzig, T. Marcin, N. Zenger, M. Dysli, L. D. Trachsel, M. Wilhelm, P. Eser. PLOS ONE \| https://doi.org/10.1371/journal.pone.0219146 July 9, 2019 References Kobayashi R, Hatakeyama H, Hashimoto Y, Okamoto T (2018) Acute effects of accumulated aerobic exercise on aortic and peripheral pulse wave velocity in young males. J Phys Ther Sci 30: 181–184. https://doi.org/10.1589/jpts.30.181 PMID: 29410594 14. Pierce DR, Doma K, Leicht AS (2018) Acute Effects of Exercise Mode on Arterial Stiffness and Wave Reflection in Healthy Young Adults: A Systematic Review and Meta-Analysis. Front Physiol 9: 73. https://doi.org/10.3389/fphys.2018.00073 PMID: 29487535 15. Naka KK, Tweddel AC, Doshi SN, Goodfellow J, Henderson AH (2006) Flow-mediated changes in pulse wave velocity: a new clinical measure of endothelial function. Eur Heart J 27: 302–309. https:// doi.org/10.1093/eurheartj/ehi619 PMID: 16267075 10 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0219146 July 9, 2019 Post-exercise peripheral PWV after myocardial infarction 16. Kamran H, Salciccioli L, Prudhvi K, Bastien C, Berman H, Sharma A, et al. (2011) Comparison of hyper- emic changes in carotid-radial pulse wave velocity by upper and lower arm cuff occlusion. Angiology 62: 409–414. https://doi.org/10.1177/0003319710389022 PMID: 21596698 17. Kamran H, Salciccioli L, Ko EH, Qureshi G, Kazmi H, Kassotis J, et al. (2010) Effect of reactive hyper- emia on carotid-radial pulse wave velocity in hypertensive participants and direct comparison with flow- mediated dilation: a pilot study. Angiology 61: 100–106. https://doi.org/10.1177/0003319709335028 PMID: 19625259 18. Rogova AN, Zairova AR, Oshchepkova EV (2008) [Changes in pulse wave velocity in the reactive hyperemia as a method of evaluation of endothelial vasomotor function in hypertensive patients]. Ter Arkh 80: 29–33. 19. Torrado J, Bia D, Zocalo Y, Farro I, Farro F, Valero M, et al. (2011) Carotid-radial pulse wave velocity as a discriminator of intrinsic wall alterations during evaluation of endothelial function by flow-mediated dilatation. Conf Proc IEEE Eng Med Biol Soc 2011: 6458–6461. https://doi.org/10.1109/IEMBS.2011. 6091594 PMID: 22255817 20. Spronck B, Heusinkveld MH, Vanmolkot FH, Roodt JO, Hermeling E, Delhaas T, et al. (2015) Pressure- dependence of arterial stiffness: potential clinical implications. J Hypertens 33: 330–338. https://doi. org/10.1097/HJH.0000000000000407 PMID: 25380150 21. Thijssen DH, Carter SE, Green DJ (2016) Arterial structure and function in vascular ageing: are you as old as your arteries? J Physiol 594: 2275–2284. https://doi.org/10.1113/JP270597 PMID: 26140618 22. Vlachopoulos C, Aznaouridis K, Stefanadis C (2010) Prediction of cardiovascular events and all-cause mortality with arterial stiffness: a systematic review and meta-analysis. J Am Coll Cardiol 55: 1318– 1327. https://doi.org/10.1016/j.jacc.2009.10.061 PMID: 20338492 23. References Matsuzawa Y, Kwon TG, Lennon RJ, Lerman LO, Lerman A (2015) Prognostic Value of Flow-Mediated Vasodilation in Brachial Artery and Fingertip Artery for Cardiovascular Events: A Systematic Review and Meta-Analysis. J Am Heart Assoc 4. 24. Sugamata W, Nakamura T, Uematsu M, Kitta Y, Fujioka D, Saito Y, et al. (2014) Combined assessment of flow-mediated dilation of the brachial artery and brachial-ankle pulse wave velocity improves the pre- diction of future coronary events in patients with chronic coronary artery disease. J Cardiol 64: 179– 184. https://doi.org/10.1016/j.jjcc.2014.01.004 PMID: 24556367 25. Walsh JH, Bilsborough W, Maiorana A, Best M, O’Driscoll GJ, Taylor RR, et al. (2003) Exercise training improves conduit vessel function in patients with coronary artery disease. J Appl Physiol (1985) 95: 20– 25. 26. Mutter AF, Cooke AB, Saleh O, Gomez YH, Daskalopoulou SS (2017) A systematic review on the effect of acute aerobic exercise on arterial stiffness reveals a differential response in the upper and lower arte- rial segments. Hypertens Res 40: 146–172. https://doi.org/10.1038/hr.2016.111 PMID: 27733765 27. Fortin J, Marte W, Grullenberger R, Hacker A, Habenbacher W, Heller A, et al. (2006) Continuous non- invasive blood pressure monitoring using concentrically interlocking control loops. Comput Biol Med 36: 941–957. https://doi.org/10.1016/j.compbiomed.2005.04.003 PMID: 16483562 28. Sung J, Yang JH, Cho SJ, Hong SH, Huh EH, Park SW (2009) The effects of short-duration exercise on arterial stiffness in patients with stable coronary artery disease. J Korean Med Sci 24: 795–799. https:// doi.org/10.3346/jkms.2009.24.5.795 PMID: 19794973 29. Halliwill JR, Buck TM, Lacewell AN, Romero SA (2013) Postexercise hypotension and sustained post- exercise vasodilatation: what happens after we exercise? Exp Physiol 98: 7–18. https://doi.org/10. 1113/expphysiol.2011.058065 PMID: 22872658 30. Williams JT, Pricher MP, Halliwill JR (2005) Is postexercise hypotension related to excess postexercise oxygen consumption through changes in leg blood flow? J Appl Physiol (1985) 98: 1463–1468. 31. Wagenseil JE, Mecham RP (2009) Vascular extracellular matrix and arterial mechanics. Physiol Rev 89: 957–989. https://doi.org/10.1152/physrev.00041.2008 PMID: 19584318 32. van der Heijden-Spek JJ, Staessen JA, Fagard RH, Hoeks AP, Boudier HA, van Bortel LM (2000) Effect of age on brachial artery wall properties differs from the aorta and is gender dependent: a population study. Hypertension 35: 637–642. PMID: 10679510 33. Bortolotto LA, Hanon O, Franconi G, Boutouyrie P, Legrain S, Girerd X (1999) The aging process modi- fies the distensibility of elastic but not muscular arteries. Hypertension 34: 889–892. PMID: 10523379 34. PLOS ONE \| https://doi.org/10.1371/journal.pone.0219146 July 9, 2019 References Green DJ, Swart A, Exterkate A, Naylor LH, Black MA, Cable NT, et al. (2010) Impact of age, sex and exercise on brachial and popliteal artery remodelling in humans. Atherosclerosis 210: 525–530. https:// doi.org/10.1016/j.atherosclerosis.2010.01.048 PMID: 20197189 35. Xu X, Wang B, Ren C, Hu J, Greenberg DA, Chen T, et al. (2017) Age-related Impairment of Vascular Structure and Functions. Aging Dis 8: 590–610. https://doi.org/10.14336/AD.2017.0430 PMID: 28966804 11 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0219146 July 9, 2019 Post-exercise peripheral PWV after myocardial infarction 36. Hofmann B, Riemer M, Erbs C, Plehn A, Navarrete Santos A, Wienke A, et al. (2014) Carotid to femoral pulse wave velocity reflects the extent of coronary artery disease. J Clin Hypertens (Greenwich) 16: 629–633. 37. Janic M, Lunder M, Sabovic M (2014) Arterial stiffness and cardiovascular therapy. Biomed Res Int 2014: 621437. https://doi.org/10.1155/2014/621437 PMID: 25170513 38. Obata Y, Mizogami M, Singh S, Nyhan D, Berkowitz DE, Steppan J, et al. (2016) The Effects of Hemo- dynamic Changes on Pulse Wave Velocity in Cardiothoracic Surgical Patients. Biomed Res Int 2016: 9640457. https://doi.org/10.1155/2016/9640457 PMID: 27900333 39. Spronck B, Delhaas T, Butlin M, Reesink KD, Avolio AP (2018) Options for Dealing with Pressure Dependence of Pulse Wave Velocity as a Measure of Arterial Stiffness: An Update of Cardio-Ankle Vas- cular Index (CAVI) and CAVI0. Pulse (Basel) 5: 106–114. 40. Bramwell J, Hill A (1922) The velocity of pulse wave in man. Proc R Soc Lond B 93: 298–306. 41. Tan I, Spronck B, Kiat H, Barin E, Reesink KD, Delhaas T, et al. (2016) Heart Rate Dependency of Large Artery Stiffness. Hypertension 68: 236–242. https://doi.org/10.1161/HYPERTENSIONAHA.116. 07462 PMID: 27245180 42. Grillo A, Parati G, Rovina M, Moretti F, Salvi L, Gao L, et al. (2017) Short-Term Repeatability of Nonin- vasive Aortic Pulse Wave Velocity Assessment: Comparison Between Methods and Devices. Am J Hypertens 31: 80–88. https://doi.org/10.1093/ajh/hpx140 PMID: 29059329 43. Ellins EA, Smith KE, Lennon LT, Papacosta O, Wannamethee SG, Whincup PH, et al. (2017) Arterial pathophysiology and comparison of two devices for pulse wave velocity assessment in elderly men: the British regional heart study. Open Heart 4: e000645. https://doi.org/10.1136/openhrt-2017-000645 PMID: 29344365 44. Hitosugi M, Kawato H, Nagai T, Ogawa Y, Niwa M, Iida N, et al. (2004) Changes in blood viscosity with heavy and light exercise. Med Sci Law 44: 197–200. https://doi.org/10.1258/rsmmsl.44.3.197 PMID: 15296241 45. Bilski J, Teleglow A, Pokorski J, Nitecki J, Pokorska J, Nitecka E, et al. PLOS ONE \| https://doi.org/10.1371/journal.pone.0219146 July 9, 2019 References (2014) Effects of a meal on the hemorheologic responses to exercise in young males. Biomed Res Int 2014: 862968. https://doi.org/ 10.1155/2014/862968 PMID: 25089277 12 / 12
https://openalex.org/W2001742138	http://pdf.blucher.com.br/marineengineeringproceedings/spolm2014/127140.pdf	Portuguese	null	ESTRATÉGIAS DE GESTÃO NO DESTINO DO LIXO TECNOLÓGICO: UM CASO DE IMPLANTAÇÃO DE UM ECOPONTO NA UNIGRANRIO	null	2,014	cc-by	5,501	RESUMO O acelerado avanço tecnológico tem causado a obsolescência dos equipamentos eletrônicos num curto espaço de tempo. Isso é o resultado do descarte de aparelhos eletrônicos, como computadores pessoais e celulares, entre outros, o lixo tecnológico é um problema cada vez mais aparente na sociedade atual. Quando não descartado adequadamente, o lixo tecnológico pode causar sérios danos à saúde e ao meio ambiente. A discussão sobre o lixo tecnológico nesta pesquisa, surgiu a partir do interesse de desenvolver um trabalho que envolvesse a Logística Reversa, buscando refletir sobre os impactos da tecnologia na sociedade. Nesse contexto, um dos objetivos é promover o uso ético e com responsabilidade social e ambiental das tecnologias. Esse artigo descreve os resultados de um estudo realizado sobre o lixo tecnológico, acerca de suas características, danos, riscos e legislação relacionada. O trabalho também trata da implantação de um Ecoponto dentro de um Campus Universitário. Ainda, apresenta-se levantamento acerca de locais para o descarte responsável. Com base nos dados obtidos, são definidas ações para o destino e reuso do lixo tecnológico, de forma a promover a sua gestão adequada. Palavras-chaves: Tecnologia, Lixo Tecnológico, Logística Reversa e Reciclagem. g , p g q avras-chaves: Tecnologia, Lixo Tecnológico, Logística Reversa e Reciclagem. Keywords: Technology, Technological Waste, Recycling and Reverse Logistics. ABSTRACT The rapid technological advancement has caused the obsolescence of electronic equipment in a short time. This is the result of the disposal of electronic equipment such as personal computers and mobile phones, among others, the technological waste is an increasingly apparent problem in society today. When not disposed of properly, the e-waste can cause serious damage to health and the environment. The discussion of technological waste this research arose from the interest of developing a job involving the Reverse Logistics, trying to reflect on the impact of technology on society. In this context, one of the goals is to promote the ethical use and social and environmental responsibility technologies. This article describes the results of a study on e-waste, about their characteristics, damages, liabilities and related legislation. The work also deals with the establishment of a Ecoponto within a University Campus. Still, presents survey about places for responsible disposal. Based on the data obtained, actions to fate and reuse of e-waste, in order to promote their proper management are defined. Keywords: Technology, Technological Waste, Recycling and Reverse Logistics. 1. INTRODUÇÃO Tanto as empresas quanto os governos não querem assumir a total responsabilidade quanto ao ciclo de vida dos equipamentos tecnológicos e, por sua vez, os consumidores não têm as informações necessárias quanto aos males do descarte indevido e também são desprovidos de alternativas para descartar equipamentos em desuso. Portanto, quais estratégias de gestão podem ser utilizadas no destino do lixo tecnológico? O lixo tecnológico é um problema de responsabilidade das empresas, dos consumidores e governos. Tanto as empresas quanto os governos não querem assumir a total responsabilidade quanto ao ciclo de vida dos equipamentos tecnológicos e, por sua vez, os consumidores não têm as informações necessárias quanto aos males do descarte indevido e também são desprovidos de alternativas para descartar equipamentos em desuso. Portanto, quais estratégias de gestão podem ser utilizadas no destino do lixo tecnológico? g O objetivo deste artigo é levantar o problema do lixo tecnológico, sua importância, impacto ambiental, consequências, situação nacional e mundial, ações e projetos preventivos e atuação das legislações, visando encontrar uma tentativa de formular estratégias de gestão e descarte do lixo tecnológico. O objetivo deste artigo é levantar o problema do lixo tecnológico, sua importância, impacto ambiental, consequências, situação nacional e mundial, ações e projetos preventivos e atuação das legislações, visando encontrar uma tentativa de formular estratégias de gestão e descarte do lixo tecnológico. 1. INTRODUÇÃO A compra facilitada de equipamentos eletrônicos, como computadores e aparelhos celulares, entre outros, nos últimos anos, tem sido uma prática frequente entre os consumidores de todo o mundo. Desta forma, o mundo tenta entender muitas questões sobre o lixo tecnológico, como descartá-lo e de quem seria a responsabilidade pela gestão desses resíduos. q como descartá-lo e de quem seria a responsabilidade pela gestão desses resíduos O lixo tecnológico recebe diferentes nomes: resíduo eletrônico, lixo eletrônico, e-waste, ou simplesmente e-lixo, sendo apenas diferentes no nome, mas configura o mesmo problema: são produtos eletrônicos sem utilidade. São, por exemplo, computadores, celulares, equipamentos de informática em geral, etc. O lixo tecnológico recebe diferentes nomes: resíduo eletrônico, lixo eletrônico, e-waste, ou simplesmente e-lixo, sendo apenas diferentes no nome, mas configura o mesmo problema: são produtos eletrônicos sem utilidade. São, por exemplo, computadores, celulares, equipamentos de informática em geral, etc. g , O problema do lixo tecnológico preocupa muito a sustentabilidade da vida no planeta, pois causa sérios danos ambientais, sendo considerado um problema de saúde pública. g O problema do lixo tecnológico preocupa muito a sustentabilidade da vida no planeta, pois causa sérios danos ambientais, sendo considerado um problema de saúde pública. Os avanços tecnológicos faz com que os equipamentos eletrônicos aumentem rapidamente, tornando-os, rapidamente, ultrapassados e ineficientes. Este rápido desuso dos eletrônicos e o aumento da produção e do consumo desses equipamentos, ajuda no crescimento do lixo tecnológico, e como consequência o problema se agrava a cada dia, deixando o planeta sem espaço para armazenamento e com pouca capacidade de reciclagem. A utilização da Logística Reversa ajudará na investigação de como é feito a descartabilidade do lixo tecnológico e quais os produtos que podem ser produzidos através da reciclagem do mesmo, que para muitos ainda é desconhecido e neste trabalho, será desenvolvido um estudo de caso na Universidade Unigranrio em seu campus de Magé no Rio de Janeiro. A implantação de Ecopontos em empresas e universidades representam ações necessárias, pois conscientiza a população, chamando a atenção para o destino correto do lixo tecnológico, tendo uma solução através da reciclagem. Assim, um maior esforço neste sentido é fundamental. ç g ç O lixo tecnológico é um problema de responsabilidade das empresas, dos consumidores e governos. 1 Lei Federal 12.305/2010 – Artigo 33, incisos I, II, III, IV, V, VI. 2. O LIXO TECNOLÓGICO Para Schneider (2004, p. 20), este tipo de lixo apresenta caráter antropogênico, pois são únicos e exclusivamente gerados pelo homem em suas atividades diárias em sociedade, além disso, apresentam caráter inesgotável, uma vez que também é ilimitada a capacidade do ser humano de crescer numericamente ou em conhecimento e inventividade, gerando a cada dia novos produtos, promovendo sempre novas transformações nas matérias-primas. O crescimento da produção de equipamentos eletrônicos é algo inevitável que logo em alguns anos o que é produzido hoje estará obsoleto num futuro bem próximo. A grande preocupação é o que fazer com a sucata gerada dia a dia e como produzir equipamentos que tenham em seus componentes, substâncias não tóxicas e recicláveis. Nesse contexto, surgem leis – federais e estaduais – que obrigam pessoas físicas e jurídicas a cuidarem do lixo tecnológico que produzem. Algumas empresas têm se antecipado e estão criando programas para recolher os produtos que produzem e que logo caem na inutilidade, reciclando-os ou dando uma disposição final correta. p ç A reciclagem consiste em transformar os materiais inúteis em novos produtos ou matérias-primas de forma a diminuir a quantidade de resíduos, poupar energia e recursos naturais valiosos, além de trazer os seguintes benefícios, como mostra o quadro 1: Quadro 1 - Benefícios da Reciclagem Benefícios da Reciclagem Desintoxicação Contribui para diminuir a poluição do solo, água e ar. Despoluição Melhora a limpeza da cidade e a qualidade de vida da população. Perenidade Prolonga a vida útil de aterros sanitários. Compostagem Melhora a produção de compostos orgânicos. Empregabilidade Gera empregos para a população não qualificada. Econômico Gera receitas com a comercialização dos recicláveis. Custo Energético Estimula a concorrência uma vez que os produtos gerados a partir dos reciclados são comercializados em paralelo àqueles gerados a partir de matérias primas virgens. Imagem Contribui para a valorização da limpeza pública e para formar uma consciência ecológica. Fonte: Próprio autor, 2014. Quadro 1 - Benefícios da Reciclagem 2.1 ASPECTOS LEGAIS Há legislações no Brasil que tratam exclusivamente do lixo tecnológico. As leis existem, desde federais (Lei Federal nº 12.305) a estaduais, pois cada estado brasileiro carece de leis específicas para a questão do lixo tecnológico. Cada estado desenvolve seus princípios e objetivos de acordo com a cultura e costumes de seus habitantes. No entanto, todos alertam para a responsabilidade dos fabricantes, importadores, distribuidores e comerciantes, a estruturação e implementação de sistemas de Logística Reversa. A Lei de Política Nacional de Resíduos Sólidos dispõe sobre os princípios, objetivos e instrumentos, bem como sobre as diretrizes relativas à gestão integrada e ao gerenciamento de resíduos sólidos, incluindo os perigosos, às responsabilidades dos geradores e do poder público e aos instrumentos econômicos aplicáveis. Estão sujeitas à observância desta Lei as pessoas físicas ou jurídicas, de direito público ou privado, responsáveis, direta ou indiretamente, pela geração de resíduos sólidos e as que desenvolvam ações relacionadas à gestão integrada ou ao gerenciamento de resíduos sólidos, somente não sendo aplicada esta Lei aos rejeitos radioativos, que são regulados por legislação específica. Leia a citação a seguir: Art. 33. São obrigados a estruturar e implementar sistemas de logística reversa, mediante retorno dos produtos após o uso pelo consumidor, de forma independente do serviço público de limpeza urbana e de manejo dos resíduos sólidos, os fabricantes, importadores, distribuidores e comerciantes de: agrotóxicos, seus resíduos e embalagens, pilhas e baterias, pneus, óleos lubrificantes, seus resíduos e embalagens, lâmpadas fluorescentes, de vapor de sódio e mercúrio e de luz mista e produtos eletroeletrônicos e seus componentes1. A Política Nacional de Resíduos Sólidos reúne o conjunto de princípios, objetivos, instrumentos, diretrizes, metas e ações adotadas pelo Governo Federal, isoladamente ou em regime de cooperação com Estados, Distrito Federal, Municípios ou particulares, com vistas à gestão integrada e ao gerenciamento ambientalmente adequado dos resíduos sólidos. 2 Pesquisa contratada pela ONU – Organizações das Nações Unidas, 2002. 3 EEE – Equipamentos Eletro Eletrônicos 3. PROCESSO DE TRATAMENTO Para Franke (2004), a tradição da remanufatura, que era ajustada aos investimentos de longa duração como no caso de máquinas operatrizes, aviões, equipamentos militares e motores de automóveis, também foi estendido para um grande número de bens de consumo com o tempo de vida útil inferior e valores relativamente baixos. Celulares, rádios e computadores pessoais seriam exemplos desses novos produtos que são reprocessados. Uma alternativa à reciclagem convencional é a remanufatura, pois constitui na missão de atender as taxas de recuperação de produto e os tratamentos especiais na legislação. O conceito de logística reversa preocupa-se com outros termos tal como a reciclagem e disposição dos resíduos, ele então destaca que a logística reversa é uma perspectiva da logística de negócios. O termo refere-se ao papel da logística no retorno de produtos, redução na fonte, reciclagem, substituição e reuso de materiais, disposição de resíduos, reformas, reparação e remanufatura, LEITE (2003). A logística Reversa tem o papel de instrumento de desenvolvimento econômico e social, caracterizada por um conjunto de ações, procedimentos e meios, destinados a facilitar a coleta e a restituição dos resíduos sólidos aos seus geradores para que sejam tratados ou reaproveitados em novos produtos, na forma de novos insumos, em seu ciclo ou em outros ciclos produtivos, visando a não geração de rejeitos. (BRASIL, Projeto de Lei nº 1991, 2007, art. 7º inciso XII, p.3). 2 Na percepção da ONU2 (2002), governantes do mundo inteiro devem adotar medidas para incentivar a reciclagem de computadores e celulares obsoletos e o prolongamento de sua vida útil, devido ao impacto destrutivo dos componentes dessas máquinas para o meio ambiente. Destaca-se também que a fabricação de um computador de 24 Kg exige 10 vezes mais o seu peso em combustível fóssil e mais produtos químicos. A fabricação de um computador e seu monitor requer aproximadamente 240 Kg de combustível e 22 Kg de produtos químicos. A legislação e os sistemas direcionados para o tratamento de Equipamentos Eletro Eletrônicos (EEE)3, são implementados nos estados brasileiros trabalhos que devem proporcionar os pré-requisitos de um tratamento adequado, ajudando a indústria, além de analisar as potências de reciclagem, reuso e remanufatura de diferentes classes de produtos para o desenvolvimento sócio-econômico. 3.1.1 O Perigo dos Componentes Tóxicos de Computadores e Celulares Os metais pesados contidos nas baterias de celulares e em computadores, quando absorvidos, são de difícil eliminação pelo organismo, podendo causar diversos efeitos nocivos ao ser humano. Observe tais informações no quadro 2, a seguir. Quadro 2: Efeitos do Cádmio, Mercúrio e Chumbo 3.1 IMPACTOS DO E-LIXO NO MEIO AMBIENTE E NO SER HUMANO O lixo tecnológico que é descartado em lugares impróprios, como aterros sanitários ou lixões ao céu aberto, constituem sérios riscos para o meio ambiente e saúde humana, pois possuem em suas composições diversas substâncias e elementos químicos extremamente nocivos à saúde, como metais pesados altamente tóxicos: mercúrio, chumbo, cádmio, etc. Esses metais em contato com o solo contaminam o lençol freático, chegando à água e consequentemente aos homens por meio da alimentação. Se queimados, poluem o ar, e também contaminam pelo contato direto, causando doenças graves. homens por meio da alimentação. Se queimados, poluem o ar, e também contaminam pelo contato direto, causando doenças graves. ç g O lixo é disposto de qualquer maneira e sem nenhum tratamento, o que acaba causando problemas ambientais. Os equipamentos rejeitados são, na maioria dos casos, reduzidos à condição de lixo tecnológico e têm como destino o lixo comum, chegando aos aterros sanitários ou lixões. Aproximadamente 50 milhões de toneladas de lixo tecnológico são gerados todo ano no mundo, representando 5% de todo o lixo gerado pela humanidade. As consequências para os seres humanos, animais e ambiente são graves, pois esses equipamentos possuem diversas substâncias e elementos químicos extremamente nocivos à saúde, principalmente os metais pesados. As pessoas podem se contaminar pelo contato direto, no caso de manipulação direta de placas eletrônicas e outros componentes perigosos dos eletroeletrônicos nos lixões a céu aberto. A contaminação pode também ocorrer indiretamente ou de forma acidental, pois quando um eletrônico é jogado em lixo comum e vai para um aterro sanitário, há grande possibilidade de que os componentes tóxicos contaminem o solo chegando até o lençol freático, afetando também a água. Se essa água for usada para irrigação ou para dessedentar o gado, os elementos chegarão ao homem através da alimentação. Medidas estão sendo tomadas e os fabricantes, cada vez mais, estão sendo pressionados a eliminar ou diminuir a quantidade de componentes tóxicos na fabricação dos produtos, entretanto, a produção de equipamentos inofensivos ao meio ambiente ou facilmente recicláveis ainda é um grande esforço para que aconteça. Além disso, os equipamentos obsoletos continuam chegando, em ritmo acelerado e sem controle, aos lixões. EFEITOS DO CÁDMIO, MERCÚRIO E CHUMBO O cádmio é um dos metais mais tóxicos. A principal via de absorção é a inalação em meios industriais ricos em fumos e poeiras de cádmio. Uma simples exposição a elevadas concentrações de óxido de cádmio pode causar graves irritações pulmonares ou mesmo a morte. O mercúrio, à medida que ele passa ao sangue, liga-se as proteínas do plasma e nos eritrócitos distribuindo-se pelos tecidos concentrando-se nos rins, fígado e sangue, medula óssea, parede intestinal, parte superior dos aparelhos respiratório, mucosa bucal, glândulas salivares, cérebro, ossos e pulmões. É um tóxico celular geral, provocando desintegração de tecidos. O chumbo é um metal pesado extremamente tóxico e que chega até os seres humanos principalmente pelo ar, água e cadeia alimentar, de forma acumulativa. Os seus efeitos tóxicos, entre outros, incluem sintomas como náusea, perda da coordenação, hiperatividade, confusão mental e perda de memória. Em casos mais severos, a incorporação deste elemento químico pode levar a pessoa ao estado de coma e à morte. Fonte: MANIFESTZINE, 2010 As baterias dos celulares são hoje um problema ambiental. Contendo resíduos perigosos e compostas de metais pesados altamente tóxicos e não-biodegradáveis, como cádmio, chumbo e mercúrio, depois de utilizadas, a maioria é jogada em lixos comuns e vai para aterros sanitários ou lixões a céu aberto. A forma como são eliminados e o consequente vazamento de seus componentes tóxicos contaminam o solo, os cursos d’água e o lençol freático, atingindo a flora e a fauna das regiões circunvizinhas. Através da cadeia alimentar essas substâncias chegam de forma acumulada aos seres humanos. Conforme apresentado no quadro 2. 4. ESTRATÉGIAS DE GESTÃO NO DESTINO DO LIXO TECNOLÓGICO Um estudo sobre estratégias de gestão do lixo tecnológico é apresentado por Arantes (1994), onde é mostrada a estratégia de gestão como um conjunto de conceitos e técnicas que auxiliam a administração a definir os procedimentos e os métodos para execução das atividades, a fixar e compartilhar os papéis e as responsabilidades entre a equipe, a promover as relações e o entendimento comum. Ele define claramente a diferença entre Administração e Estratégia de Gestão, e a situar este como um elemento útil e necessário para que a Administração execute sua tarefa empresarial. A Estratégia de Gestão é um conjunto de ferramentas que auxiliam o sistema a executar seus procedimentos de forma eficiente e eficaz (CHIAVENATO, 2000). As estratégias de gestão propostas neste trabalho se estrutura num conjunto de procedimentos compondo etapas estruturadas, objetivando o tratamento adequado de lixo tecnológico, de modo a evitar impacto no meio ambiente, preservando o ser humano e a disponibilidade de recursos naturais. O fluxograma a seguir, traz estratégias de tratamento do lixo tecnológico na figura 1: O fluxograma a seguir, traz estratégias de tratamento do lixo tecnológico na Figura 1: Fluxograma Estratégia de Gestão do destino do Lixo Tecnológico Fonte: Beiriz 2005 Figura 1: Fluxograma Estratégia de Gestão do destino do Lixo Tecnológico Fonte: Beiriz, 2005. Analisando o ciclo de vida útil do EEE constata-se que cada vez mais são encurtados o tempo de uso, sendo esses rapidamente substituídos por novos modelos para poder atender às demandas da sociedade mundial. Tem-se como consequência uma maior geração de resíduos por parte do mercado. Gera-se um consenso para lidar com o problema de lixo tecnológico, adotando processos de logística reversa. A seguir será apresentado um conjunto de estratégias de gestão de destino do lixo eletrônico: Figura 1: Fluxograma Estratégia de Gestão do destino do Lixo Tecnológico Figura 1: Fluxograma Estratégia de Gestão do destino do Lixo Tecnológico Fonte: Beiriz, 2005. Analisando o ciclo de vida útil do EEE constata-se que cada vez mais são encurtados o tempo de uso, sendo esses rapidamente substituídos por novos modelos para poder atender às demandas da sociedade mundial. Tem-se como consequência uma maior geração de resíduos por parte do mercado. Gera-se um consenso para lidar com o problema de lixo tecnológico, adotando processos de logística reversa. 4. ESTRATÉGIAS DE GESTÃO NO DESTINO DO LIXO TECNOLÓGICO A seguir será apresentado um conjunto de estratégias de gestão de destino do lixo eletrônico: Analisando o ciclo de vida útil do EEE constata-se que cada vez mais são encurtados o tempo de uso, sendo esses rapidamente substituídos por novos modelos para poder atender às demandas da sociedade mundial. Tem-se como consequência uma maior geração de resíduos por parte do mercado. Gera-se um consenso para lidar com o problema de lixo tecnológico, adotando processos de logística reversa. A seguir será apresentado um conjunto de estratégias de gestão de destino do lixo eletrônico: 1ª Estratégia: Agente/Gerador – os fabricantes detêm a propriedade dos equipamentos eletrônicos e ao término da vida útil estes retornam às fábricas nas quais seus componentes são reaproveitados ou reciclados, em projetos pré-concebidos para tal procedimento, dentro de uma política industrial. Atende-se a isto a necessidade de uma política voltada para o desenvolvimento de uma tecnologia de vida útil prolongada e ecológica, com uso de fontes de alimentação de maior autonomia. 2ª Estratégia: Coleta/Transporte – com a obsolescência os equipamentos começam a convergir para o desmanche/remanufatura. O consumo dos EEE se dá de maneira a construir um cenário de grande dispersão dos insumos. Nessa estratégia tem-se uma política de logística reversa que envolve a sociedade com conscientização e incentivos, os fabricantes ou importadores com a responsabilidade sobre o tratamento do lixo tecnológico, inclusive transporte e armazenagem, e o governo evoluindo junto com as mudanças com a legislação e incentivos em prol do crescimento do mercado. 3ª Estratégia: Reaproveitamento – nos Centros de Coleta, é proporcionada a convergência da segunda estratégia, onde é viabilizado o tratamento da terceira estratégia, o reaproveitamento, para atendimento de demanda de menor poder aquisitivo e inclusão da tecnologia. Com o processo de reaproveitamento pode-se recondicionar os EEE obsoletos de forma a recolocá-los no mercado. O aproveitamento de componentes é o principal foco na utilização de partes que compõe um equipamento, por sua difícil utilização, seja econômica ou técnica. p q p q p p ç j Após o tratamento, o que for reutilizável segue para realimentar o mercado, sobre a forma de peças e componentes para manutenção. Para viabilizar a inclusão digital a um custo acessível, passa a ser fundamental a preocupação em direcionar as remanufaturas. 4. ESTRATÉGIAS DE GESTÃO NO DESTINO DO LIXO TECNOLÓGICO 4ª Estratégia: Desmanche/Processamento de Insumos – ao receber os insumos, ocorre o desmanche para melhor identificação das partes dos equipamentos, logo depois separados segundo o interesse de reciclagem, seguindo para a fase de trituração, sendo quebrados em pequenos pedaços e depois moídos. Os resíduos não aptos à reciclagem, ou seja, os tóxicos são compactados e encapsulados para impedir o seu contato com o ecossistema é armazenado em aterros ecológicos. Tem-se um controle de entrada de resíduos industriais autorizados e compatíveis com as suas instalações e licenciamento ambiental do mesmo. 5ª Estratégia: Reciclagem – nesta estratégia pretende-se reciclar, ou seja, objetiva-se reaver insumos em escassez na natureza, tendo em vista os custos destes insumos encontrados na natureza e sua reserva, e visando ainda processos sustentáveis, reduzindo o impacto na geração do lixo tecnológico. Na atualidade, os processos requerem aprimoramento em escalas industriais e menor consumo de energia, sendo classificados em metalúrgicos, hidrometalúrgicos e pirometalúrgicos. 6. METODOLOGIA Para esta pesquisa, os objetivos são classificados como conceitual, qualitativa, exploratória e bibliográfica. Sua característica conceitual é demonstrada quando obtém-se respostas às questões formuladas. É uma pesquisa exploratória, na medida em que, tem por objetivo verificar em que medida a obra consultada interessa à pesquisa (GIL, 2002). Trata-se basicamente de uma pesquisa bibliográfica. Por se tratar de um estudo qualitativo, a identificação dos dados e informações foi adquirida a partir do estudo no contexto a ser desenvolvido, permitindo focalizar com maior precisão as questões a serem investigadas e formular mais as respostas (VERGARA, 1997). A pesquisa de campo foi baseada no método Pesquisa-Ação, como cita Thiollent (1985) é um tipo de pesquisa social que é concebida e realizada em estreita associação entre os pesquisadores e os participantes. 5. A IMPLANTAÇÃO DE UM ECOPONTO NA UNIGRANRIO A ideia de implantação de um Ecoponto para a coleta de materiais eletrônicos – microcomputadores e celulares – em um campus universitário da Universidade Unigranrio, no município de Magé, RJ, surgiu a partir da construção do trabalho de conclusão de curso. O Ecoponto em questão consiste num espaço com containers individualizados para o recolhimento de computadores e celulares. O projeto visa contribuir para a conscientização da população mageense, ensinando-os que o lixo tecnológico não deve ser jogado na rua entre outros locais, mas sim numa estação de tratamento adequado. O Ecoponto em questão consiste num espaço com containers individualizados para o recolhimento de computadores e celulares. O projeto visa contribuir para a conscientização da população mageense, ensinando-os que o lixo tecnológico não deve ser jogado na rua entre outros locais, mas sim numa estação de tratamento adequado. 7. APRESENTAÇÃO DO PROCESSO DE PESQUISA A pesquisa de campo foi estruturada a partir dos seguintes passos, como ilustra a linha do tempo na figura 2: Figura 2: Linha do Tempo Fonte: Próprio autor, 2014. Procurar local dentro da Universidade para implantação do Eco- Ponto; 1º Passo Montagem de formulário contendo nome, matrícula, curso e quantidade doada de lixo eletrônico; 2º Passo Criar tabela com horas complementares, que serão dadas aos alunos pela coordenação da Universidade em troca das doações de lixo eletrônico; 3º Passo Montagem de banners para colocar no Eco- Ponto; 4º Passo Passar nas salas de aula avisando os alunos que estávamos recolhendo lixo eletrônico em troca os mesmos receberiam horas complementares; 5º Passo Selecionar empresas para o destino do lixo eletrônico; 7º Passo Acionar a coordenação para transformar a pesquisa em projeto de extensão; 6º Passo Encaminhar para empresa todo lixo eletrônico contabilizado; 9º Passo Contabilizar todo material arrecadado; 8º Passo Fazer a análise e discussão dos dados obtidos. 10º Passo Figura 2: Linha do Tempo Criar tabela com horas complementares, que serão dadas aos alunos pela coordenação da Universidade em troca das doações de lixo eletrônico; Procurar local dentro da Universidade para implantação do Eco- Ponto; Selecionar empresas para o destino do lixo eletrônico; Passar nas salas de aula avisando os alunos que estávamos recolhendo lixo eletrônico em troca os mesmos receberiam horas complementares; Encaminhar para empresa todo lixo eletrônico contabilizado; 3º Passo 7º Passo 1º Passo 5º Passo 9º Passo 3º Passo 6º Passo 2º Passo 8º Passo Contabilizar todo material arrecadado; Montagem de banners para colocar no Eco- Ponto; Acionar a coordenação para transformar a pesquisa em projeto de extensão; Fazer a análise e discussão dos dados obtidos. Montagem de formulário contendo nome, matrícula, curso e quantidade doada de lixo eletrônico; Fonte: Próprio autor, 2014. 8. ANÁLISE E DISCUSSÃO DOS DADOS OBTIDOS Concluídos todos os passos estabelecidos para a pesquisa de campo, foram transcritas todas as informações obtidas, os dados coletados foram agrupados de modo a orientar a elaboração de tabelas, gráficos e análises que visam fornecer os subsídios necessários para atingir os objetivos deste estudo. Para colocar em prática a implantação do Eco-Ponto que “são estações de entrega voluntária de inservíveis, ou seja, de descarte de lixo de maneira totalmente gratuita e legalizada”, foi discutida a aplicação do método Pesquisa-Ação tendo coparticipação da Universidade na criação da estrutura do Eco-Ponto (THIOLLENT, 1985). A localização do Eco-Ponto teria que ser um local onde todos passassem sempre todos os dias, de forma que não deixassem de ver, lembrar, perguntar e participar. Por isso, foi escolhido no pátio da Universidade. Para iniciar a parte prática foi necessário pensar em como seria armazenado todos os dados obtidos. Desta forma, foram criados formulários para mostrar aos alunos a quantidade de horas complementares e formulário para preenchimento do material coletado, veja: quadro 3, 4 e tabela 1. Quadro 3: Formulário para Cadastrar as Doações de Lixo Eletrônico dos Alunos Formulário de Doações de Lixo Eletrônico Nome do Aluno: Curso: Matrícula: CPU Celular Smartphone Notebook Monitor Mouse Teclado Placa-mãe Baterias Memória Processador Fonte: Elaboração Própria, 2014. Quadro 4: Exemplo de Formulário de Doações Preenchido Formulário de Doações de Lixo Eletrônico Nome do Aluno: Curso: Matrícula: CPU Celular Smartphone Notebook Monitor Mouse Teclado Placa-mãe Baterias Memória Processador 1 1 5 3 Fonte: Elaboração Própria, 2014. Tabela 1: Horas Complementares Tabela de Horas Complementares CPU e Notebook 15 horas complementares Celular e Smartphone 10 horas complementares Monitor, Mouse, Teclado, Placa-mãe, Memória, Processador e Baterias 5 horas complementares Fonte: Elaboração Própria, 2014. Quadro 3: Formulário para Cadastrar as Doações de Lixo Eletrônico dos Alunos Quadro 4: Exemplo de Formulário de Doações Preenchido Quadro 4: Exemplo de Formulário de Doações Preenchido Formulário de Doações de Lixo Eletrônico Foi criado também um banner para fazer a propaganda que para (KOTLER & KELLER, 2006) “é uma tarefa específica de comunicação e um nível de sucesso a ser atingido em meio a determinado público, em um prazo estabelecido”. Conforme figura 3. A empresa que nos ajudou, recolhendo todo o lixo coletado, tem sua Razão Social como: Perterson Reciclagem em Geral. Essa empresa recebe os compostos eletrônicos e envia para outras empresas que trabalham com partes de computadores e celulares. 8. ANÁLISE E DISCUSSÃO DOS DADOS OBTIDOS A empresa que nos ajudou, recolhendo todo o lixo coletado, tem sua Razão Social como: Perterson Reciclagem em Geral. Essa empresa recebe os compostos eletrônicos e envia para outras empresas que trabalham com partes de computadores e celulares. Figura 3: Banner Eco-Ponto. Fonte: Elaboração Própria, 2014. Fonte: Elaboração Própria, 2014. Antes de enviar todo o material para a empresa escolhida, foi feita a contagem de todo material arrecadado, como é demonstrado na tabela 2 e no gráfico 1 a seguir. Tabela 2: Total do Material Coletado TOTAIS ITENS UNIDADES CPU 24 Notebook 5 Celular 755 Smartphone 204 Monitor 38 Mouse 153 Teclado 135 Placa-mãe 77 Memória 170 Processador 63 Baterias 1064 Fonte: Elaboração Própria 2014 Tabela 2: Total do Material Coletado TOTAIS ITENS UNIDADES CPU 24 Notebook 5 Celular 755 Smartphone 204 Monitor 38 Mouse 153 Teclado 135 Placa-mãe 77 Memória 170 Processador 63 Baterias 1064 Fonte: Elaboração Própria, 2014. Gráfico 1: Total do Material Coletado Fonte: Elaboração Própria, 2014. Gráfico 1: Total do Material Coletado G á co : ota do ate a Co etado Fonte: Elaboração Própria, 2014. Fonte: Elaboração Própria, 2014. Fonte: Elaboração Própria, 2014. O recolhimento do material eletrônico foi feito por todos os componentes do grupo. Cada dia da semana ficou uma dupla envolvida na arrecadação de material. A contagem foi feita por todos os componentes. Esse projeto foi uma experiência muito importante em nossas vidas, pois tivemos a chance de constatar que toda a parte teórica se transformou em realidade. E que tudo que foi dito e pesquisado para o artigo é uma verdade que todos vivemos, resultado da metodologia de Pesquisa-Ação (THIOLLENT, 1985). A sociedade tem descartado seus equipamentos eletrônicos com muita facilidade em locais inadequados, e isso traz para o meio ambiente graves prejuízos, que só um processo de conscientização, de empresas e instituições comprometidas com o descarte ecologicamente correto do lixo eletrônico pode fazer toda a diferença. 9. CONCLUSÃO O crescente interesse das organizações em introduzir a Logística Reversa em vários segmentos foi a motivação para tratar do tema do lixo tecnológico e seu significado e tendência na Era Digital, além das transformações que os dispositivos eletrônicos têm na vida diária de todos em uma sociedade. O levantamento do problema de pesquisa estruturou os insumos tecnológicos mais utilizados e sua posição atual no mercado mundial, visando propiciar uma proposta de estratégia de gestão do lixo tecnológico. O levantamento do problema de pesquisa estruturou os insumos tecnológicos mais utilizados e sua posição atual no mercado mundial, visando propiciar uma proposta de estratégia de gestão do lixo tecnológico. Com a estratégia de gestão formulada, entende-se que deverá ser usada no sentido de resolver o descarte de lixo tecnológico de forma ecologicamente correta diminuindo e evitando a contaminação do meio ambiente com consequências positivas para o ser humano, viabilizando uma indústria lucrativa de reciclagem e aproveitamento de matéria-prima. Portanto, é necessário o aperfeiçoamento quanto à legislação nacional atual, estabelecendo que os agentes responsáveis pelo recolhimento do lixo tecnológico, criem incentivos para o agente gerador, melhorando a rede de coleta, não existindo empecilhos legais no transporte do lixo tecnológico, definindo a estratégia, de forma que não ofereça riscos à sociedade. A criação do Ecoponto incorporará na Universidade e na população mageense uma política de sensibilização a cerca da importância da adoção de procedimentos de controle e monitoração, criando-se uma cultura de preocupação no destino de produtos tecnológicos ao final de sua vida útil. A todos envolvidos neste trabalho nosso agradecimento deixando claro que se cada um fizer a sua parte, teremos uma sociedade cada vez melhor! BRASIL. Projeto de Lei nº 1991, 2007, art. 7º inciso XII, p.3). BEIRIZ, Fernando Antônio Santos. Gestão Ecológica de Resíduos Eletrônicos. Dissertação (Mestrado em Sistemas de Gestão) – Universidade Federal Fluminense, Niterói, 2005. CHIAVENATO, Idalberto. Introdução à teoria geral da administração. 6. ed. Rio de Janeiro: Campus, 2000. CHIAVENATO, Idalberto. Introdução à teoria geral da administração. 6. ed. Rio de Janeiro: Campus, 2000. FRANKE, Carsten. Tratamento de lixo tecnológico no Brasil e na União Europeia. Ambiente Brasil, São Paulo, 2004. GIL, Antônio Carlos. Como elaborar projetos de pesquisa. 4. ed. São Paulo: Atlas, 2002, p. 18. 10. REFERÊNCIAS BIBLIOGRÁFICAS ARANTES, Nélio. Sistemas de gestão empresarial. São Paulo: Atlas, 1994 BEIRIZ, Fernando Antônio Santos. Gestão Ecológica de Resíduos Eletrônicos. Dissertação (Mestrado em Sistemas de Gestão) – Universidade Federal Fluminense, Niterói, 2005. BRASIL. Projeto de Lei nº 1991, 2007, art. 7º inciso XII, p.3). GIL, Antônio Carlos. Como elaborar projetos de pesquisa. 4. ed. São Paulo: Atlas, 2002, p. 18. LEI FEDERAL 12.305/2010 – Artigo 33, incisos I, II, III, IV, V, VI. LEI FEDERAL 12.305/2010 – Artigo 33, incisos I, II, III, IV, V, VI. KOTLER, Philip; KELLER, K. L. Administração de Marketing. 12. ed. São Paulo: Pearson Prentice Hall, 2006. LEITE, P. R. (2003) Logística Reversa: meio ambiente e competitividade. São Paulo: Pearson Prentice Hall. MANIFESTZINE. Lixo Eletrônico. Disponível em: http://manifestzine.blogspot.com.br. Acesso em: 01 de julho de 2010. ização das Nações Unidas. ONU e o Meio Ambiente, 2002. ONU – Organização das Nações Unidas. ONU e o Meio Ambiente, 2002. SCHNEIDER, Vania Elizabeth. Manual de gerenciamento de resíduos sólidos em serviços de saúde. 2. ed. rev. e ampl. Caxias do Sul, RS: EDUCS, 2004. THIOLLENT, M. (1997). Pesquisa-ação nas organizações. São Paulo: Atlas. VERGARA, Sylvia Constante. Projetos e Relatórios de Pesquisa em Administração. 13ª ed. São Paulo: Atlas, 2011.
https://openalex.org/W2479269165	https://epub.ub.uni-muenchen.de/37675/1/10.3389_fnins.2016.00356.pdf	English	null	AAV Vectors for FRET-Based Analysis of Protein-Protein Interactions in Photoreceptor Outer Segments	Frontiers in neuroscience	2,016	cc-by	11,305	Edited by: Hildegard Büning, University of Cologne, Germany Edited by: Hildegard Büning, University of Cologne, Germany Reviewed by: Peter Christian Kloehn, University College London, UK Jiajie Diao, University of Cincinnati, USA Correspondence: Elvir Becirovic elvir.becirovic@cup.uni-muenchen.de Stylianos Michalakis michalakis@lmu.de Reviewed by: Peter Christian Kloehn, University College London, UK Jiajie Diao, University of Cincinnati, USA Correspondence: Elvir Becirovic elvir.becirovic@cup.uni-muenchen.de Stylianos Michalakis michalakis@lmu.de Correspondence: Elvir Becirovic elvir.becirovic@cup.uni-muenchen.de Stylianos Michalakis michalakis@lmu.de Keywords: ﬂuorescence resonance energy transfer, FRET, adeno-associated viral vectors, AAV, protein-protein interaction, photoreceptor, outer segment Specialty section: This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience Specialty section: This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience Received: 20 April 2016 Accepted: 14 July 2016 Published: 28 July 2016 Specialty section: This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience PROTOCOLS published: 28 July 2016 doi: 10.3389/fnins.2016.00356 PROTOCOLS AAV Vectors for FRET-Based Analysis of Protein-Protein Interactions in Photoreceptor Outer Segments Elvir Becirovic 1, 2, Sybille Böhm 1, 2, Ong N. P. Nguyen 1, 2, Lisa M. Riedmayr 1, 2, Verena Hammelmann 1, 2, Christian Schön 1, 2, Elisabeth S. Butz 1, 2, Christian Wahl-Schott 1, 2, Martin Biel 1, 2 and Stylianos Michalakis 1, 2* 1 Department of Pharmacy – Center for Integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität München, Munich, Germany, 2 Department of Pharmacy – Center for Drug Research, Ludwig-Maximilians-Universität München, Munich, Germany Fluorescence resonance energy transfer (FRET) is a powerful method for the detection and quantiﬁcation of stationary and dynamic protein-protein interactions. Technical limitations have hampered systematic in vivo FRET experiments to study protein-protein interactions in their native environment. Here, we describe a rapid and robust protocol that combines adeno-associated virus (AAV) vector-mediated in vivo delivery of genetically encoded FRET partners with ex vivo FRET measurements. The method was established on acutely isolated outer segments of murine rod and cone photoreceptors and relies on the high co-transduction efﬁciency of retinal photoreceptors by co-delivered AAV vectors. The procedure can be used for the systematic analysis of protein-protein interactions of wild type or mutant outer segment proteins in their native environment. Conclusively, our protocol can help to characterize the physiological and pathophysiological relevance of photoreceptor speciﬁc proteins and, in principle, should also be transferable to other cell types. INTRODUCTION The outer segments (OS) of rod and cone photoreceptors are highly specialized cilia harboring all proteins involved in light-induced phototransduction (Arshavsky and Burns, 2012). Mutations in genes encoding these proteins lead to untreatable diseases that severely impair rod and/or cone structure and/or functionality (Berger et al., 2010). The majority of these genes encode proteins that are organized in large macromolecular complexes assembled by networks of homomeric and/or heteromeric protein-protein interactions (Roepman and Wolfrum, 2007). To decipher pathomechanisms underlying the retinal disorders and to develop appropriate treatments, the identiﬁcation and characterization of these networks is inevitable. So far, the vast majority of OS protein-protein interaction studies in photoreceptors were performed using classical biochemical techniques (Goldberg et al., 1995; Loewen and Molday, 2000; Poetsch et al., 2001; Jastrzebska et al., 2004; Michalakis et al., 2011; Knepp et al., 2012). However, the biochemical techniques have several limitations: (i) They strongly rely on the availability of antibodies with high aﬃnity and speciﬁcity, (ii) they do not necessarily reﬂect “true” interactions since, due to the tissue homogenization, some Received: 20 April 2016 Accepted: 14 July 2016 Published: 28 July 2016 Keywords: ﬂuorescence resonance energy transfer, FRET, adeno-associated viral vectors, AAV, protein-protein interaction, photoreceptor, outer segment Citation: Becirovic E, Böhm S, Nguyen ONP, Riedmayr LM, Hammelmann V, Schön C, Butz ES, Wahl-Schott C, Biel M and Michalakis S (2016) AAV Vectors for FRET-Based Analysis of Protein-Protein Interactions in Photoreceptor Outer Segments. Front. Neurosci. 10:356. doi: 10.3389/fnins.2016.00356 July 2016 \| Volume 10 \| Article 356 Frontiers in Neuroscience \| www.frontiersin.org AAVs for FRET in Photoreceptors Becirovic et al. that yields high titers of approx. 1013 vector genomes (vg) per ml. For FRET measurements, 1 × 109 total vg of each construct is delivered into the subretinal space of anesthetized mice. For co-transduction experiments of two titer-matched constructs a total number of 2 × 109 total vg is injected. The co-transduction eﬃciency was up to 90% in rods and up to 30% in cones after co-delivery of mixtures of two rAAVs. The isolation of rod or cone OS was performed 10–14 days post-injection using a modiﬁed version of the mechanical share method (Mitchell et al., 2009). Three cube FRET measurements were carried out using a slightly modiﬁed version of the protocol described previously (Erickson et al., 2003; Shaltiel et al., 2012; Ben Johny et al., 2013). The isolated OS were stable for several hours after isolation, which in principle also allows for dynamic FRET measurements of single interactions. proteins may randomly interact even though they are spatially separated in vivo, (iii) they do not allow for dynamic measurements, and (iv) exact quantiﬁcation of interactions is rather diﬃcult as many diﬀerent technical parameters which might inﬂuence the interaction must be controlled rigorously. g g y With regard to these obstacles, FRET is superior to the biochemical approaches. One important constraint of FRET is the fact that it requires the co-expression of the ﬂuorescently tagged molecules in a given cell type. This can be easily handled in heterologous expression systems, however, in complex organisms like mammals the application of FRET has been hampered by the laborious and time consuming generation of transgenic animals. Therefore, only a few FRET-based studies were performed in vivo, most of them using FRET biosensors for analysis of spatiotemporal dynamics of small signaling molecules (Hovan et al., 2010; Hirata et al., 2012; Wen et al., 2013; Kumagai et al., 2014). Here, we describe a protocol for systematic monitoring of protein-protein interactions without the necessity of generating transgenic animals. Advantages of the Method Compared to FRET and other ex vivo measurements of protein- protein interactions, which are currently widely used, this method has several advantages: (i) It can be performed within 3 weeks including the production of speciﬁc rAAVs. The isolation of OS can be carried out within a few minutes and yields a high quantity of OS of adequate quality and purity. (ii) It allows for systematic analysis of protein-protein interactions ex vivo in a specialized small compartment with preserved native morphology. (iii) It is principally transferable to analyses of protein-protein interactions in ciliary compartments of other tissues (e.g., respiratory cilia and olfactory cilia). This is of special importance for deciphering the disease mechanisms of a large group of diseases known as ciliopathies. Citation: The technique utilizes adeno- associated virus (AAV) vectors that are valuable tools for in vivo gene transfer into brain and retinal neurons (Schön et al., 2013; Murlidharan et al., 2014; Trapani et al., 2014; Zacchigna et al., 2014). AAVs lead to long-term ectopic expression of the transgene in mammals and thus can be used as a rapid alternative to the generation of transgenic animals. We have already used AAV mediated viral gene expression in retinal neurons in our previous studies (Becirovic et al., 2014, 2016; Nguyen et al., 2016). Using this method, we were able to conﬁrm several homomeric and heteromeric protein-protein interactions in rod OS that had been previously observed using other (biochemical) methods. In addition, we also identiﬁed novel protein-protein interactions in OS of rods and cones. Furthermore, we demonstrate that FRET is applicable to quantify the eﬀects of disease associated point mutations on protein-protein interactions in these compartments. Conclusively, we show that our FRET protocol enables robust qualitative and quantitative measurements of protein-protein interactions in small cellular compartments like photoreceptor OS. The protocol allows for rapid and robust measurements of sensitized acceptor emission FRET on isolated virally transduced murine photoreceptor OS. The complete procedure can be performed within 3 weeks on wild type animals and requires only limited expertise and special equipment (Figure 1). We show that this method is suitable for the detection and quantiﬁcation of protein-protein interactions of proteins involved in the pathogenesis of hereditary degenerative retinal diseases. We propose the application of this method for FRET-based protein-protein interaction studies in other primary cilia of many cell types to study the pathogenesis of other ciliopathies. Frontiers in Neuroscience \| www.frontiersin.org (viii) In principle, it can be adapted for the establishment of FRET measurements on retinas of living animals. (viii) In principle, it can be adapted for the establishment of FRET measurements on retinas of living animals. citrine-tagged proteins could have toxic eﬀects that inﬂuence photoreceptor function and morphology. Therefore, to exclude any toxic eﬀects, we suggest using histological analysis of retinal cross sections prior to the actual FRET measurements for each construct. (ix) Due to its easy handling our protocol should also be transferable to other tissues or organisms. (ix) Due to its easy handling our protocol should also be transferable to other tissues or organisms. (iv) Experimental Procedure The genes of interest encoding each of the protein-protein interaction partners are C- and/or N-terminally fused to the genes encoding citrine or cerulean, two FRET-optimized derivatives of YFP and CFP, respectively (Griesbeck et al., 2001; Rizzo et al., 2004). Each of these constructs is then cloned into the multiple cloning site of a rAAV cis-plasmid containing the rod photoreceptor speciﬁc human rhodopsin (hRHO) (Allocca et al., 2007) or murine short wavelength opsin (mSWS) promoter (Michalakis et al., 2010a), a Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and a bovine growth hormone polyadenylation signal (BGH pA). Subsequently, each rAAV cis plasmid is co-transfected with the trans (AAV helper and Adeno helper) plasmids into HEK293T cells using triple Ca2+ phosphate transfection. rAAV particles are then puriﬁed in a multistep procedure (iv) As isolated OS are stable for up to 6 h, the method can also be used for dynamic FRET measurements. (v) The protocol was established in wild type mice. Thus, it allows for systematic analysis of disease mechanisms of single disease associated point mutations without the time consuming generation of transgenic animals. (vi) It can in principle be used to determine the relative binding aﬃnities of single interactions. (vii) The co-transduction eﬃciency for two constructs was up to 90% for rod OS and up to 30% for cone OS. Hence, a suﬃcient number of OS co-expressing both proteins of interest can be isolated from a single animal. (vii) The co-transduction eﬃciency for two constructs was up to 90% for rod OS and up to 30% for cone OS. Hence, a suﬃcient number of OS co-expressing both proteins of interest can be isolated from a single animal. July 2016 \| Volume 10 \| Article 356 Frontiers in Neuroscience \| www.frontiersin.org 2 AAVs for FRET in Photoreceptors Becirovic et al. FIGURE 1 \| Schematic overview of the main steps of the procedure. AAV vectors encoding the FRET fusion proteins are produced in HEK293T cells (day 1–10). Single AAV vectors and combinations of AAVs encoding the FRET partners are delivered into the subretinal space of wildtype mice (day 11). Ten days later, OS are isolated for ex vivo FRET measurements. FIGURE 1 \| Schematic overview of the main steps of the procedure. AAV vectors encoding the FRET fusion proteins are produced in HEK293T cells (day 1–10). Limitations of the Method (iv) In the case of three cube FRET, in addition to the simultaneous delivery of two FRET partners, injections of single FRET constructs are also necessary. They serve as bleed-through and crosstalk controls for the determination of the FRET eﬃciencies. There are a few limitations of the method, which can be overcome by appropriate techniques or conditions: (i) With respect to the packaging capacity of rAAVs (5 kb) (Trapani et al., 2014), this method seems less suitable for analysis of protein-protein interactions of large proteins. However, there are several approaches showing that larger transcripts can also be packaged into rAAVs, even though with lower eﬃciencies (Trapani et al., 2014). (v) Overexpression of the tagged proteins by strong promoters might result in an artefactual FRET signal. To exclude this possibility, additional controls might be necessary. In this context, the most eligible control is to use point mutations within the protein-protein interaction site of one (or both) FRET partners. If the protein interaction interface is not known, artefactual FRET signal can be excluded by overexpressing other proteins not expected to interact with the respective FRET partner. (ii) The endogenous wild type proteins in the OS of transduced photoreceptors interfere with the labeled constructs and lead to overall lower FRET eﬃciencies than expected from in vitro experiments (incomplete labeling) (Becirovic et al., 2014). This limitation should be of relevance only if weak interactions are expected. This issue could be overcome by using appropriate single or double knock- out animals, which lack expression of the endogenous (unlabeled) proteins of interest. (vi) Bulky ﬂuorescent tags could possibly hinder proper traﬃcking or localization of the fusion protein (Feilmeier et al., 2000). In addition, they might inﬂuence the proper folding and the stability of the protein. In turn, incorrect folding may also aﬀect the ﬂuorophore itself and lead to little or no ﬂuorescence (Waldo et al., 1999). If these problems occur, they can be overcome by varying the position of the ﬂuorescent tag within the protein or by placing a short linker sequence between tag and protein. (iii) So far, we found no short or long term toxic eﬀects of the ﬂuorophores on the structural integrity of the retina, which is consistent with previous studies (Bennett et al., 1999; Becirovic et al., 2014). Frontiers in Neuroscience \| www.frontiersin.org Experimental Procedure Single AAV vectors and combinations of AAVs encoding the FRET partners are delivered into the subretinal space of wildtype mice (day 11). Ten days later, OS are isolated for ex vivo FRET measurements. (viii) In principle, it can be adapted for the establishment of FRET measurements on retinas of living animals. Limitations of the Method However, we cannot exclude that large amounts of speciﬁc cerulean- or July 2016 \| Volume 10 \| Article 356 Frontiers in Neuroscience \| www.frontiersin.org 3 AAVs for FRET in Photoreceptors Becirovic et al. Animals • Glass pipettes All procedures were performed on P14-21 C57BL/6J mice with permission from local authorities (District Government of Upper Bavaria). Anesthesia was performed by subcutaneous injection of ketamine (40 mg/kg body weight) and xylazine (20 mg/kg body weight). Euthanasia was performed by cervical dislocation. p p • Peristaltic pump MINIPULS 3 (Gilson, cat. no. n/a) • Peristaltic pump MINIPULS 3 (Gilson, cat. no. n/a) • Quick-Seal Cordless Tube Topper kit, 50 Hz (Beckman, cat. no. 358313) • Quick-Seal Cordless Tube Topper kit, 50 Hz (Beckman, cat. no. 358313) • Beckman Coulter J2-MC high speed centrifuge (Beckman, cat. no. n/a) • JA-10 Rotor, Fixed Angle, Aluminum, 6 × 500 ml, 10,000 rpm, 17,700 × g (Beckman, cat. no. 369687) MATERIALS AND EQUIPMENT • Quick-Seal polypropylene tubes, 39 ml (Beckman, cat. no. 342141) • Quick-Seal polypropylene tubes, 39 ml (Beckman, cat. no. 342141) Reagents • Beckman Coulter Optima LE-80K ultracentrifuge (Beckman, cat. no. n/a) • Beckman Coulter Optima LE-80K ultracentrifuge (Beckman, cat. no. n/a) g • Sodium chloride (NaCl; VWR, cat. no. 27810.364) • Sodium chloride (NaCl; VWR, cat. no. 27810.364) • 70 Ti Rotor, Fixed Angle, Titanium, 8 × 39 mm, 70,000 rpm, • 70 Ti Rotor, Fixed Angle, Titanium, 8 × 39 mm, 70,000 rpm, 504,000 × g (Beckman, cat. no. 337922) • 70 Ti Rotor, Fixed Angle, Titanium, 8 × 39 m 504,000 × g (Beckman, cat. no. 337922) • Potassium chloride (KCl; Carl Roth, cat. no. 6781.1) • Potassium chloride (KCl; Carl Roth, cat. no. 6781.1) 504,000 × g (Beckman, cat. no. 337922) • Magnesium chloride hexahydrate (MgCl2 × 6H2O; Carl Roth, cat. no. 2189.1) • Magnesium chloride hexahydrate (MgCl2 × 6H2O; Carl Roth, cat. no. 2189.1) • Syringe needle, 21-gauge • Syringe, 5 ml • Calcium chloride dihydrate (CaCl2 × 2H2O; VWR, cat. no. 22317.230) • Sterile syringe ﬁlters with acrylic housing, 0.2 µm, cellulose acetate (VWR, cat. no. 28145-477) • Sterile syringe ﬁlters with acrylic housing, 0.2 µm, cellulose acetate (VWR, cat. no. 28145-477) • Glucose monohydrate (Carl Roth, cat. no. 6887.1) • Glucose monohydrate (Carl Roth, cat. no. 6887.1) • HiTrap Q FF sepharose column, 5 ml (GE Healthcare, cat. no. 17-5156-01) • HiTrap Q FF sepharose column, 5 ml (GE Healthcare, cat. no. 17-5156-01) • HEPES sodium salt (VWR, cat. no. A16516.22) • HEPES sodium salt (VWR, cat. no. A16516.22) • Sodium hydroxide, 5 M (NaOH; VWR, 28244.295 • Sodium hydroxide, 5 M (NaOH; VWR, 28244.295) • Superloop, 50 ml (GE Healthcare, cat. no. 19-7850-01) • Superloop, 50 ml (GE Healthcare, cat. no. 19-7850-01) • KAPA SYBR FAST Universal x2 qPCR MasterMix (peqlab, cat. no. 07-KK4600-01) • Poly-L-lysine, 1 mg/ml (Sigma, cat. no. P2636) • Polybrene/Hexadimethrine bromide (Sigma cat no 107689) • KAPA SYBR FAST Universal x2 qPCR MasterMix (peqlab, cat. no. 07-KK4600-01) • KAPA SYBR FAST Universal x2 qPCR MasterMix (peqlab, cat. no. 07-KK4600-01) • Poly-L-lysine, 1 mg/ml (Sigma, cat. no. P2636) • ÄKTAprime plus chromatography system (GE Healthcare, cat. no. 11-0013-13) • PrimeView 5.31 software (GE Healthcare, cat. no. 28-9949-61) • ÄKTAprime plus chromatography system (GE Healthcare, cat. no. 11-0013-13) • PrimeView 5.31 software (GE Healthcare, cat. no. 28-9949-61) • Poly-L-lysine, 1 mg/ml (Sigma, cat. no. P2636) • Polybrene/Hexadimethrine bromide (Sigma, cat. no. 107689) • Amicon Ultra-4 centrifugal ﬁlter units, 100 kDa (Millipore, cat. Reagents no. UFC810024) • Amicon Ultra-4 centrifugal ﬁlter units, 100 kDa (Millipore, cat. no. UFC810024) • Dextran (Sigma, cat. no. 95771) • Dextran (Sigma, cat. no. 95771) • BES sodium salt (Sigma, cat. no. B2891) • BES sodium salt (Sigma, cat. no. B2891) • LightCycler 480 multiwell plate 96 (Roche, cat. no. 04729692001) • LightCycler 480 multiwell plate 96 (Roche, cat. no. 04729692001) • AAV cis (for cloning of the expression cassette) and trans plasmids (encoding for Cap, Rep and adenoviral helper genes) can be obtained from various sources including Penn Vector Core (http://www.med.upenn.edu/gtp/vectorcore) or from UNC Vector Core (http://www.med.unc.edu/genetherapy/ vectorcore,seealsoRef. Grieger et al., 2006) • AAV cis (for cloning of the expression cassette) and trans plasmids (encoding for Cap, Rep and adenoviral helper genes) can be obtained from various sources including Penn Vector Core (http://www.med.upenn.edu/gtp/vectorcore) or from UNC Vector Core (http://www.med.unc.edu/genetherapy/ vectorcore,seealsoRef. Grieger et al., 2006) • LightCycler 480 Sealing Foil (Roche, cat. no. 04729757001) • LightCycler 480 Sealing Foil (Roche, cat. no. 04729757001) • LightCycler 480 Instrument II real-time PCR ampliﬁcation and detection instrument (Roche, cat. no. 05015278001) • LightCycler 480 Instrument II real-time PCR ampliﬁcation and detection instrument (Roche, cat. no. 05015278001) • NanoFil syringe, 10 µl (World Precision Instruments, cat. no. NANOFIL) • NanoFil syringe, 10 µl (World Precision Instruments, cat. no. NANOFIL) • Benzonase (VWR, cat. no. 1.01695.0001) • Benzonase (VWR, cat. no. 1.01695.0001) • NanoFil 34-gauge beveled needle (World Precision Instruments, cat. no. NF34BV-2) • NanoFil 34-gauge beveled needle (World Precision Instruments, cat. no. NF34BV-2) • OptiPrep (Progen, cat. no. 1114542) • Disodium hydrogen phosphate (Na2HPO4; Sigma, cat. no. S9763) • Nanodrop 2000c UV-Vis spectrophotometer (peqlab, ca. no. 91-ND-2000C) • Nanodrop 2000c UV-Vis spectrophotometer (peqlab, ca. no. 91-ND-2000C) • Sodium dihydrogen phosphate (NaH2PO4; Sigma, cat. no. S5011) • Dexpanthenol eye and nose salve, 5% (Bepanthen, Bayer Vital GmbH, cat. no. 1578681) • Dexpanthenol eye and nose salve, 5% (Bepanthen, Bayer Vital GmbH, cat. no. 1578681) • Tris(hydroxymethyl)aminomethane (TRIS; VWR, cat. no. 71003-490) • OPMI 1 FR pro surgical microscope (Zeiss, cat. no. n/a) • OPMI 1 FR pro surgical microscope (Zeiss, cat. no. n/a) • Gentamicin 5 mg/g and dexamethasone 0.3 mg/g eye salve (Dexamytrex, Dr. Gerhard Mann GmbH, cat. no. 2747789) • Gentamicin 5 mg/g and dexamethasone 0.3 mg/g eye salve (Dexamytrex, Dr. Gerhard Mann GmbH, cat. no. 2747789) • Phenol red (Sigma, cat. no. P3532) • Tween 20 (Sigma, cat. no. P2287). Equipment for rAAV Preparation and Subretinal Injection • Epiﬂuorescence microscope Axioplan 2 imaging (Zeiss, cat. no. n/a). • Epiﬂuorescence microscope Axioplan 2 imaging (Zeiss, cat. no. n/a). Reagents • Tween 20 (Sigma, cat. no. P2287). • Dissection stereomicroscope Stemi 2000 (Zeiss, cat. no. 495005-0022-000) • Dissection stereomicroscope Stemi 2000 (Zeiss, cat. no. 495005-0022-000) Equipment for rAAV Preparation and Subretinal Injection • Vortexer • Microcentrifuge tubes, 1.5 ml • Microcentrifuge tubes with screw cap, 1.5 ml • ibidi µ-dish, 35 mm, low (ibidi, cat. no. 80131) • Room temperature bench top centrifuge (1.5 ml tube rotor) • Petri dishes, 3 cm • Forceps • Polystyrene tubes, 50 ml • Cell culture dishes, 15 cm • Cell scraper (VWR, cat. no. 734-1111) • Liquid nitrogen Buffers and Solutions Excitation ﬁlter, emission ﬁlter, and dichroic mirror The following ﬁlter cubes are present in our microscope: A donor cube containing a cerulean excitation ﬁlter, T455lp dichroic mirror and a cerulean emission ﬁlter; an acceptor cube with a citrine excitation ﬁlter, T515lp dichroic mirror, and a citrine emission ﬁlter. Furthermore a FRET cube containing a cerulean excitation ﬁlter, T455lp dichroic mirror and a citrine emission ﬁlter. In case of using a monochromator as excitation source, the excitation ﬁlters are optional. 2x BBS transfection solution Final component concentrations are 45 mM BES, 280 mM NaCl, 1.5 mM Na2HPO4. Adjust the pH to 6.95 and sterile ﬁltrate. Prepare 50 ml aliquots and store them at 4◦C for up to 1 year. Lysis Buﬀer Final component concentrations are 150 mM NaCl, 50 mM TRIS. Adjust the pH to 8.5 and sterile ﬁltrate. Prepare freshly before use. 15% iodixanol solution Final component concentrations are 1x PBS (phosphate buﬀered saline), 1 mM MgCl2, 2.5 mM KCl, 1 M NaCl, 15% Optiprep. Add phenol red (use 1% solution) ad libitum until the solution is visibly colored. Store at 4◦C for up to 1 week. Software and data acquisition For photometric FRET measurements, we employ the three cube FRET method (Erickson et al., 2003; Shaltiel et al., 2012; Ben Johny et al., 2013) that utilizes the detection of ﬂuorescence intensities from a specimen using three ﬁlter cubes. The excitation source (DeltaRam monochromator) and appendant excitation parameters (e.g., wavelength, pulse duration) are thereby controlled via the software FelixGX, whereas the ﬁlter switch is done manually. Fluorescence intensity signals are acquired with the FelixGX software and deciphered with a custom-written MATLAB script. Subsequent analysis is performed using Excel or an adequate data processing software. 25% iodixanol solution Final component concentrations are 1x PBS, 1 mM MgCl2, 2.5 mM KCl, 25% Optiprep. Add phenol red (use 1% solution) ad libitum until the solution is visibly colored. Store at 4◦C for up to 1 week. 25% iodixanol solution Final component concentrations are 1x PBS, 1 mM MgCl2, 2.5 mM KCl, 25% Optiprep. Add phenol red (use 1% solution) ad libitum until the solution is visibly colored. Store at 4◦C for up to 1 week. 40% iodixanol solution Final component concentrations are 1x PBS, 1 mM MgCl2, 2.5 mM KCl, 1 M NaCl, 40% Optiprep. The solution remains uncolored. Store at 4◦C for up to 1 week. Equipment for Three Cube FRET Measurements • Inverted ﬂuorescent microscope (Leica, cat. no. DMI6000B) • HC PL APO 63x/1.40-0.60 oil immersion objective (Leica, cat. no. 11506349) • Cerulean ﬁlter cube, excitation: ET436/20x, Dichroic: T455lp, emission: ET480/40m, transmission rate 93–97% (Chroma Technology, cat. no. 49001) July 2016 \| Volume 10 \| Article 356 Frontiers in Neuroscience \| www.frontiersin.org Frontiers in Neuroscience \| www.frontiersin.org 4 AAVs for FRET in Photoreceptors Becirovic et al. • Citrine ﬁlter cube, excitation: ET500/20x, Dichroic: T515lp, emission: ET535/30m, transmission rate 93–97% (Chroma Technology, cat. no. 49003) • Citrine ﬁlter cube, excitation: ET500/20x, Dichroic: T515lp, emission: ET535/30m, transmission rate 93–97% (Chroma Technology, cat. no. 49003) should be coated with Poly-L-lysine to ensure complete adherence and immobilization of OS. In our hands, the use of pre-coated dishes (e.g., ibiTreat, ibidi) resulted in insuﬃcient OS adherence. • FRET ﬁlter cube, excitation: ET436/20x, Dichroic: T455lp, emission: ET535/30m, transmission rate 93–97% (Chroma Technology, cat. no. 49052) • FRET ﬁlter cube, excitation: ET436/20x, Dichroic: T455lp, emission: ET535/30m, transmission rate 93–97% (Chroma Technology, cat. no. 49052) Inverted epiﬂuorescent microscope, excitation source, and detection All measurements were performed using a Leica DMI6000B inverted ﬂuorescent microscope equipped with a motorized turret and a 63x oil objective. The microscope contains a motorized ﬁlter-wheel enabling rapid switching of ﬁlter cubes within 300 ms. Of note, the 40x oil objective is also suitable for FRET measurements on isolated OS. The ﬂuorescent intensity is detected by a photomultiplier detection system including a photomultiplier tube (Horiba). As excitation source, we use a DeltaRam monochromator containing a 150W xenon high stability lamp. The use of a conventional LED lamp is also suitable. However, it is crucial to achieve a stable uniform illumination. gy • DeltaRamX monochromator (Horiba, cat. no. n/a) • DeltaRamX monochromator (Horiba, cat. no. n/a) • D-104 B microscope photometer (Horiba, cat. no. n/a) • D-104 B microscope photometer (Horiba, cat. no. n/a) • Xenon short arc lamp type UXL-75XE (Ushio Inc. Japan, cat. no. n/a) • Xenon short arc lamp type UXL-75XE (Ushio Inc. Japan, cat. no. n/a) • PMT housing 914 (Horiba, cat. no. n/a) • R1527 photomultiplier (Horiba, cat. no. n/a) • FelixGX software (Horiba, cat. no. n/a) • MATLAB R⃝R2014b (MathWorks. Inc., cat. no. n/a) • Excel software (Microsoft Corporation, cat. no. n/a). • Excel software (Microsoft Corporation, cat. no. n/a). Buffers and Solutions 60% iodixanol solution Final component concentrations are 1 mM MgCl2, 2.5 mM KCl, 60% Optiprep. Add phenol red (use 1% solution) ad libitum until the solution is visibly colored. Store at 4◦C for up to 1 week. PBS-MK solution Final component concentrations are 1x PBS, 1 mM MgCl2, 2.5 mM KCl. Sterile ﬁltrate and store at 4◦C for up to 1 week. Production and Titer Determination of rAAVs p Buﬀer A Final component concentrations are 20 mM TRIS, 15 mM NaCl. Adjust the pH to 8.5. Store at 4◦C for up to 1 week. Transfection, Harvesting, and Lysis of HEK293T Cells The production and handling of recombinant pseudotyped AAV2-derived vectors is subject to biosafety level 1. For a detailed protocol on AAV production, see ref. (Grieger et al., 2006). 24 h before transfection, conﬂuent 15-cm-dishes of HEK293T cells were split into 15 15-cm-dishes for each construct. For calcium phosphate transfection, the components listed in Table 1 (the amounts are adjusted for transfection of 15 15-cm-dishes) were added in a 50-ml polystyrene tube. The following two equations are used to calculate the amount of pAd Helper plasmid and AAV2/8 Cap/Rep plasmid needed for the pre-transfection mix described in Table 1, respectively: Buﬀer B Final component concentration is 2.5 M NaCl. Adjust the pH to 8.5. Store at 4◦C for up to 1 week. FRET imaging solution Final component concentrations are 140 mM NaCl, 5 mM KCl, FRET imaging solution Final component concentrations are 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM glucose, 10 mM HEPES sodium salt. Adjust the pH with 5 M NaOH to 7.4. Prepare 50 ml aliquots and store them at −20◦C for up to 1 year. Store thawed aliquots at 4◦C for up to 1 week. g 2 2 g sodium salt. Adjust the pH with 5 M NaOH to 7.4. Prepare 50 ml aliquots and store them at −20◦C for up to 1 year. Store thawed aliquots at 4◦C for up to 1 week. MM = molar mass of double stranded plasmid. were ﬁlled up and accurately balanced with PBS-MK. Prior to centrifugation, the tubes were sealed with the Beckman Tube Topper. Centrifugation was performed at 361.000 × g for 1 h 45 min at 18◦C (70.000 rpm in an Optima LE-80K Beckman ultracentrifuge using a 70 Ti rotor). During centrifugation, the virus-containing fraction should accumulate in the 40% phase. After centrifugation, the tube was pierced at the top near the seal for pressurization. To collect the virus-containing phase, a 21-gauge-needle was used with a 5-ml-syringe to pierce the tube through the side at the lower end of the 40–60% interface. During this step, the open site of the needle tip should face the 40% phase. Approximately 5 ml of the 40% phase were collected. The virus- containing phase can be stored in a 15-ml-polystyrene-tube at −80◦C until proceeding with the puriﬁcation step. MM = molar mass of double stranded plasmid. While vortexing the pre-transfection mix in the polystyrene tube at maximum speed, 15 ml 2x BBS was added dropwise. The mixture was incubated at room temperature for 4–5 min and 2 ml of the transfection mix was added dropwise to each 15- cm-dish of ∼80% conﬂuent HEK293T cells. Transfected cells were then incubated at 37◦C and 5% CO2 atmosphere for 24 h. Subsequently, the medium from transfected cells was replaced and the cells were incubated at 37◦C and 10% CO2 atmosphere for additional 24 h. 48 h post transfection, the cells were harvested by scraping them from each 15-cm-dish. Cell suspensions were collected from 15 dishes in a 500-ml-centrifuge-tube. The cells were centrifuged at 2000 × g for 15 min at 4◦C (4000 rpm in a J2-MC Beckman centrifuge using a JA-10 rotor). After medium removal, the cell pellet was gently resuspended in 7.5 ml lysis buﬀer (volume for cell pellet harvested from 15 15-cm-dishes) and transferred into 50-ml-polystyrene-tubes. The cell suspension was shock-freezed in liquid nitrogen and subsequently thawed at 37◦C. Freeze-thaw cycle was repeated two additional times. The resulting cell suspension can be stored at −80◦C overnight. For puriﬁcation, the virus-containing phase was thawed on ice. HiTrap Q FF sepharose column and superloop were connected with the ÄKTAprime plus chromatography system. For collection of the puriﬁed fractions, 1.5-ml-centrifuge-tubes were loaded into the tube rack. Dropsync unit was adjusted to position 1. FRET Imaging Setup Imaging dishes We recommend the use of commercially available glass-bottomed cell-culture dishes for imaging. They July 2016 \| Volume 10 \| Article 356 Frontiers in Neuroscience \| www.frontiersin.org 5 AAVs for FRET in Photoreceptors Becirovic et al. MM = molar mass of double stranded plasmid. The HiTrap Q FF sepharose column was equilibrated with 25 ml of buﬀer A at 10 ml/min ﬂow rate. Manual run mode was selected with 1.0 ml/min ﬂow rate, 1 ml fraction size, and the measuring curves were put to starting point via autozero. Virus phase was diluted 1:1 with buﬀer A prior to application to the ÄKTAprime plus system with a 10 ml syringe to ﬁll the superloop. The virus dilution from the superloop was injected into the ÄKTAprime plus system and 1 ml fractions were collected in 1.5-ml-tubes. During this step, the UV and conductance curves should be monitored via the Äktaprime software. When the conductance curve has reached basal values, the ÄKTAprime plus system was switched to 100% buﬀer B at 10 ml/min ﬂow rate and 0 ml fraction size to purge the sepharose column from the remaining virus dilution. To wash out the remaining salt from the system, sterile, double-distilled water was used at 10 ml/min ﬂow rate. When the conductance curve reached zero, the washing step was continued for additional 5 min. All of the collected 1-ml fractions within the plateau phase of the conductance curve were pooled. The puriﬁed and pooled virus fractions can be stored in a 15-ml-polystyrene-tube at −80◦C until continuing with the virus concentration step. Gradient Centrifugation and Puriﬁcation of rAAVs Gradient Centrifugation and Puriﬁcation of rAAVs Benzonase was added to the thawed cell suspension to a ﬁnal concentration of 50 U/ml followed by an incubation at 37◦C for 30 min. Subsequently, the cells were pelleted via centrifugation at 2000 × g for 25 min at 4◦C. The virus-containing supernatant was transferred into a Beckman Quick-Seal polypropylene tube. To form the iodixanol gradient, a sterile, long glass pipette, and a Gilson MINIPULS3 pump were used. First, the virus- containing phase was underlaid with 7 ml 15% iodixanol, then with 5 ml 25% iodixanol, followed by 5 ml 40% iodixanol, and at last with 6 ml 60% iodixanol. To avoid mixing of the layers, the pump should be run at the slowest speed. Polypropylene tubes TABLE 1 \| Reagents for a pre-transfection mix of one transgene AAV construct. Reagent Amount Transgene AAV cis plasmid (Michalakis et al., 2010b; Koch et al., 2012) 270 µg pAd Helper plasmid (pAdDeltaF6 Auricchio et al., 2001) x µg AAV2/5 (Hildinger et al., 2001) or AAV2/8 (Gao et al., 2002) Rep/Cap plasmid y µg Polybrene (8 mg/ml) 15 µl Dextran (10 mg/ml) 1500 µl 2.5 M CaCl2 1500 µl Double-distilled water ad 15 ml The listed amounts are for transfection of 15 15-cm-dishes of HEK293T cells. TABLE 1 \| Reagents for a pre-transfection mix of one transgene AAV construct. Reagent Amount Transgene AAV cis plasmid (Michalakis et al., 2010b; Koch et al., 2012) 270 µg pAd Helper plasmid (pAdDeltaF6 Auricchio et al., 2001) x µg AAV2/5 (Hildinger et al., 2001) or AAV2/8 (Gao et al., 2002) Rep/Cap plasmid y µg Polybrene (8 mg/ml) 15 µl Dextran (10 mg/ml) 1500 µl 2.5 M CaCl2 1500 µl Double-distilled water ad 15 ml The listed amounts are for transfection of 15 15-cm-dishes of HEK293T cells. TABLE 1 \| Reagents for a pre-transfection mix of one transgene AAV construct. Titer Determination of rAAVs For titer determination, the virus suspension was diluted 1:500 with double-distilled water. 5 µl of virus dilution, a standard sample, and double-distilled water were used for quantitative real-time PCR according to Tables 2, 3. For standard preparation, a small fragment within the WPRE (woodchuck hepatitis virus posttranscriptional regulatory element) of the pAAV2.1 vector was ampliﬁed via PCR. The corresponding primers are listed in Table 4. The PCR product was puriﬁed after gel electrophoresis and DNA concentration was determined with a spectrophotometer (Nanodrop 2000c). The concentration of the standard for 1010 copies per 5 µl was calculated according to the following equation: Dissection of Murine Retinas and Isolation of Photoreceptor Outer Segments c pg µl = 1010 × 660 × 1012 pg mol × fragment size 6.022 × 1023 1 mol × 5µl The mouse was sacriﬁced 10 days post injection and the injected eye was proptosed by placing blunt forceps around the optic nerve close to its exit from the eye. The globe was then transected along the equator with a sharp razor blade or scalpel and the vitreous body was removed by carefully pushing it out of the incision with a thin needle. The forceps was pushed upwards to detach the retina from the optic nerve and from the pigment epithelium. The upward movement was gradually continued until the retina lay free on the forceps. The isolated retina was transferred into a petri dish ﬁlled with phosphate buﬀered saline (PBS). The expression or co-expression of ﬂuorescent fusion proteins was analyzed by an epiﬂuorescence microscope (Axioplan 2 imaging, Zeiss). As described in Figure 2A, the retina was transferred into a 1.5-ml-microcentrifuge-tube containing 100 µl PBS. In case of weak expression or in case of isolating cone OS, two (or more) retinas can be pooled at this stage c is the concentration of the standard for 1010 copies per 5 µl; 660 × 1012 pg/mol is the mean molar mass of a base pair (deoxyribosyladenosine with deoxyribosylthymidine or deoxyribosylcytidine with deoxyribosylguanosine); 6.022 × 1023/mol is the Avogadro constant; The size of the ampliﬁed WPRE fragment is given in base pairs. A 10-fold serial dilution was made to obtain standards at diﬀerent concentrations. 5 µl of a standard were used to determine the virus titer via quantitative real-time PCR. Standards were stored at −20◦C until use. Concentration of rAAVs For virus concentration, the puriﬁed virus fraction was ﬁrst thawed on ice. 4 ml of the fraction was then transferred to an Amicon centrifugal ﬁlter unit and was centrifuged at 2000 × g for 10 min at 20◦C (4000 rpm in a Beckman centrifuge using a JA-10 rotor). The ﬂow-through was discarded and the cycles of 4 ml virus fraction transfer and subsequent centrifugation were repeated until the complete virus was loaded to the column and ∼500 µl remained in the ﬁlter unit. The concentrated virus was July 2016 \| Volume 10 \| Article 356 Frontiers in Neuroscience \| www.frontiersin.org 6 AAVs for FRET in Photoreceptors Becirovic et al. was placed on a 37◦C heating plate. The eye was dilated with 1% atropine and 0.5% tropicamide solution. The eye fundus was focused with a stereomicroscope until blood vessels became clearly visible. The outer layers of the eye (sclera, choroidea, and retinal pigment epithelium) were then penetrated with the needle in a 60◦angle. When the needle was visible beneath the retina, 1 µl (containing approx. 109 rAAV particles) of virus suspension was gradually applied freehand. For the co-delivery of two constructs, a 1:1 mixture of titer matched rAAVs was used. The formation of a clearly visible injection bleb indicated a correct application to the subretinal space. The needle was slowly removed from the eye, which was subsequently treated with gentamicin 5 mg/g and dexamethasone 0.3 mg/g eye salve. The anesthetized mouse was placed under a heat lamp and was monitored until awaking from narcosis. If available, optical coherence tomography (OCT) was conducted immediately after subretinal injection to monitor the injection procedure and the degree of subretinal detachment (Mühlfriedel et al., 2013). washed with 1 ml 0.014% Tween/PBS-MK by pipetting up and down ﬁve times. The virus suspension was then centrifuged at 2000 × g at 20◦C until 100 µl of concentrated virus suspension remained in the ﬁlter unit. The supernatant was aliquoted and stored in 1.5-ml screw cap tubes. Virus suspensions were stored at −80◦C until determination of virus titer and subretinal injection. July 2016 \| Volume 10 \| Article 356 Subretinal Injection of rAAV Vectors The procedure of subretinal rAAV injection was carried out as described by (Mühlfriedel et al., 2013) with slight modiﬁcations. The procedure of subretinal rAAV injection was carried out as described by (Mühlfriedel et al., 2013) with slight modiﬁcations. The NanoFil 34-gauge beveled needle was sterilized before each experiment. The 10-µl NanoFil syringe was preloaded with the virus suspension under exclusion of air bubbles. The mouse was anesthetized via intraperitoneal injection of xylazine (20 mg/kg) and ketamine (40 mg/kg). Five percent dexpanthenol eye salve was applied to the non-injected eye and the anesthetized mouse TABLE 3 \| Program steps of quantitative real-time PCR for titer determination. Step Phase Temperature (◦C) Time Cycle numbers 1 Initial Denaturation 95 10 min 1 2 Denaturation 95 10 s 40 3 Annealing 60 5 s 4 Elongation 72 20 s 5 Final elongation 72 5 min 1 TABLE 4 \| Primer sequences for quantitative real-time PCR and standard preparation. Primer for ampliﬁcation of standard fragment Sequence (5′ to 3′) WPREqF AGTTCCGCCGTGGCAATAGG WPREqR CAAGGAGGAGAAAATGAAAGCC July 2016 \| Volume 10 \| Article 356 TABLE 3 \| Program steps of quantitative real-time PCR for titer determination. TABLE 2 \| Pipetting scheme for quantitative real-time PCR. Reagent Volume (µl) Template plasmid (50–100 ng)/standard/double-distilled water (negative control) 5,0 KAPA SYBR FAST Universal x2 qPCR MasterMix 10,0 WPREqF (10 pmol/µl) 1,0 WPREqR (10 pmol/µl) 1,0 Double-distilled water ad 20,0 4 Elongation 72 20 s 5 Final elongation 72 5 min 1 TABLE 4 \| Primer sequences for quantitative real-time PCR and standard preparation. Primer for ampliﬁcation of standard fragment Sequence (5′ to 3′) WPREqF AGTTCCGCCGTGGCAATAGG WPREqR CAAGGAGGAGAAAATGAAAGCC Frontiers in Neuroscience \| www.frontiersin.org 7 July 2016 \| Volume 10 \| Article 356 TABLE 2 \| Pipetting scheme for quantitative real-time PCR. Reagent Volume (µl) Template plasmid (50–100 ng)/standard/double-distilled water (negative control) 5,0 KAPA SYBR FAST Universal x2 qPCR MasterMix 10,0 WPREqF (10 pmol/µl) 1,0 WPREqR (10 pmol/µl) 1,0 Double-distilled water ad 20,0 5 F TABLE 4 \| preparatio Primer for fragment WPREqF WPREqR Frontiers in Neuroscience \| www.frontiersin.org 7 TABLE 2 \| Pipetting scheme for quantitative real-time PCR. Reagent Volume (µl) Template plasmid (50–100 ng)/standard/double-distilled water (negative control) 5,0 KAPA SYBR FAST Universal x2 qPCR MasterMix 10,0 WPREqF (10 pmol/µl) 1,0 WPREqR (10 pmol/µl) 1,0 Double-distilled water ad 20,0 Frontiers in Neuroscience \| www.frontiersin.org TABLE 2 \| Pipetting scheme for quantitative real-time PCR. AAVs for FRET in Photoreceptors Becirovic et al. Subretinal Injection of rAAV Vectors FIGURE 2 \| OS preparation and FRET. (A) Overview of the protocol for the OS preparation and for the subsequent FRET measurements. (B) Representative images of isolated OS at different time points after the isolation. Scale bar, 3 µm. FIGURE 2 \| OS preparation and FRET. (A) Overview of the protocol for the OS preparation and for the subsequent FRET measurements. (B) Representative images of isolated OS at different time points after the isolation. Scale bar, 3 µm. imaged as well. For optimized signal acquisition, we recommend to start the FRET measurements with OS co-expressing both cerulean- and citrine-tagged FRET partners (hereinafter referred to as FRET sample). to ensure a suﬃcient number of ﬂuorescent OS. The OS were separated from the retina by vortexing for 15–30 s. Vortexing should not surpass 30 s as excessive shearing disrupts the shape of OS. Afterwards, centrifugation was conducted at 500 × g for 30 s and the supernatant was carefully transferred to a fresh 1.5-ml-microcentrifuge-tube without disturbing the pellet. The supernatant contained the OS fraction suitable for FRET measurements. If desirable, the quality and ﬂuorescence of puriﬁed OS can be analyzed (Figure 2B) on a standard epiﬂuorescence or confocal microscope by transferring 5–10 µl of supernatant on microscope slides topped with cover slips. Puriﬁed OS solution was diluted in 400 µl FRET imaging solution and was transferred to the center of an imaging dish. FRET measurements should be performed on single OS as ﬂuorescence from adjacent OS might interfere with the measurements. Therefore, the density of OS should be adjusted in order to allow for detection of single ﬂuorescent OS within the deﬁned region of interest on the imaging setup (image-plane pinhole). As the density of transduced OS varies with transduction and puriﬁcation eﬃciency, it is necessary to individually adjust the amount of OS for each preparation. The OS were allowed to sediment to the bottom for 10–15 min at room temperature. The ﬂuorescence lamp was turned on at least 30 min prior to the experiment to ensure stable illumination. The image-plane pinhole was set to an appropriate size that allows monitoring of a single OS, which was positioned in the center of the axial beam path. This position must not be changed throughout the entire FRET measurements performed on the same day. The FelixGX software was started to acquire the Data Analysis The data set consists of three data points per OS, i.e., one intensity value for each ﬁlter cube. As controls, we additionally included data points measured from non-ﬂuorescent OS for background subtraction as well as from cerulean-only and citrine-only expressing OS for donor bleed-through and cross- excitation corrections, respectively. The data was transferred to an Excel sheet or an adequate data processing program. For the εcitrine and εcerulean are the FRET setup speciﬁc average molar extinction coeﬃcients for citrine and cerulean, respectively. FRET Measurements on Isolated Outer Segments Setup of FRET Microscope, Software, and OS Sample Calculation of three cube sensitized acceptor emission FRET requires data for FRET donor (cerulean) only and acceptor (citrine) only samples (Erickson et al., 2003; Shaltiel et al., 2012; Ben Johny et al., 2013). Therefore, in addition to the ﬂuorescence signals from OS co-expressing cerulean and citrine constructs, OS expressing cerulean- or citrine-tagged proteins only must be July 2016 \| Volume 10 \| Article 356 Frontiers in Neuroscience \| www.frontiersin.org 8 AAVs for FRET in Photoreceptors Becirovic et al. 10 non-ﬂuorescent OS measured for each of the preparations, i.e. FRET, cerulean-only and citrine-only, the mean intensity values were calculated. These mean values were subtracted from each data point of the particular preparation to adjust for background ﬂuorescence. In order to correct for donor bleed- through, the correction constant RD1 for each of the single data points gathered from the cerulean-only expressing preparation was calculated by dividing the intensities captured with the FRET-cube by those obtained from the cerulean-cube. In order to correct for acceptor cross-excitation, the correction constant RA for each single data point gathered from the citrine-only expressing preparation was calculated by dividing the intensities captured with the FRET-cube by those obtained from the citrine-cube. The mean value for both constants, RD1 and RA, was calculated. Usually, a value of ∼0.25 for cerulean (donor- bleed through) and 0.03 for citrine (acceptor cross-excitation) was obtained in our setup. However, these values might diﬀer with ﬂuorophore variant, protein tag, and imaging setup. The calculated constants were used for donor bleed-through and acceptor cross-excitation to evaluate the FRET ratio (FR) for each single measurement according to the following equation: ﬂuorescence intensities. An oil-immersion and high-resolution objective (40x or higher) was used and immersion medium was applied. The imaging dish was placed onto the sample holder and bright ﬁeld illumination was used to focus on OS. Individual OS were identiﬁed based on their characteristic morphology as shown in Figure 2B. Citrine-only Expressing Preparation OS with varying expression levels of citrine were chosen. For each OS, the ﬂuorescence intensity was measured with the cerulean, FRET, and citrine ﬁlter cube according to the same protocol as described above for the FRET sample. Data from at least 15 OS were collected. To subtract the OS autoﬂuorescence, the cerulean, FRET, and citrine cube were measured for ∼10 OS that do not express citrine within the same sample. EA = [FR −1] · εcitrine(436) εcerulean(436) Cerulean-only Expressing Preparation OS with varying expression levels of cerulean were used. For each OS, the ﬂuorescence intensity was measured with the cerulean, FRET, and citrine ﬁlter cube according to the same protocol as described above for the FRET sample. Data from at least 15 OS were collected. To subtract the OS autoﬂuorescence, the cerulean, FRET, and citrine cube were measured for ∼10 OS that do not express cerulean within the same sample. FR = SFRET −RD1 · Scerulean RA · (Scitrine −RD2 · Scerulean) In our FRET setup, RD2 was negligibly small and could thus be ignored. However, it should be rigorously tested for each individual imaging setup. The mean value of the previously obtained data was calculated. The mean value represents the overall FR. Note that the FR values are instrumentation dependent. FRET eﬃciencies (EA) (which are independent of the setup used) can be easily calculated from the FR using the following equation: Measurements on the FRET Sample For measurements of FRET samples, OS that express suﬃcient levels of both citrine and cerulean were used. A ﬂuorescent OS was placed in the center of the optic ﬁeld and ﬂuorescence from adjacent cells invading the optic ﬁeld was excluded. Gain and ﬂuorescence intensity settings were adjusted in order to achieve an appropriate signal prior to the ﬁrst signal acquisition. All subsequent data must be acquired under the same conditions. Fluorescence intensities were collected with the cerulean cube, FRET cube, and citrine cube. The settings for the single cubes are as follows: For cerulean cube: 426–446 nm excitation ﬁlter, T455lp dichroic mirror, and 460–500 nm emission ﬁlter; for citrine cube: 490–510 nm excitation ﬁlter, T515lp dichroic mirror, and 510– 550 nm emission ﬁlter; for FRET cube: 426–446 nm excitation ﬁlter, T455lp dichroic mirror, and 510–550 nm emission ﬁlter. FR = SFRET −RD1 · Scerulean RA · Scitrine FR = SFRET −RD1 · Scerulean RA · Scitrine The data for a single OS was acquired according to the following sequence: cerulean excitation was started and the signals with the cerulean cube followed by the FRET cube were obtained. Then, citrine excitation was carried out and the signal with the citrine cube was obtained. 25–35 individual OS were measured. To subtract the OS autoﬂuorescence, the same three cube protocol as described above was applied to ∼10 OS that do not express citrine or cerulean within the corresponding sample. Scerulean, Scitrine, and SFRET are intensity signals acquired with respective ﬁlter cubes (FRET, cerulean, and citrine) in a FRET specimen and RA, RD1 are predetermined factors for calibration issues (RA: Acceptor cross-excitation, RD1: Donor bleed-through). Depending on the imaging setup and, in particular, the ﬁlter set used for experiments, a considerable amount of cerulean ﬂuorescence may be detected by the citrine cube. To correct for this, an additional donor bleed-trough correction constant RD2 should be calculated by dividing the cerulean ﬂuorescence intensity obtained with the citrine cube by the one detected with the cerulean cube. Accordingly, the FR equation was adjusted to: (Erickson et al., 2003) Frontiers in Neuroscience \| www.frontiersin.org • Transfection of HEK293T cells: 48 h • Transfection of HEK293T cells: 48 h • Transfection of HEK293T cells: 48 h • Isolation of rAAV: 2–4 h (varies depending on number of dishes to be harvested) • Gradient puriﬁcation and rAAV concentration: 6 h (for 2 diﬀerent rAAV constructs) • Preparation of standards for titer determination: 2 h • Subretinal injection: 20 min (per mouse) • rAAV transduction of murine retina and protein expression: 10 days • Isolation of photoreceptor OS: 10 min (per eye) • FRET imaging: 5–9 h (depending on number of retinas to be analyzed) • Data analysis: 2–3 h (depending on number of measurements to be processed). Solution Test transfection reagents for their efﬁciencies. Requantify plasmid concentration used for transfection. Remove HEK293T dishes from incubator immediately before transfection. Check for the presence of SmaI and/or Eam1105I restriction sites within the palindromic ITR sequence. In case of unexpected restriction fragment band pattern, repeat cloning using an intact rAAV vector. Make sure to choose only mice with clear, intact eyes for subretinal injection. Check the eyes under the stereomicroscope. Dilute with 0.014% Tween/PBS-MK to adjust virus particle concentration. If the suspension is too viscous, dilute with double-distilled water at a maximum ratio of 1:2 (water-to-virus suspension). Proceed with the next step without any removal of undesirable tissue. Proceed with the next step without any removal of undesirable tissue. Make sure to see a clear injection bleb as this indicates the correct injection to the subretinal space. An injection into the upper layers of the eye such as sclera or choroidea causes a bleb outside at the globe. If the needle is intravitreal, it can be clearly and sharply seen under the stereomicroscope. By contrast, a needle in the subretinal space (injection angle at ∼60◦) appears rather blurred. Keep the injection needle for at least 20 s in the bleb to ensure proper delivery of the desired volume. If possible, perform OCT measurements on the anesthetized mouse after injection to conﬁrm detachment of the retina at the injection position. Virus titer has not been determined correctly. Double check the virus titer. Otherwise, repeat the virus production. Time Schedule interactions of diﬀerent membrane and soluble proteins in OS of rod and cone photoreceptors. Representative results are found in our previous publications (Becirovic et al., 2014; Nguyen et al., 2016). Selected data from these publications are presented in the modiﬁed graphs shown in Figure 3. FRET is given as FRET eﬃciencies (EA) which can be easily calculated from the FRET ratios (FR) as shown in the “Data Analysis” Section. It is noteworthy to mention that the mean value of EA may not represent the maximal FRET eﬃciency (EA max). EA max can be calculated if a relatively high variability of the cerulean/citrine molar ratios is present in FRET measurements. If this is the case, single FRET values can be plotted against the cerulean/citrine intensity ratios to obtain the binding curves and to calculate EA max. A comparison of EA max between diﬀerent constructs allows for the determination of relative binding aﬃnities. Since EA max is directly proportional to the binding aﬃnity, our method in principle should also allow for comparisons of relative binding aﬃnities of single protein-protein interactions in an isolated subcellular compartment. Troubleshooting Problems, possible underlying reasons and advices concerning critical steps of the procedure can be found in Table 5. July 2016 \| Volume 10 \| Article 356 Frontiers in Neuroscience \| www.frontiersin.org 9 AAVs for FRET in Photoreceptors Becirovic et al. TABLE 5 \| Troubleshooting. Problem Possible Reason Solution Low titer. Transfection reagents do not have sufﬁcient transfection efﬁciency. Test transfection reagents for their efﬁciencies. Requantify plasmid concentration used for transfection. pH of HEK293T cell medium is not optimal for transfection. Remove HEK293T dishes from incubator immediately before transfection. The sequence of the ITRs (inverted terminal repeats) within the plasmid is not intact, thus replication and packaging of the rAAV vector is inefﬁcient. Check for the presence of SmaI and/or Eam1105I restriction sites within the palindromic ITR sequence. In case of unexpected restriction fragment band pattern, repeat cloning using an intact rAAV vector. The injection bleb in the subretinal space is not visible under the surgical/stereomicroscope. The eye shows damages or opacity of the cornea. Make sure to choose only mice with clear, intact eyes for subretinal injection. Check the eyes under the stereomicroscope. Virus suspensions diluted with double-distilled water to adjust the particle concentration do not show optical refraction as clear as the virus suspension in 0.014% Tween/PBS-MK. Dilute with 0.014% Tween/PBS-MK to adjust virus particle concentration. If the suspension is too viscous, dilute with double-distilled water at a maximum ratio of 1:2 (water-to-virus suspension). The injection angle is not optimal. Adjust the injection angle to ∼60◦. Loss of many OS. Removal of the pigment epithelium and ciliary body after retina isolation with foreceps may result in loss of many OS. Proceed with the next step without any removal of undesirable tissue. Protein expression level is too low, thus leading to a too weak or absent ﬂuorescence signal. Virus suspension is not (completely) injected into the subretinal space. Make sure to see a clear injection bleb as this indicates the correct injection to the subretinal space. An injection into the upper layers of the eye such as sclera or choroidea causes a bleb outside at the globe. If the needle is intravitreal, it can be clearly and sharply seen Solution ANTICIPATED RESULTS AND DISCUSSION In this protocol, we demonstrate that robust FRET signals can be measured for well-known homomeric and heteromeric Taken together, our protocol enables the measurement of robust FRET signals ex vivo in small and highly specialized July 2016 \| Volume 10 \| Article 356 Frontiers in Neuroscience \| www.frontiersin.org 10 AAVs for FRET in Photoreceptors Becirovic et al. FIGURE 3 \| FRET measurements in isolated photoreceptor OS. (A) Schematic depiction of the single constructs used for the subretinal injection and for the determination of the FRET efﬁciencies (EA) shown in (C). (B) Representative confocal images of single isolated rod OS co-expressing C-terminally tagged peripherin-2 and rhodopsin. The excitation wavelength and emission ﬁlter settings used to obtain the single channels (Donor, FRET and Acceptor) are summarized in the “Stepwise Procedures” section. Scale bar, 1.5 µm. (C) Results of the FRET measurements for different FRET pair combinations given as mean values ± standard error of the mean (SEM). Numbers of independent measurements (n) are given in brackets. EA values for the single FRET pairs are as follows: Rho-Rho, EA = 10.45 ± 0.78; Rho-citr-P, EA = 4.52 ± 0.82; Rho-P-citr, EA = 2.76 ± 0.65; Rho-GARP2, EA = 0.26 ± 0.65; (D) Schematic view of the single constructs used for the subretinal injection and for the determination of the FRET efﬁciencies (EA) shown in (F). (E) Representative confocal images of single isolated cone OS co-expressing C-terminally tagged peripherin-2 and cone opsin. Scale bar, 1.5 µm. (F) Results of the FRET measurements (given as mean values ± SEM) for different FRET pair combinations as indicated. Numbers of independent measurements (n) are given in brackets. EA values for the single FRET pairs are described elsewhere (Nguyen et al., 2016). FIGURE 3 \| FRET measurements in isolated photoreceptor OS. (A) Schematic depiction of the single constructs used for the subretinal injection and for the determination of the FRET efﬁciencies (EA) shown in (C). (B) Representative confocal images of single isolated rod OS co-expressing C-terminally tagged peripherin-2 and rhodopsin. The excitation wavelength and emission ﬁlter settings used to obtain the single channels (Donor, FRET and Acceptor) are summarized in the “Stepwise Procedures” section. Scale bar, 1.5 µm. (C) Results of the FRET measurements for different FRET pair combinations given as mean values ± standard error of the mean (SEM). Numbers of independent measurements (n) are given in brackets. ANTICIPATED RESULTS AND DISCUSSION EA values for the single FRET pairs are as follows: Rho-Rho, EA = 10.45 ± 0.78; Rho-citr-P, EA = 4.52 ± 0.82; Rho-P-citr, EA = 2.76 ± 0.65; Rho-GARP2, EA = 0.26 ± 0.65; (D) Schematic view of the single constructs used for the subretinal injection and for the determination of the FRET efﬁciencies (EA) shown in (F). (E) Representative confocal images of single isolated cone OS co-expressing C-terminally tagged peripherin-2 and cone opsin. Scale bar, 1.5 µm. (F) Results of the FRET measurements (given as mean values ± SEM) for different FRET pair combinations as indicated. Numbers of independent measurements (n) are given in brackets. EA values for the single FRET pairs are described elsewhere (Nguyen et al., 2016). multiphoton FLIM-FRET, or near infrared-FRET ﬂuorescence lifetime imaging (Abe et al., 2013; Johnsson et al., 2014). cellular compartments of mammalian cells like OS of murine photoreceptors. This allows for the systematic analysis of protein- protein interactions in a physiological or pathophysiological context of the photoreceptor biology. In particular, it can be used to analyze the eﬀects of disease-associated mutations on protein- protein interactions. OS of photoreceptors are modiﬁed cilia. Ciliopathies encompass a very large group of genetic disorders compromising the functional or structural integrity of cilia. Since the isolation of ciliary compartments from other tissues (i.e. kidney, lung, brain, olfactory epithelium) is well-established (Mitchell et al., 2009), our protocol should also be transferable to analyses of protein-protein interactions in any other ciliated tissue. In non-dividing cells, rAAVs lead to an episomal and long-term expression of the respective gene for up to several years (Trapani et al., 2014). Thus, FRET measurements can be performed on several time points during the development of the tissue. Finally, our protocol could also provide an experimental basis for the establishment of FRET measurements in the retina of living animals by means of fundus ophthalmoscopy combined with e.g., conventional ﬂuorescence imaging, 2-photon imaging, FUNDING This work was supported by the Deutsche Forschungsgemeinschaft (DFG, BE 4830/1 1). This work was supported by the Deutsche Forschungsgemeinschaft (DFG, BE 4830/1 1). AUTHOR CONTRIBUTIONS EB designed the protocol with contribution from SM. EB, SB, ON, LR, VH, CS, and ESB performed the experiments, EB, SB, ON, and ESB analyzed the results. EB, ON, ESB, and SM wrote the manuscript with contribution from CW and MB. EB designed the protocol with contribution from SM. EB, SB, ON, LR, VH, CS, and ESB performed the experiments, EB, SB, ON, and ESB analyzed the results. EB, ON, ESB, and SM wrote the manuscript with contribution from CW and MB. Frontiers in Neuroscience \| www.frontiersin.org REFERENCES Hovan, S. C., Howell, S., and Park, P. S. (2010). Forster resonance energy transfer as a tool to study photoreceptor biology. J. Biomed. Opt. 15, 067001. doi: 10.1117/1.3505023 Abe, K., Zhao, L., Periasamy, A., Intes, X., and Barroso, M. (2013). Non- invasive in vivo imaging of near infrared-labeled transferrin in breast cancer cells and tumors using ﬂuorescence lifetime FRET. PLoS ONE 8:e80269. doi: 10.1371/journal.pone.0080269 Jastrzebska, B., Maeda, T., Zhu, L., Fotiadis, D., Filipek, S., Engel, A., et al. (2004). Functional characterization of rhodopsin monomers and dimers in detergents. J. Biol. Chem. 279, 54663–54675. doi: 10.1074/jbc.M4086 91200 Allocca, M., Mussolino, C., Garcia-Hoyos, M., Sanges, D., Iodice, C., Petrillo, M., et al. (2007). Novel adeno-associated virus serotypes eﬃciently transduce murine photoreceptors. J. Virol. 81, 11372–11380. doi: 10.1128/JVI.01 327-07 Johnsson, A. K., Dai, Y., Nobis, M., Baker, M. J., McGhee, E. J., Walker, S., et al. (2014). The Rac-FRET mouse reveals tight spatiotemporal control of Rac activity in primary cells and tissues. Cell Rep. 6, 1153–1164. doi: 10.1016/j.celrep.2014.02.024 Arshavsky, V. Y., and Burns, M. E. (2012). Photoreceptor signaling: supporting vision across a wide range of light intensities. J. Biol. Chem. 287, 1620–1626. doi: 10.1074/jbc.R111.305243 Knepp, A. M., Periole, X., Marrink, S. J., Sakmar, T. P., and Huber, T. (2012). Rhodopsin forms a dimer with cytoplasmic helix 8 contacts in native membranes. Biochemistry 51, 1819–1821. doi: 10.1021/bi30 01598 Auricchio, A., Hildinger, M., O’Connor, E., Gao, G. P., and Wilson, J. M. (2001). Isolation of highly infectious and pure adeno-associated virus type 2 vectors with a single-step gravity-ﬂow column. Hum. Gene Ther. 12, 71–76. doi: 10.1089/104303401450988 Koch, S., Sothilingam, V., Garcia Garrido, M., Tanimoto, N., Becirovic, E., Koch, F., et al. (2012). Gene therapy restores vision and delays degeneration in the CNGB1(−/−) mouse model of retinitis pigmentosa. Hum. Mol. Genet. 21, 4486–4496. doi: 10.1093/hmg/dds290 Becirovic, E., Bohm, S., Nguyen, O. N., Riedmayr, L. M., Koch, M. A., Schulze, E., et al. (2016). In vivo analysis of disease-associated point mutations unveils profound diﬀerences in mRNA splicing of peripherin-2 in rod and cone photoreceptors. PLoS Genet. 12:e1005811. doi: 10.1371/journal.pgen.1 005811 Kumagai, Y., Naoki, H., Nakasyo, E., Kamioka, Y., Kiyokawa, E., and Matsuda, M. (2014). Heterogeneity in ERK activity as visualized by in vivo FRET imaging of mammary tumor cells developed in MMTV-Neu mice. Oncogene 34, 1051–1057. doi: 10.1038/onc.2014.28 Becirovic, E., Nguyen, O. N., Paparizos, C., Butz, E. S., Stern-Schneider, G., Wolfrum, U., et al. (2014). REFERENCES Peripherin-2 couples rhodopsin to the CNG channel in outer segments of rod photoreceptors. Hum. Mol. Genet. 23, 5989–5997. doi: 10.1093/hmg/ddu323 Loewen, C. J., and Molday, R. S. (2000). Disulﬁde-mediated oligomerization of Peripherin/Rds and Rom-1 in photoreceptor disk membranes. Implications for photoreceptor outer segment morphogenesis and degeneration. J. Biol. Chem. 275, 5370–5378. doi: 10.1074/jbc.275.8.5370 Ben Johny, M., Yang, P. S., Bazzazi, H., and Yue, D. T. (2013). Dynamic switching of calmodulin interactions underlies Ca2+ regulation of CaV1.3 channels. Nat. Commun. 4, 1717. doi: 10.1038/ncomms2727 Michalakis, S., Muhlfriedel, R., Tanimoto, N., Krishnamoorthy, V., Koch, S., Fischer, M. D., et al. (2010a). Restoration of cone vision in the CNGA3−/− mouse model of congenital complete lack of cone photoreceptor function. Mol. Ther. 18, 2057–2063. doi: 10.1038/mt.2010.149 Bennett, J., Maguire, A. M., Cideciyan, A. V., Schnell, M., Glover, E., Anand, V., et al. (1999). Stable transgene expression in rod photoreceptors after recombinant adeno-associated virus-mediated gene transfer to monkey retina. Proc. Natl. Acad. Sci. U.S.A. 96, 9920–9925. doi: 10.1073/pnas.96.1 7.9920 Michalakis, S., Mühlfriedel, R., Tanimoto, N., Krishnamoorthy, V., Koch, S., Fischer, M. D., et al. (2010b). Restoration of cone vision in the CNGA3−/− mouse model of congenital complete lack of cone photoreceptor function. Mol. Ther. 18, 2057–2063. doi: 10.1038/mt.2010.149 Berger, W., Kloeckener-Gruissem, B., and Neidhardt, J. (2010). The molecular basis of human retinal and vitreoretinal diseases. Prog. Retin. Eye Res. 29, 335–375. doi: 10.1016/j.preteyeres.2010.03.004 Michalakis, S., Zong, X., Becirovic, E., Hammelmann, V., Wein, T., Wanner, K. T., et al. (2011). The glutamic acid-rich protein is a gating inhibitor of cyclic nucleotide-gated channels. J. Neurosci. 31, 133–141. doi: 10.1523/JNEUROSCI.4735-10.2011 Erickson, M. G., Liang, H., Mori, M. X., and Yue, D. T. (2003). FRET two- hybrid mapping reveals function and location of L-type Ca2+ channel CaM preassociation. Neuron 39, 97–107. doi: 10.1016/S0896-6273(03)00 395-7 Mitchell, K. A., Szabo, G., and Otero Ade, S. (2009). Methods for the isolation of sensory and primary cilia–an overview. Methods Cell Biol. 94, 87–101. doi: 10.1016/S0091-679X(08)94004-8 Feilmeier, B. J., Iseminger, G., Schroeder, D., Webber, H., and Phillips, G. J. (2000). Green ﬂuorescent protein functions as a reporter for protein localization in Escherichia coli. J. Bacteriol. 182, 4068–4076. doi: 10.1128/JB.182.14.4068- 4076.2000 Mühlfriedel, R., Michalakis, S., Garrido, M. G., Biel, M., and Seeliger, M. W. (2013). Optimized technique for subretinal injections in mice. Methods Mol. Biol. 935, 343–349. doi: 10.1007/978-1-62703-080-9_24 Murlidharan, G., Samulski, R. J., and Asokan, A. (2014). ACKNOWLEDGMENTS We thank Elisabeth Schulze and Berit Noack for excellent technical support. July 2016 \| Volume 10 \| Article 356 Frontiers in Neuroscience \| www.frontiersin.org 11 AAVs for FRET in Photoreceptors Becirovic et al. REFERENCES Biology of adeno- associated viral vectors in the central nervous system. Front. Mol. Neurosci. 7:76. doi: 10.3389/fnmol.2014.00076 Gao, G. P., Alvira, M. R., Wang, L., Calcedo, R., Johnston, J., and Wilson, J. M. (2002). Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc. Natl. Acad. Sci. U.S.A. 99, 11854–11859. doi: 10.1073/pnas.182412299 Nguyen, O. N., Bohm, S., Giessl, A., Butz, E. S., Wolfrum, U., Brandstatter, J. H., et al. (2016). Peripherin-2 diﬀerentially interacts with cone opsins in outer segments of cone photoreceptors. Hum. Mol. Genet. doi: 10.1093/hmg/ddw103. [Epub ahead of print]. Goldberg, A. F., Moritz, O. L., and Molday, R. S. (1995). Heterologous expression of photoreceptor peripherin/rds and Rom-1 in COS-1 cells: assembly, interactions, and localization of multisubunit complexes. Biochemistry 34, 14213–14219. doi: 10.1021/bi00043a028 Poetsch, A., Molday, L. L., and Molday, R. S. (2001). The cGMP-gated channel and related glutamic acid-rich proteins interact with peripherin-2 at the rim region of rod photoreceptor disc membranes. J. Biol. Chem. 276, 48009–48016. doi: 10.1074/jbc.M108941200 Grieger, J. C., Choi, V. W., and Samulski, R. J. (2006). Production and characterization of adeno-associated viral vectors. Nat. Protoc. 1, 1412–1428. doi: 10.1038/nprot.2006.207 Rizzo, M. A., Springer, G. H., Granada, B., and Piston, D. W. (2004). An improved cyan ﬂuorescent protein variant useful for FRET. Nat. Biotechnol. 22, 445–449. doi: 10.1038/nbt945 Griesbeck, O., Baird, G. S., Campbell, R. E., Zacharias, D. A., and Tsien, R. Y. (2001). Reducing the environmental sensitivity of yellow ﬂuorescent protein. Mechanism and applications. J. Biol. Chem. 276, 29188–29194. doi: 10.1074/jbc.M102815200 Roepman, R., and Wolfrum, U. (2007). Protein networks and complexes in photoreceptor cilia. Subcell. Biochem. 43, 209–235. doi: 10.1007/978-1-4020- 5943-8_10 Hildinger, M., Auricchio, A., Gao, G., Wang, L., Chirmule, N., and Wilson, J. M. (2001). Hybrid vectors based on adeno-associated virus serotypes 2 and 5 for muscle-directed gene transfer. J. Virol. 75, 6199–6203. doi: 10.1128/JVI.75.13.6199-6203.2001 Schön, C., Biel, M., and Michalakis, S. (2013). Gene replacement therapy for retinal CNG channelopathies. Mol. Genet. Genomics 288, 459–467. doi: 10.1007/s00438-013-0766-4 Hirata, E., Yukinaga, H., Kamioka, Y., Arakawa, Y., Miyamoto, S., Okada, T., et al. (2012). In vivo ﬂuorescence resonance energy transfer imaging reveals diﬀerential activation of Rho-family GTPases in glioblastoma cell invasion. J. Cell Sci. 125(Pt 4), 858–868. doi: 10.1242/jcs.089995 Shaltiel, L., Paparizos, C., Fenske, S., Hassan, S., Gruner, C., Rotzer, K., et al. (2012). Frontiers in Neuroscience \| www.frontiersin.org July 2016 \| Volume 10 \| Article 356 REFERENCES Complex regulation of voltage-dependent activation and inactivation properties of retinal voltage-gated Cav1.4 L-type Ca2+ channels Frontiers in Neuroscience \| www.frontiersin.org July 2016 \| Volume 10 \| Article 356 12 Becirovic et al. AAVs for FRET in Photoreceptors by Ca2+-binding protein 4 (CaBP4). J. Biol. Chem. 287, 36312–36321. doi: 10.1074/jbc.M112.392811 by Ca2+-binding protein 4 (CaBP4). J. Biol. Chem. 287, 36312–36321. doi: 10.1074/jbc.M112.392811 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. Trapani, I., Puppo, A., and Auricchio, A. (2014). Vector platforms for gene therapy of inherited retinopathies. Prog. Retin. Eye Res. 43C, 108–128. doi: 10.1016/j.preteyeres.2014.08.001 Despite hosting the research topic together with one of the authors of this manuscript, the handling Editor state that the process met the standards of a fair and objective review. Waldo, G. S., Standish, B. M., Berendzen, J., and Terwilliger, T. C. (1999). Rapid protein-folding assay using green ﬂuorescent protein. Nat. Biotechnol. 17, 691–695. doi: 10.1038/10904 Copyright © 2016 Becirovic, Böhm, Nguyen, Riedmayr, Hammelmann, Schön, Butz, Wahl-Schott, Biel and Michalakis. 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. Wen, L., Thunemann, M., Feil, S., Hillenbrand, M., Vachaviolos, A., Ott, T., et al. (2013). Analysis of cGMP signalling with transgenic mice expressing FRET- based cGMP sensors. BMC Pharmacol. Toxicol. 14, 1–2. doi: 10.1186/2050- 6511-14-s1-p76 Zacchigna, S., Zentilin, L., and Giacca, M. (2014). Adeno-associated virus vectors as therapeutic and investigational tools in the cardiovascular system. Circ. Res. 114, 1827–1846. doi: 10.1161/CIRCRESAHA.114.302331 July 2016 \| Volume 10 \| Article 356 Frontiers in Neuroscience \| www.frontiersin.org 13
https://openalex.org/W2057970678	https://hal.science/hal-01893669/file/journal.pbio.1001177.PDF	English	null	A Holistic Approach to Marine Eco-Systems Biology	PLoS biology	2,011	cc-by	4,384	A Holistic Approach to Marine Eco-Systems Biology Eric Karsenti, Silvia G. Acinas, Peer Bork, Chris Bowler, Colomban de Vargas, Jeroen Raes, Matthew Sullivan, Detlev Arendt, Francesca Benzoni, Jean-Michel Claverie, et al. To cite this version: Eric Karsenti, Silvia G. Acinas, Peer Bork, Chris Bowler, Colomban de Vargas, et al.. A Holistic Approach to Marine Eco-Systems Biology. PLoS Biology, 2011, 9 (10), pp.e1001177. 10.1371/jour- nal.pbio.1001177. hal-01893669 Distributed under a Creative Commons Attribution 4.0 International License Introduction With biology becoming quantitative, systems-level studies can now be per- formed at spatial scales ranging from molecules to ecosystems. Biological data generated consistently across scales can be integrated with physico-chemical contex- tual data for a truly holistic approach, with a profound impact on our understanding of life [1–5]. Marine ecosystems are crucial in the regulation of Earth’s biogeochemi- cal cycles and climate [6,7]. Yet their organization, evolution, and dynamics remain poorly understood [8,9]. The Tara Oceans project was launched in Septem- ber 2009 for a 3-year study of the global ocean ecosystem aboard the ship Tara. A unique sampling programme encompass- ing optical and genomic methods to describe viruses, bacteria, archaea, pro- tists, and metazoans in their physico- chemical environment has been imple- mented. Starting as a grassroots initiative of a few scientists, the project has grown into a global consortium of over 100 specialists from diverse disciplines, includ- ing oceanography, microbial ecology, genomics, molecular, cellular, and systems biology, taxonomy, bioinformatics, data management, and ecosystem modeling. This multidisciplinary community aims to generate systematic, open access datasets Viruses, bacteria, archaea, protists, and planktonic metazoans form the bulk of biomass throughout the oceans and drive the global biogeochemical cycles that regulate the Earth system [6,9,10]. For instance, marine microbes produce nearly as much oxygen through primary produc- tion as land plants [11]. This system is driven by a complex ecological network of autotrophic, heterotrophic, and mixo- trophic organisms, where trophodynamics and biogeochemical interdependencies are determining factors for primary production rates in marine systems. In addition, ocean viruses modulate primary production by inducing organism mortality and by en- coding core photosynthesis genes that are expressed during infection [12–14]. There- fore, only an ecosystem-wide approach, A global-scale study of morphological, genetic, and functional biodiversity of plankton organisms in relation to the changing physico-chemical parameters of the oceans [8,16–18] is now critical to understanding and managing our fragile oceans. Specifically, such a dataset will improve our understanding of the princi- ples governing marine ecosystems and the evolution of life in the ocean, thus enhancing our capacity of assessing ecosys- tem services and enabling a better predic- tion of fish stock distribution and impacts of Copyright: 2011 Karsenti 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. HAL Id: hal-01893669 https://hal.science/hal-01893669v1 Submitted on 14 Dec 2018 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 Community Page A Holistic Approach to Marine Eco-Systems Biology Eric Karsenti1, Silvia G. Acinas2, Peer Bork1, Chris Bowler3,4, Colomban De Vargas3,5,6, Jeroen Raes7,8, Matthew Sullivan9, Detlev Arendt1, Francesca Benzoni10, Jean-Michel Claverie3,11, Mick Follows12, Gaby Gorsky3,6,13, Pascal Hingamp3,11, Daniele Iudicone14, Olivier Jaillon15, Stefanie Kandels-Lewis1, Uros Krzic1, Fabrice Not3,5,6, Hiroyuki Ogata3,11, Ste´phane Pesant16,17, Emmanuel Georges Reynaud18, Christian Sardet3,6,19, Michael E. Sieracki20, Sabrina Speich21, Didier Velayoudon22, Jean Weissenbach15, Patrick Wincker15, the Tara Oceans Consortium" 1 European Molecular Biology Laboratory (EMBL), Heidelberg, Germany, 2 Institut de Cie`ncies del Mar (ICM), Consejo Superior de Investigaciones Cientı´ficas (CSIC), Barcelona, Spain, 3 CNRS, Paris, France, 4 Institut de Biologie de l’Ecole Normale Supe´rieure (IBENS), Paris, France, 5 Station Biologique de Roscoff (SBR), Evolution du Plancton et Paleo Oceans (EPPO), Roscoff, France, 6 Universite´ Pierre et Marie Curie (UPMC), Paris, France, 7 VIB, Brussels, Belgium, 8 Vrije Universiteit Brussel, Brussels, Belgium, 9 University of Arizona, Tucson, Arizona, United States of America, 10 University of Milano-Bicocca, Milan, Italy, 11 Aix-Marseille Universite´, Marseilles, France, 12 MIT, Cambridge, Massachusetts, United States of America, 13 Laboratoire d’Oceanographie de Villefranche (LOV), Villefranche-sur-Mer, France, 14 Stazione Zoologica Anton Dohrn, Naples, Italy, 15 Commissariat a` l’e´nergie atomique et aux e´nergies alternatives (CEA), Genoscope, Evry, France, 16 Universitaet Bremen, Bremen, Germany, 17 MARUM Center for Marine Environmental Sciences, Bremen, Germany, 18 University College Dublin, Dublin, Ireland, 19 BioDev, Villefranche-sur-Mer, France, 20 Bigelow Laboratory for Ocean Sciences, West Boothbay Harbor, Maine, United States of America, 21 Laboratoire de Physique des Oce´ans (LPO), CNRS/Institut franc¸ais de recherche pour l’exploitation de la mer (IFREMER)/Institut de Recherche pour le De´veloppement (IRD)/Universite´ de Bretagne Occidentale (UBO), Brest, France, 22 DVIPC, Paris, France from viruses to metazoans, will enable us to start disentangling the functioning of the Earth system. This approach ranges from mapping organismal diversity across scales spanning five orders of magnitude to developing empirical datasets that inform conceptual models about the complex interplay between organisms driving fluxes of energy, biogeochemical, and molecular ‘‘currencies’’ in ocean ecosystems [15]. usable for probing the morphological and molecular makeup, diversity, evolution, ecology, and global impacts of plankton on the Earth system. Citation: Karsenti E, Acinas SG, Bork P, Bowler C, De Vargas C, et al. (2011) A Holistic Approach to Marine Eco- Systems Biology. PLoS Biol 9(10): e1001177. doi:10.1371/journal.pbio.1001177 Copyright: 2011 Karsenti 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. The Community Page is a forum for organizations and societies to highlight their efforts to enhance the dissemination and value of scientific knowledge. E-mail: karsenti@embl.de Introduction Funding: The Tara Oceans expedition is generously funded by CNRS, EMBL, Genoscope/CEA, VIB, Stazione Zoologica Anton Dohrn, UNIMIB, ANR, FWO, BIO5, Biosphere 2, agne`s b., the Veolia Environment Foundation, Region Bretagne, World Courier, Illumina, Cap L’Orient, the EDF Foundation EDF Diversiterre, FRB, the Prince Albert II de Monaco Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The Community Page is a forum for organizations and societies to highlight their efforts to enhance the dissemination and value of scientific knowledge. Competing Interests: The authors have declared that no competing interests exist. * E-mail: karsenti@embl.de " Membership of the Tara Oceans Consortium is provided in the Acknowledgments. " Membership of the Tara Oceans Consortium is provided in the Acknowledgments. PLoS Biology \| www.plosbiology.org October 2011 \| Volume 9 \| Issue 10 \| e1001177 October 2011 \| Volume 9 \| Issue 10 \| e1001177 October 2011 \| Volume 9 \| Issue 10 \| e1001177 1 fish larvae. While such a ‘‘study it all’’ approach is not novel (e.g., NSF Long Term Ecological Research sites), it has remained science fiction until technology and informatics became enabling. Now, high throughput sequencing, quantitative imaging methods, dedicated bio-informat- ics and modeling tools are poised to assess the complexity of the global ocean systems. To achieve such integration, Tara Oceans is driven by researchers with expertise in biological and physical oceanography, ecology, microbiology, systematics, molec- ular, cellular and systems biology, bioinfor- matics, data management, and modeling. observation sites, and arrays of remote sensors on floats that provide physical, chemical, and biological data [15]. Other global genomics studies have been launched, e.g., Global Ocean Sampling (GOS) expedition [22] and the Earth Microbiome [23] project, as well as inte- grative projects focusing on specific biomes (e.g., Malaspina, http://www.expedicion malaspina.es/). However, Tara Oceans takes such investigations one step further by integrating the genetic, morphological, and functional diversity in its environmen- tal context at global ocean scale and at multiple depths (Figure 1), from viruses to global climate variations [19]. Planktonic organisms are also an enormous but largely untapped source [8,20] of bio-active com- pounds for the pharmaceutical, food, and cosmetics industries, as well as metabolic pathways that may provision our future energy needs [21]. In this context, the Tara Oceans consortium was founded, which embarked on a 3-year research cruise across the worlds’ oceans. Introduction Tara Oceans is not the first group to explore global ocean biodiversity. For example, previous global initiatives include satellite-based chlorophyll measurements, the Census of Marine Life, long-term Figure 1. The Tara Oceans cruise. (A) Route of the Tara Oceans expedition. Sampling stations from surface to 1,000 m are carried out between ports of call guided by satellite data about the basin to sub-mesoscale structures. (B) Tara Oceans sampling sites in the Mozambique Channel and South Atlantic. The images show near real time sea surface height (SSH) from satellite. Each sampling station is indicated by a black diamond; those that are currently being targeted for priority studies are encircled by a white halo. The altimetry data is from September 16, 2011, when Tara was sampling inside an Agulhas ring. Several other rings are also apparent in the figure, as is the Malvinas Current off the Argentinean coast that injects cold Antarctic water into the resident waters. doi:10.1371/journal.pbio.1001177.g001 Figure 1. The Tara Oceans cruise. (A) Route of the Tara Oceans expedition. Sampling stations from surface to 1,000 m are carried out between ports of call guided by satellite data about the basin to sub-mesoscale structures. (B) Tara Oceans sampling sites in the Mozambique Channel and South Atlantic. The images show near real time sea surface height (SSH) from satellite. Each sampling station is indicated by a black diamond; those that are currently being targeted for priority studies are encircled by a white halo. The altimetry data is from September 16, 2011, when Tara was sampling inside an Agulhas ring. Several other rings are also apparent in the figure, as is the Malvinas Current off the Argentinean coast that injects cold Antarctic water into the resident waters. doi:10.1371/journal.pbio.1001177.g001 October 2011 \| Volume 9 \| Issue 10 \| e1001177 PLoS Biology \| www.plosbiology.org 2 Pragmatically, to accomplish such an ambitious goal, Tara Oceans consortium scientists have necessarily been intimately involved in every aspect of the expedition. This includes planning, preparation, and running of the on-board sampling proto- cols, as well as the development of sample analysis and bioinformatics pipelines, data management, and modeling projects. This involvement ensures a coherent worldwide data collection and analysis strategy, which is reinforced through regular work- shops. The consortium has an open access policy concerning the data that will be made available to the scientific community as soon as possible after validation. Introduction Finally, a significant outreach effort is made to show life on board of Tara as well as translate results to the broader public (see http://oceans.taraexpeditions.org/ and http://www.planktonchronicles.org). tic modelling [28–31], as well as mapping functional gene ecology and activities throughout the world’s oceans. This pro- cess has been initiated, predominantly for 0.1–0.8-mm-sized surface ocean microbes, using data provided by the GOS expedi- tion [22,27,32,33], but will be dramatical- ly extended here by sampling throughout the water column, across organismal size scales and beyond metagenomic sequenc- ing, tightly coupled to global ocean- modelling efforts. strategy and downstream analysis pipeline to deeply characterize the biology of these ecosystems. Through systematic study of such heterogenous systems coupled to broader ‘‘normal’’ ocean sampling, we hope to unveil the rules that govern the structure and dynamics of ocean ecosys- tems and to extrapolate such local obser- vations to develop basin-scale process models as predictive tools [26]. Undoubt- edly, such measurements and predictions will provide a starting point for hypothesis testing by more focused, follow-up cam- paigns. These data will transform our ability to link species diversity and metabolic poten- tial/activity to environmental conditions and ecosystem outputs and promises to lead to the discovery of emergent ecolog- ical principles (Figure 2C). They will allow a deeper understanding of biodiversity gradients within and among systems and contrasting environments. We also antic- ipate establishing rules governing the self- organization of organism networks (e.g., to address which types of viruses, bacteria, protists, and zooplankton live together in a given environment) [16,27], and develop predictions about how these rules and communities will be affected by a chang- ing environment. The Tara Oceans Integrated Pipeline: Towards Eco-Systems Biology The scientific programme of Tara Oceans requires an integrated pipeline that combines semi- or fully automated data acquisition methods, new bioinfor- matics tools, and standardized data orga- nisation (Figure 2C). The high throughput imaging platform includes instruments tuned to organisms of particular size classes. They include (i) on-board and on-land flow cytometers to monitor virus particles, bacteria, and small protists, (ii) on-land digital and confocal microscopy for detailed 2D/3D imaging of cells within the 5–20-mm range, (iii) on-board and on- land FlowCams and ZooScans for quan- titative recognition of organisms ranging from 20 mm to a few cm, light sheet and confocal microscopes for 3D imaging, and (iv) on-land electron microscopes for detailed ultrastructural analyses of small protists and viruses. In parallel, we use high throughput sequencing methods to obtain both deep phylogenetic rDNA/ rRNA tag data and metagenomic and metatranscriptomic functional profiles from size fractions covering the entire plankton community from viruses to fish larvae (Figure 2B and 2C). To bring all these data together for analyses, we leverage recently developed, dedicated computational approaches [27]. In addi- tion, Tara Oceans is archiving meteoro- logical, oceanographic, biogeochemical, and plankton morphology data in the PANGAEA database (http://www.pangaea. de/), linking to larger European and inter- national data infrastructures. Thus, Tara Oceans can visualize, quantify, and ge- netically characterize ocean biodiversity within entire plankton ecosystems, as well as find patterns across unprecedentedly comprehensive data types. The Expedition and Sampling Strategy To collect and fractionate plankton communities on the basis of organism size, spanning five orders of magnitude (Figure 2A), the sampling combines tradi- tional (Niskin bottles and plankton net tows) and novel methods (e.g., a gravity- driven plankton sieve and chemistry-based concentration of viruses [24]) that feed into analytical pipelines appropriate for each size class. These analyses range from immediate visualization and quantification on-board Tara with a diversity of imaging tools, to collection and preservation of samples for genomic and morphological analysis back on land. In summary, the Tara Oceans project leverages powerful new technologies and analytical tools to develop the first plane- tary-scale data collection effort that links biogeography with ecology, genetics, and morphology. Guided by the cross-disci- plinary philosophy the pay-offs can be immense, considering the massive number of samples and data that have been collected, archived, and interconnected for scientific study, only half-way through the expedition. A lesson from this project is that, when it comes to addressing broad and complex issues of general interest to mankind, competition between scientists may not be the best model. The Tara Oceans project is a pioneering enterprise towards a truly worldwide, systems-level characterization of the largest and most fundamental ecosystem on our planet. Given the global nature of the expedi- tion and spatial heterogeneity in the oceans, a concern is the ‘‘snapshot’’ sampling strategy employed here at a single time point and at relatively sparse stations relative to the global ocean, which represents the inherent challenge of global ocean studies. In Tara Oceans we leverage near real-time remote sensing and other data to locate oceanographically interest- ing features (e.g., eddies, fronts, upwell- ings) and strengthen ecosystem compari- sons (Figures 1B and 2A). The vast ocean basins are relatively homogeneous on seasonal to decadal time scales, whereas smaller-scale systems are more dynamic. For example, the Agulhas leakage system (Figure 1B) transports water from the Indian to the Atlantic Ocean and across to South America leaving heterogeneous ocean features behind that persist for weeks to months [25]. The Agulhas system represents an ideal case for applying the Tara Oceans near real-time sampling Acknowledgments We are keen to thank the commitment of the following people and sponsors who made this singular expedition possible: CNRS, EMBL, Genoscope/CEA, VIB, Stazione Zoologica Anton Dohrn, UNIMIB, ANR (projects PO- SEIDON, BIOMARKS, PROMETHEUS, and TARA-GIRUS), FWO, BIO5, Biosphere 2, agne`s b., the Veolia Environment Founda- tion, Region Bretagne, World Courier, Illu- mina, Cap L’Orient, the EDF Foundation EDF Diversiterre, FRB, the Prince Albert II de Monaco Foundation, Etienne Bourgois, the Tara schooner and its captain and crew. Tara Oceans would not exist without the continuous With such a powerful dataset and toolkit, we anticipate testing the predic- tions of biodiversity hotspots from stochas- PLoS Biology \| www.plosbiology.org October 2011 \| Volume 9 \| Issue 10 \| e1001177 PLoS Biology \| www.plosbiology.org 3 Figure 2. The Tara Oceans model. (A) Methods for sampling organisms by size classes and abundance. The blue background indicates the filt volume required to obtain sufficient organism numbers for analysis. Actual volumes from which organisms are sampled are always recorded Methods for analyzing samples. Data on the right are from Tara Oceans sampling stations. (C) Models that will benefit from Tara Oceans data. throughput genome sequencing and quantitative image analysis provide evolution, metabolic, and interaction data to build commu metabolome maps, taxa/gene networks, and spatial ecosystem models. doi:10.1371/journal.pbio.1001177.g002 PLoS Biology \| www.plosbiology.org 4 October 2011 \| Volume 9 \| Issue 10 \| e100 Figure 2. The Tara Oceans model. (A) Methods for sampling organisms by size classes and abundance. The blue background indicates the filtered volume required to obtain sufficient organism numbers for analysis. Actual volumes from which organisms are sampled are always recorded. (B) Methods for analyzing samples. Data on the right are from Tara Oceans sampling stations. (C) Models that will benefit from Tara Oceans data. High throughput genome sequencing and quantitative image analysis provide evolution, metabolic, and interaction data to build community metabolome maps, taxa/gene networks, and spatial ecosystem models. doi:10 1371/journal pbio 1001177 g002 Figure 2. The Tara Oceans model. (A) Methods for sampling organisms by size classes and abundance. The blue background indicates the filtered volume required to obtain sufficient organism numbers for analysis. Actual volumes from which organisms are sampled are always recorded. (B) Methods for analyzing samples. Data on the right are from Tara Oceans sampling stations. (C) Models that will benefit from Tara Oceans data. References Arrigo KR (2005) Marine microorganisms and global nutrient cycles. Nature 437: 349–355. 19. Smith J, Wigginton N, Ash C, Fahrenkamp- Uppenbrink J, Pennisi E (2010) Changing oceans. Introduction. Science 328: 1497–1528. 29. Tittensor DP, Mora C, Jetz W, Lotze HK, Ricard D, et al. (2010) Global patterns and predictors of marine biodiversity across taxa. Nature 466: 1098–1101. 7. Falkowski PG, Katz ME, Knoll AH, Quigg A, Raven JA, et al. (2004) The evolution of modern eukaryotic phytoplankton. Science 305: 354–360. 20. Sarmento H, Montoya JM, Va´zquez-Domı´nguez E, Vaque´ D, Gasol JM (2010) Warming effects on marine microbial food web processes: how far can we go when it comes to predictions? Philos Trans Royal Soc London B Biol Sci 365: 2137–2149. 8. Bowler C, Karl DM, Colwell RR (2009) Micro- bial oceanography in a sea of opportunity. Nature 459: 180–184. 30. Barton AD, Dutkiewicz S, Flierl G, Bragg J, Follows MJ (2010) Patterns of diversity in marine phytoplankton. Science 327: 1509–1511. 9. Falkowski PG, Fenchel T, Delong EF (2008) The microbial engines that drive Earth’s biogeochem- ical cycles. Science 320: 1034–1039. 31. Bragg JG, Dutkiewicz S, Jahn O, Follows MJ, Chisholm SW (2010) Modeling selective pressures on phytoplankton in the global ocean. PLoS ONE 5: e9569. doi:10.1371/journal.pone.0009569. 21. Arnaud-Haond S, Arrieta JM, Duarte CM (2011) Global genetic resources. Marine biodiversity and gene patents. Science 331: 1521–1522. 10. Karl DM (2007) Microbial oceanography: para- digms, processes and promise. Nat Rev Microbiol 5: 759–769. 22. Rusch DB, Halpern AL, Sutton G, Heidelberg KB, Williamson S, et al. (2007) The Sorcerer II Global Ocean Sampling expedition: northwest Atlantic through eastern tropical Pacific. PLoS Biol 5: e77. doi:10.1371/journal.pbio.0050077. 32. Gianoulis TA, Raes J, Patel PV, Bjornson R, Korbel JO, et al. (2009) Quantifying environ- mental adaptation of metabolic pathways in metagenomics. Proc Natl Acad Sci U S A 106: 1374–1379. 11. Field CB, Behrenfeld MJ, Randerson JT, Falkowski P (1998) Primary production of the biosphere: integrating terrestrial and oceanic components. Science 281: 237–240. 23. Gilbert JA, Meyer F, Antonopoulos D, Balaji P, Brown CT, et al. (2010) Meeting report: the terabase metagenomics workshop and the vision of an Earth microbiome project. Stand Genomic Sci 3: 243–248. 33. Temperton B, Gilbert JA, Quinn JP, McGrath JW (2011) Novel analysis of oceanic surface water metagenomes suggests importance of polypho- sphate metabolism in oligotrophic environments. PLoS ONE 6: e16499. doi:10.1371/journal. pone.0016499. 12. Acknowledgments High throughput genome sequencing and quantitative image analysis provide evolution, metabolic, and interaction data to build community metabolome maps, taxa/gene networks, and spatial ecosystem models. doi:10.1371/journal.pbio.1001177.g002 PLoS Biology \| www.plosbiology.org October 2011 \| Volume 9 \| Issue 10 \| e1001177 October 2011 \| Volume 9 \| Issue 10 \| e1001177 4 G7,8, Mahe´ F3,5,6, Mazzocchi MG14, Montresor M14, Morin P3,6,30, Noel B15, Pedro´s-Alio´ C2, Pelletier E15, Perez Y31,35, Picheral M3,6,13, Piganeau G3,6,27, Poirot O3,11, Poulain J15, Poulton N20, Prejger F3,6,13, Prihoda J3,4, Probert I3,5,6, Rampal J31, Reverdin G3,6,34, Romac S3,5,6, Romagnan JB3,6,13,26, Roullier F3,6,13, Rouviere C3,6,13,26, Samson G15, Santini S3,11, Sarmento H2, Sciandra A3,6,13, Solonenko S9, Stemmann L3,6,13, Subirana L3,6,27, Suna- gawa S1, Tanaka A3,4, Testor P6,29, Thompson A12, Tichanne´-Seltzer V3,11, Tirichine L3,4,27, Toulza E3,6,27, Tozzi S33, Veluchamy A3,4, Zingone A14 support of the participating 23 institutes (see http://oceans.taraexpeditions.org). Special thanks also goes to Fabrice Not and Noan Le Bescot for creating the figures of this manu- script. This is contribution number 0001 of the Tara Oceans Expedition 2009–2012. 27 Observatoire Oce´anologique de Banyuls sur mer (OOB) Banyuls-sur-Mer, France, support of the participating 23 institutes (see http://oceans.taraexpeditions.org). Special thanks also goes to Fabrice Not and Noan Le Bescot for creating the figures of this manu- script. This is contribution number 0001 of the Tara Oceans Expedition 2009–2012. References synthetic reaction center genes and transcripts in the marine environment. ISME J 1: 492–501. efficient method for concentration of ocean viruses by chemical flocculation. Environ Micro- biol Rep 3: 195–202. 1. Karsenti E (2008) Self-organization in cell biology: a brief history. Nat Rev Mol Cell Biol 9: 255–262. synthetic reaction center genes and transcripts in the marine environment. ISME J 1: 492–501. efficient method for concentration of ocean viruses by chemical flocculation. Environ Micro- biol Rep 3: 195–202. 14. Sullivan MB, Lindell D, Lee J, Thompson LR, Bielawski JP, et al. (2006) Prevalence and evolution of core photosystem II genes in marine cyanobacterial viruses and their hosts. PLoS Biol 4: e234. doi:10.1371/journal.pbio.0040234. 2. Keurentjes JJ, Angenent GC, Dicke M, Santos VA, Molenaar J, et al. (2011) Redefining plant systems biology: from cell to ecosystem. Trends Plant Sci 16: 183–190. p 25. Beal LM, De Ruijter WPM, Biastoch A, Zahn R (2011) On the role of the Agulhas system in ocean circulation and climate. Nature 472: 429–436. ( ) g y circulation and climate. Nature 472: 429–436. 26. Follows MJ, Dutkiewicz S, Grant S, Chisholm SW (2007) Emergent biogeography of microbial communities in a model ocean. Science 315: 1843–1846. j p 15. Lupp C (2009) Microbial oceanography. Nature 459: 179. 3. Krakauer DC, Collins JP, Erwin D, Flack JC, Fontana W, et al. (2011) The challenges and scope of theoretical biology. J Theor Biol 276: 269–276. 16. Fuhrman JA (2009) Microbial community struc- ture and its functional implications. Nature 459: 193–199. 27. Raes J, Letunic I, Yamada T, Jensen LJ, Bork P (2011) Toward molecular trait-based ecology through integration of biogeochemical, geograph- ical and metagenomic data. Mol Syst Biol 7: 473. 4. Ne´de´lec F, Surrey T, Karsenti E (2003) Self- organisation and forces in the microtubule cytoskeleton. Curr Opon Cell Biol 15: 118–124. 17. Irigoien X, Huisman J, Harris RP (2004) Global biodiversity patterns of marine phytoplankton and zooplankton. Nature 429: 863–867. 5. Raes J, Bork P (2008) Molecular eco-systems biology: towards an understanding of community function. Nat Rev Microbiol 6: 693–699. 28. d’Ovidio F, De Monte S, Alvain S, Dandonneau Y, Le´vy M (2010) Fluid dynamical niches of phytoplankton types. Proc Natl Acad Sci U S A 107: 18366–18370. p 18. Rohwer F, Thurber RV (2009) Viruses manipu- 18. Rohwer F, Thurber RV (2009) Viruses manipu- late the marine environment. Nature 459: 207–212. 6. Acknowledgments 28 Centre de Formation et de Recherche sur les Environnements Me´diterrane´ens (CE- FREM), Universite´ de Perpignan, Perpignan, France, In addition to the main authors, the Tara Oceans consortium includes the following individuals: 29 Laboratoire d’Oce´anographie et du Cli- mat (LOCEAN), Paris, France, 30 Station Biologique de Roscoff (SBR), Roscoff, France, g Abergel C3,11, Arslan D3,11, Audic S3,5,6, Aury JM15, Babic N3,5,6, Beaufort L3,11,23, Bittner L3,5,6, Boss E24, Boutte C3,5,6, Brum J9, Carmichael M3,5,6, Casotti R14, Chambouvet A25, Chang P3,6,13,26, Chica C15, Clerissi C3,6,27, Colin S3,5,6, Cornejo-Castillo FM2, Da Silva C15, De Monte S3,4, Decelle J3,5,6, Desdevises Y3,6,27, Dimier C3,5,6, Dolan J3,6,13, Duhaime M9, Durrieu de Madron X3,28, d’Ortenzio F3,6,13, d’Ovidio F6,29, Ferrera I2, Garczarek L3,6,30, Garet-Delmas MJ3,5,6, Gasmi S31, Gasol JM2, Grimsley N3,6,27, Heilig R15, Ignacio-Espinoza J9, Jamet JL32, Karp-Boss L24, Katinka M15, Khalili H15, Kolber Z33, Le Bescot N3,5,6, Le Gofff H3,6,34, Lima-Mendez 31 Universite´ de Provence, Marseille, France, 32 PROcessus de Transferts et d’Echanges dans l’Environnement (PROTEE), Universite´ du Sud Toulon-Var (USTV), Toulon, France, 23 Centre Europe´en de Recherche et d’En- seignement des Ge´osciences de l’Environne- ment (CEREGE), Marseille, France, 33 University of California Santa Cruz, Santa Cruz, California, United States of America, 24 The University of Maine, Orono, Maine, United States of America, 34 Laboratoire d’Oce´anographie Dynami- que et de Climatologie (LODYC), Paris, France, 25 Natural History Museum, London, Unit- ed Kingdom, 35 Institut Me´diterrane´en d’Ecologie et de Pale´oe´cologie (IMEP), Marseille, France 26 Biologie de Developpement (BioDev), Villefranche-sur-Mer, France, References Lindell D, Jaffe JD, Johnson ZI, Church GM, Chisholm SW (2005) Photosynthesis genes in marine viruses yield proteins during host infec- tion. Nature 438: 86–89. 24. John SG, Mendez CB, Deng L, Poulos B, Kauffman AKM, et al. (2010) A simple and 13. Sharon I, Tzahor S, Williamson S, Shmoish M, Man-Aharonovich D, et al. (2007) Viral photo- PLoS Biology \| www.plosbiology.org October 2011 \| Volume 9 \| Issue 10 \| e1001177 PLoS Biology \| www.plosbiology.org 5
https://openalex.org/W4229374324	https://zenodo.org/record/6532323/files/IJDOS-2377-8075-09-403.pdf	English	null	A Conception versus Contention of Mandibular Axis	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	4,609	A Conception versus Contention of Mandibular Axis Research Article Manoj Shetty1, NandithaVenkatesh2, Ganaraj Shetty3 1 Professor and Head, Department of oral Implantology, AB Shetty Memorial Institute Of Dental Sciences, NITTE (Deemed to be University), Der- alakatte Mangalore 575018, India. 2 Post Graduate, Department of Prosthodontics and Crown & Bridge, AB Shetty Memorial Institute Of Dental Sciences, NITTE(Deemed to be Uni- versity), Deralakatte Mangalore 575018, India. 3 A i t t P f D t t f P th d ti d C & B id AB Sh tt M i l I tit t Of D t l S i NITTE(D d t b 1 Professor and Head, Department of oral Implantology, AB Shetty Memorial Institute Of Dental Sciences, NITTE (Deemed to be University), Der- alakatte Mangalore 575018, India. alakatte Mangalore 575018, India. 2 Post Graduate, Department of Prosthodontics and Crown & Bridge, AB Shetty Memorial Institute Of Dental Sciences, NITTE(Deemed to be Uni- versity), Deralakatte Mangalore 575018, India. 3 Assistant Professor, Department of Prosthodontics and Crown & Bridge, AB Shetty Memorial Institute Of Dental Sciences, NITTE(Deemed to be University), Deralakatte Mangalore, India. 2 Post Graduate, Department of Prosthodontics and Crown & Bridge, AB Shetty Memorial Institute Of Dental Scien versity), Deralakatte Mangalore 575018, India. g 3 Assistant Professor, Department of Prosthodontics and Crown & Bridge, AB Shetty Memorial Institute Of Dental Sciences, NITTE(Deemed to be University), Deralakatte Mangalore, India. Abstract The mandibular axis is considered to be the axis along which the temporomandibular joint(mandibular condyle) in its terminal, and the retruded position has a purely rotational movement without any translation. Its significance in prosthodontics has been associated with the centric relation position and the fabrication of prosthesis with regards to this position. Its role during functional mandibular movements has therefore been questioned. This has led to various schools of thought on the presence/ absence, location, and the number of the hinge axis with proponents for and against this. This review article aims to discuss the concepts and controversies surrounding the hinge axis and the current clinical significance of this concept. Keywords: Hinge Axis; Terminal Hinge Axis; Centric Relation; Temporomandibular Joint; Condylar Rotation. combination of the gliding and hinge factors making the under- standing of mandibular dynamics confusing. The only position in which the condyle can have a pure rotational or hinge movement is when the condyle is as far as it can go by its muscular power into the glenoid fossa. This is a repeatable and reproducible posi- tion that can be achieved by training the patient andused to deter- mine thecentric relation.[1, 2] https://scidoc.org/IJDOS.php International Journal of Dentistry and Oral Science (IJDOS) ISSN: 2377-8075 Introduction The temporomandibular joint (TMJ) is a complex joint in the head and neck region with the two articular surfaces being formed by the temporal bone and the mandible. The articular eminence and anterior mandibular fossa of the temporal bone form the superior articular surface and the mandibular condyle forms the inferior articular surface. The articular surfaces have a fibrocarti- laginous covering.[1] Any three-dimensional object that moves in a coordinated rota- tional path of motion, which is part of a circle or ellipse, has an axis of rotation and the motion is perpendicular to this axis. (Weinberg, 1959). The imaginary line or axis around which the condyles have pure rotational or hinge motion without translation is known as the hinge axis.[3] The TMJ being aginglymodiarthrodial joint (gliding hinge), is the only one of its type in the body differing in its form and function from other joints. Effectively the meniscus or articular disc sepa- rates it into two joints. The gliding movement takes place in the superior compartment, above the meniscus while the hinge move- ment takes place in the inferior compartment, below the menis- cus. The center of rotation of the condyle and the articular disc coincides such that the meniscus moves along with thecondyle as it traverses anteriorly, posteriorly, or laterally.[2] In a three-dimensional view, there are different axes of rotation of the mandibular condyles based on the plane from which it is viewed and the movement of the condyle is perpendicular to the axis of rotation. In the vertical or sagittal plane, the axis which passes through both condyles is associated with rotation of the mandible and is termed the transverse hinge axis. Rotation in the The opening and closing movement of the mandible is always a Corresponding Author: Prof. (Dr.) Manoj Shetty, Professor and Head, Department of oral Implantology, AB Shetty Memorial Institute Of Dental Sciences, NITTE (Deemed to be University), Deralakatte Mangalore 575018, India. Tel: 09845267087 E-mail: drmanojshetty@nitte.edu.in Received: June 28, 2021 Accepted: April 16, 2022 Published: April 22, 2022 noj Shetty, NandithaVenkatesh, Ganaraj Shetty. A Conception versus Contention of Mandibular Axis. Int J Dentistry Oral Sci. 2022;9(4):5288-5292. Prof. (Dr.) Manoj Shetty, Professor and Head, Department of oral Implantology, AB Shetty Memorial Institute Of Dental Sciences, NITTE (Deemed to be University), Deralakatte Mangalore 575018, India. OPEN ACCESS OPEN ACCESS Clinical Significance As stated by Cohen recording the hinge axis can help to mount study casts to determine if the patient’s centric relation is coin- ciding with centric occlusion. Working casts can be mounted in the best relationship for the teeth or the denture bases. Since the hinge is a fixed component of every closing position of the man- dible, it is necessary to reproduce it on the appropriate instrument if the occlusion is to be rehabilitated. It is possible to increase or decrease the vertical dimension on the instrument without dis- turbing centric relation.[1] Weinberg on the other hand stated that the recording of the hinge axis only helps in orienting the maxilla and determine the static starting point for functional mandibular movements. If the path of motion of a body, in this case, the mandibular condyle, is part of a circle, the axis of rotation itself is not mov- ing. This is observed clinically when it is closing as if on a hinge such as during minimal mouth opening. If the path of motion is an ellipse, then the axis itself moves i.e., when the condyles are translating such as in the wide opening of the mouth. No purpose is served by recording this translatory axis or position without first locating the terminal hinge position which is considered the “starting point”.[3] He emphasized that recording this axis or position does not help in recording either the condylar movements or centric relation.3 There has been much criticism concerning this trained hinge movement as patient function during opening is usually accompa- nied by condylar translation as well; therefore it can be used only to determine the starting point of mandibular opening and not the path of the condyle. [3] Hinge Axis And Centric Relation The loss of teeth in a patient leads to the loss of periodontal proprioception and thereby the loss of the guiding signals to the mandibular musculature during the closure of the jaw. Locating The Hinge Axis [6] Locating The Hinge Axis [6] • Arbitrary hinge axis location by the use of arbitrary face bows. • Location of true hinge axis with kinematic face bows The pattern of proprioceptive stimuli must be reestablished by teaching the patient to move the mandibleas posteriorly as pos- sible, into a repeatable border position. the mandible is out of centric. [5] transverse or horizontal plane and is by the working side condyle and is along the physiologic vertical axis of rotation. The sagittal axis goes through the working side condyle and rotation along this axis is by the balancing condyle with rotation in the frontal plane.[4] transverse or horizontal plane and is by the working side condyle and is along the physiologic vertical axis of rotation. The sagittal axis goes through the working side condyle and rotation along this axis is by the balancing condyle with rotation in the frontal plane.[4] Excessive pressure on the borders of the disc which are innervat- ed can also lead to pain in case of eccentric condylar-disc relation. It can also lead to muscular spasms due to the excess propriocep- tive stimulation by the PDL and TMJ. Multiple authors have defined the hinge axis. According to GPT -9, the Transverse horizontal axis is defined as an imaginary line around which the mandible may rotate within the sagittal plane. Boucher defined it as “An imaginary line between the mandibular condyles around which the mandible can rotate without transla- tory movement” while Heartwell defines it as “an imaginary line around which the condyles can rotate without translation. The opening axis is an imaginary line around which the condyles may rotate during the opening & closing movements of the mandible.” “The hinge position or the terminal hinge position is that position of the mandible from which or in which pure hinge movement of a variably wide range is possible” as described by Sicher.[9] Clinical Significance This is done when the centric relation is recorded as it is assumed that in the retruded position (terminal hinge position) the anteroposterior relation of the mandible to maxilla is the same as the centric relation.[1] Introduction Tel: 09845267087 E-mail: drmanojshetty@nitte.edu.in Received: June 28, 2021 Accepted: April 16, 2022 Published: April 22, 2022 Citation: Manoj Shetty, NandithaVenkatesh, Ganaraj Shetty. A Conception versus Contention of Mandibular Axis. Int J Dentistry Oral Sci. 2022;9(4):5288-5292. doi: http://dx.doi.org/10.19070/2377-8075-220001059 Copyright: Manoj Shetty©2022. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribu- tion and reproduction in any medium, provided the original author and source are credited. Citation: Manoj Shetty, NandithaVenkatesh, Ganaraj Shetty. A Conception versus Contention of Mandibular Axis. Int J Dentistry Oral Sci. 2022;9(4):5288-5 doi: http://dx.doi.org/10.19070/2377-8075-220001059 Copyright: Manoj Shetty©2022. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribu- tion and reproduction in any medium, provided the original author and source are credited. Manoj Shetty, NandithaVenkatesh, Ganaraj Shetty. A Conception versus Contention of Mandibular Axis. Int J Dentistry Oral Sci. 2022;9(4):5288-5292. 5288 https://scidoc.org/IJDOS.php OPEN ACCESS the mandible is out of centric. [5] Manoj Shetty, NandithaVenkatesh, Ganaraj Shetty. A Conception versus Contention of Mandibular Axis. Int J Dentistry Oral Sci. 2022;9(4):5288-5292. Absolute Location of the Hinge Axis [2] • Occlusal discrepancies are well visualized, corrected, and kept to a minimum especially in cases of full mouth rehabilitation, thus increasing the prognosis and patient comfort. This school of thought is based on the philosophy that there is a definite transverse axis that can be located accurately. This can be done with a face bow and is used to mimic the relation of the maxillary cast to the articulator and its transverse similar to how the maxillae are related to the mandibular condyles and the temi- nal axis. [ McCollum (1955), Lucia (1960)][3] Arbitrary Hinge Axis Location As the condyles do not lie in a common plane of orientation with a single possessing of the intercondylar shaft.[2, 13] Beyron found that approximately 87% of the located points were within a 5 mm radius of the arbitrary points.Lauritizen and Bod- ner found only 33% of the true axis points to be located within a 5 mm radius of the arbitrary points.[16] Teteruck and Lundeen found similar results.Walker found that 20% of the true axis points were located within 5 mm from the arbitrarily selected point.[17] DISADVANTAGES[7] • Patient comfort is compromised while recording because of the armamentarium used. • Patient comfort is compromised while recording because of the armamentarium used. • The insertion of clutches might lead to altered position of con- dyle which might interfere with the absolute location. • The insertion of clutches might lead to altered position of con- dyle which might interfere with the absolute location. This will also lead to a similar path of closure intraorally and on the articulator. Some articulators such as Gnathoscope, Hanau, Gnatholator House, Dentatus, Terrell, and Bergstrom Arcon used this priniciple.[10] • It is technique sensitive and warrants remaking. • It can be used only with a fully adjustable articulator. • The procedure is time-consuming Schools Of Thought Schools Of Thought Schools Of Thought Schools Of Thought • The hinge axis location is exact. This leads to decreased chair sie time required for trimming. Manoj Shetty, NandithaVenkatesh, Ganaraj Shetty. A Conception versus Contention of Mandibular Axis. Int J Dentistry Oral Sci. 2022;9(4):5288-5292. CONTROVERSIES [7]  Existence and accurate location of Hinge axis.  A single or Multiple hinge axis exists.  Clinical usefulness regarding the location of the hinge axis.  Clinical usefulness regarding the location of the hinge axis. Movement from the terminal hinge positionis always less than the maximal mouth opening and is a conditioned response. As dis- cussed previously this position is significant because it is a learn- able, repeatable, and recordable position.  Whether an arbitrary point can be substituted for a kinematic axis (Gordon, 1984).  Whether an arbitrary point can be substituted for a kinematic axis (Gordon, 1984). Sloane (1952), Granger (1952), Thompson (1954), Kornfeld (1955), Aull (1963) suggested that there existed only one hinge axis. However, other authors argued the presence of multiple axes. Kurth and Feinstein (1951) said that multiple points may act as hinge points and Beck (1959), Trapozzano, and Lazzari (1967) claimed the presence of multiple hinge axis.[7, 8] The extent of hinge movement, while the condyles are in this position, is approximately 12 to 15 degrees from maximum inter- cuspation or approximately 19 to 20 mm between the upper and lower incisal edges. The condyles occupy a definite position in the mandibularfossae during these terminal hinge movements. [1] The proponents of Gnathology, claimed there was one transverse hinge axis for both condyles that could be determined accurately. The proponents of Transographics, on the other hand, claimed that there is a different transverse hinge axis for each condyle which could only be recorded by a transograph.[7] The chewing cycle cannot occur in centric relation but when the bolus is being prepared for swallowing, maceration of it by the teeth needs a strong muscular force. The condyles, at this point, traverse the path that the fossa anatomy dictates (upward and backward) and try to seat themselves as far as they will go by these muscular forces into the glenoid fossa. The intervention of teeth at this point such as any premature contacts will generate a lateral force proportional to the muscular force and the extent to which Still, others claimedthe impossibility of exact duplication of movements of the mandible and instead to make use of an articu- lator, that utilizes a face bow transfer and several average values to 5289 5289 https://scidoc.org/IJDOS.php Arbitrary Hinge Axis Location Granger (1954) The mandible is capable of an infinite variety of paths of movement; one condyle could be undergoing only rota- tional movement while the other condyle was both rotating and gliding, or both could be rotating and gliding simultaneously. The split hinge rotation was discarded as the condyles were positioned in centric relation only when the mandible was in the most retrud- ed or backward position. Successful treatment depended upon the correct orientation of the teeth to each other and the hinge-axis. [11, 12] The Arbitrary Hinge Axis location is also known as Anatomic technique of locating the position of hinge axis. It is the most commonly used method especially in complete dentures because of the ease of technique. Proponents of this theory said that the determination of the true hinge axis is not essential when one looks at the effort required to find it. This method in conjunction described adequate accuracy for rehabilitation of the oral cavity, without hampering the vertical dimension at occlusion signifi- cantly. The study by Aull in 1963 is representative of the design of single- axis "proof". His design employed 4 styli from one mandibular clutch supporting rod. The proof was demonstrated by the fact that all 4 points located (2 on each side) lay in a straight line and therefore, both condyles must have a common collinear axis.8, 13 Also supported by Brotman (1960), Cohen (1961), Weinberg. [1, 3, 5] The hinge axis was pinpointed arbitrarily based on anatomical marker. Scallhorn found that the hinge axis points were located 13 mm anterior to the distal marginal border of the tragus muscle on the line between tragus – distal orbital line angle within a 5 mm radius of the kinematically located axis in 95% of the individuals. [15] The theory was criticized as they concluded the articulators were designed based on imaginary lines drawing the same midpoint on either side. However, the claim was discarded as mandibular ana- tomical apparatus are bilaterally asymmetrical in size and shape. Non-Believers in Transverse Axis Location As it isn’t an absolute location, a 5mm error around this true hinge axis might lead to an array of occlusal discrepancies, which tend to considerably increase the chairside time. Proponents of this theory stated that the concept of transverse axis is only theoretical and not practical as the location of the transverse hinge axis with accuracy is impossible.[25] Beck (1957) made a comparison of four axes of rotation:[19] Bohr and Posselt were unable to record the hinge axis on a modi- fied Hanau H articulator without any errors. Errors estimated to 1-1.5 at an opening of 10-15 degrees. • Bergstrom's axis: 10 mm anterior to the center of the auditory meatus and 7 mm below Frankfort plane. • Bergstrom's axis: 10 mm anterior to the center of the auditory meatus and 7 mm below Frankfort plane. • Arbitrary axis is given by Gysi: lies online from upper border of external auditory meatus to canthus of the eye, and 13 mm ant of margin of the meatus. Authors like Beck suggested that the opening and closing move- ments of an articulator cannot be replicate the actual movement of a mandible, as the articulator moves only along a single axis • Arbitrary axis is given by Beyron: 13 mm anterior to posterior Margin of tragus, on the tragus-canthus line. In 1962, Shanahan postulated that the artificially produced jaw movements, an axis of the mandible, position of the jaw are not physiologic. No evidence of rotation about a single mandibular axis in the condylar region along with translation anteriorly was found in these studies of the masticating movements as well as opening and closing movements.[23] • The kinematic axis is given by McCollum Within radius of 5 mm, the Bergstrom point is the most favorable with the kinematic points, Next came Beyron's axis points, whereas from the kinematic points, the Gysi point showed a rather greater deviation from the true hinge axis. Non-Believers in Transverse Axis Location Kurth and Feinstein in 1951 mathematically investigated the de- termination of the hinge-axis concluding that owing to all the variables like anatomy, physiology, the ability of the patient to fol- low instructions, operator's prejudice, and perception, it was less likely that the location of the hinge-axis could be accurate.[24] This theory received considerable critics owing to the failure to recognize that in case the hinge axis of the articulator and patient don't coincide, then the path of closure wouldn’t be the same.[2] The critics of this group claimed that the primary motion is pure- ly rotational along with some amount oftranslation, thus adding up to a common center of rotation. The repeatability of this mo- tion makes it a reliable orientation point. [24] Advantages Critics say that unidirectional movement in a single plane can only have one rotational axis as the concept of having two axial centers for the same direction and plane seem contradictory. • Less time-consuming procedure. • The technique is very simple to practice. • The uncomplicated procedure leads to a reduction in errors in location. • The uncomplicated procedure leads to a reduction in errors in location. Considering the anatomy and physiology of the TMJ , the vertical height of the translating condyle would have to change in case the presence of two independent axes should be considered accept- able.[2, 13] • Records almost 5mm around the absolute location by kinematic hinge axis. • Can be used with a semi-adjustable articulator. Split Axis Rotation Proponents of this school of thought follow the ‘Transographic theory’ proposed by Page. The supporters believe in the ‘Split axes where each condyle rotates independently of each other. Owing to the asymmetry present in the mandible the axes are not bilaterally symmetrical and the terminal hinge position mark on either side of the face is slightly higher than its position on the other side, thus concluding that there cannot be a common axis. [20, 21] that no one condyle was placed in symmetry to its opponent.[20] a spherical insert for the auditory meatus and 7 mm below the Frankfort horizontal plane. a spherical insert for the auditory meatus and 7 mm below the Frankfort horizontal plane. that no one condyle was placed in symmetry to its opponent.[20] Harry Page brought a big challenge to the traditional concept of only one inter condylar axis proposing his transographic concept. He hypothesized that every condyle has its axis of rotation and that there exists two, noncolinear, mutually independent axes. Page stated that such independence from the mutual axis is al- lowable anatomically and mechanically possible which can be at- tributed to the flexibility of the mandible.[22]  Beyron’s point - A point 13 mm anterior to the posterior mar- gin of the tragus of the ear on a line from the center of the tragus to the outer canthus of the eye.  Gysi point -10mm anterior to posterior margin of tragus on a line from the center of tragus to the outer canthus of the eye Manoj Shetty, NandithaVenkatesh, Ganaraj Shetty. A Conception versus Contention of Mandibular Axis. Int J Dentistry Oral Sci. 2022;9(4):5288-5292. True Hinge Axis Location Palik, Nelson, and White found that the earpiece face-bow related the maxillary cast to the hinge axis only 50% of the time. 92% of the time the arbitrary axis was located anterior to the terminal hinge axis. Kinematic facebow uses the terminal hinge axis and inferior or- bital rim as reference points.The area of the true hinge axis is located by palpating the subject's condyles during the opening and closing of the mandible.The kinematic method is not the commonly used method of locating hinge axis because of the complexity of the procedure. It is used only in fixed prostheses warranting a reorganized approach.[1] According to Weinberg (1959), the anatomic transverse hinge axis location and the subsequent face bow transfer within a 5 mm error is a practical and dependable method for orientating the maxillary cast. Inter-occlusal centric relation records that limit the interocclusal opening to 6 mm at the incisors produce a negligible error (0.1044 mm at the incisors). The hinge bow or kinematic facebow is used to locate the true hinge axis. A clutch and assembly which has two adjustable pins near the condyles are attached to mandibular teeth. The patient opens and closes in a trained (unstrained) rotational path of motion and when this path of motion is part of a circle (if the condyles do not translate), the pin assembly can be manipulated and adjusted for only rotatory movement. A graph paper is then placed [5, 14] Based on these different authors suggested the use of various anatomical landmarks as posterior reference points to locate the hinge axis arbitrarily with facebows. These reference points in- clude:[18]  Bergstrom’s point - A point 11 mm anterior to the center of j Shetty, NandithaVenkatesh, Ganaraj Shetty. A Conception versus Contention of Mandibular Axis. Int J Dentistry Oral Sci. 2022;9(4):5288-5292. 5290 5290 OPEN ACCESS References g [15]. Schalhorn RG. A study of the arbitrary center and kinematic center of rota- tion for facebow mounting. J Prosthet Dent. 1957;7(2):162-9. g 5]. Schalhorn RG. A study of the arbitrary center and kinematic cen tion for facebow mounting. J Prosthet Dent. 1957;7(2):162-9. [1]. Cohen R. The hinge axis and its practical application in the determination of centric relation. J Prosthet Dent. 1960 Mar 1;10(2):248-57. g [16]. Lauritzen AG, Bodner GH. Variations in location of arbitrary and true hinge axis points. J Prosthet Dent. 1961 Mar 1;11(2):224-9. hinge axis points. J Prosthet Dent. 1961 Mar 1;11(2):224-9. [17]. Walker PM. Discrepancies between arbitrary and true hinge axes. J Prosthet Dent. 1980 Mar;43(3):279-85. PubMed PMID: 6928196. [2]. Singh S, Rehan S, Palaskar J, Mittal S. Hinge axis-location, clinical use and controversies. J Res Dent. 2017;4(6):158-61. h [18]. Kalavathy K, Ananthraj A, Premanth K, Kumar CS. Face bow a caliper- Review article. SRM Journal of Research in Dental Sciences. 2011 Jan 1;2(1):37. [3]. Weinberg LA. The transverse hinge axis: real or imaginary. J Prosthet Dent. 1959 Sep 1;9(5):775-87. [4]. Okeson JP. Management of temporomandibular disorders and occlusion. Mosby. St Louis. 2013:103-22. [19]. Beck HO. A clinical evaluation of the arcon concept of articulation. J Pros- thet Dent. 1959 May 1;9(3):409-21. [5]. Brotman DN. Hinge axes: Part I. The transverse hinge axis. J Prosthet Dent. 1960 May 1;10(3):436-40. [20]. Trapozzano VR. Discussion of the transograph and transographic articula- tion by Jerome M. Schweitzer, DDS. J Prosthet Dent. 1957 Sep 1;7(5):622- 4. y [6]. N Gowri, N Gopichander, E Solomon. The Factor Concept. The Internet Journal of Dental Science. 2008;7(2). Journal of Dental Science. 2008;7(2). [21]. Trapozzano VR, Lazzari JB. The physiology of the terminal rotational posi- tion of the condyles in the temporomandibular joint. J Prosthet Dent. 1967 Feb;17(2):122-33. PubMed PMID: 5225392. [7]. Bainiwal D, Dhiman M. Hinge Axis Recording “The Essential Need In Prosthodontics”. Int J Curr Res. 2018;10:71178-84. g gh Prosthodontics”. Int J Curr Res. 2018;10:71178-84. [8]. Aull AE. A study of the transverse axis. J Prosthet Dent. 1963 May 1;13(3):469-79. hi [22]. Page H. Hinge axis, arguments and typical examples–Proof. Dent Dig. 1960:368. fh [9]. Sicher H. The biologic significance of hinge axis determination. J Prosthet Dent. 1956 Sep 1;6(5):616-20. [23]. Shanahan TE, Leff A. Mandibular and articulator movements: Part III. The mandibular axis dilemma. J Prosthet Dent. 1962 Mar 1;12(2):292-7. [10]. Brekke CA. Jaw function: Part II. Conclusion Despite the numerous studies on the hinge axis, its concept is one of the most discussed controversies in the literature. The varying schools of thought with regards to its existence and its actual location often generate doubt regarding the application of this concept in clinical practice. There have been two axes that are parallel to one another with both axes at right angles to opening and closing movements of the mandible. Owing to the irregular morphology of the condyles they do not have a commonpoint of rotation. Frank in his study of condylar positions using Roentgenographic reports, concluded As Cohen correctly stated that the accurate value of an individ- ual’s work can only be measured in terms of fineness that is re- 5291 5291 OPEN ACCESS https://scidoc.org/IJDOS.php flected in our practice of dentistry rather than which school of thought do we prefer over the other. [13]. Preston JD. A reassessment of the mandibular transverse horizontal axis the- ory. J Prosthet Dent. 2004 Jun;91(6):505-12. PubMed PMID: 15211289. y [14]. Winstanley RB. Hinge axis location on the articulator. J Prosthet Dent. 1979 Aug;42(2):135-44. PubMed PMID: 287795. Manoj Shetty, NandithaVenkatesh, Ganaraj Shetty. A Conception versus Contention of Mandibular Axis. Int J Dentistry Oral Sci. 2022;9(4):5288-5292. References Hinge axis, hinge axes. J Prosthet Dent. 1959 Nov 1;9(6):936-40. [24]. Kurth LE, Feinstein IK. The hinge axis of the mandible. J Prosthet Dent. 1951 May;1(3):327-32. PubMed PMID: 14832862. [11]. Granger ER. Functional relations of the stomatognathic system. JADA. 1954 Jun 1;48(6):638-47. y [25]. Rathee M, Singla S, Kumar A. Philosophy and Physics of Mandibular Hinge Axis: A Review. Med Sci. 2014 Apr;3(4). [12]. Granger ER. Centric Relation. J Prosthet Dent. 1952;2:160-171. Manoj Shetty, NandithaVenkatesh, Ganaraj Shetty. A Conception versus Contention of Mandibular Axis. Int J Dentistry Oral Sci. 2022;9(4):5288-5292. 5292
https://openalex.org/W2170633216	https://europepmc.org/articles/pmc5456173?pdf=render	English	null	Preparation and Characterization of Bioplastic-Based Green Renewable Composites from Tapioca with Acetyl Tributyl Citrate as a Plasticizer	Materials	2,014	cc-by	7,006	Materials 2014, 7, 5617-5632; doi:10.3390/ma7085617 Materials 2014, 7, 5617-5632; doi:10.3390/ma7085617 materials ISSN 1996-1944 www.mdpi.com/journal/materials OPEN ACCESS Preparation and Characterization of Bioplastic-Based Green Renewable Composites from Tapioca with Acetyl Tributyl Citrate as a Plasticizer Chi-Hui Tsou 1,2,3,, Maw-Cherng Suen 4, Wei-Hua Yao 3, Jen-Taut Yeh 5, Chin-San Wu 6, Chih-Yuan Tsou 1, Shih-Hsuan Chiu 1, Jui-Chin Chen 3, Ruo Yao Wang 3, Shang-Ming Lin 3, Wei-Song Hung 2,, Manuel De Guzman 2, Chien-Chieh Hu 2 and Kueir-Rarn Lee 2 Chi-Hui Tsou 1,2,3,, Maw-Cherng Suen 4, Wei-Hua Yao 3, Jen-Taut Yeh 5, Chin-San Wu 6, Chih-Yuan Tsou 1, Shih-Hsuan Chiu 1, Jui-Chin Chen 3, Ruo Yao Wang 3, Shang-Ming Lin 3, Wei-Song Hung 2,, Manuel De Guzman 2, Chien-Chieh Hu 2 and Kueir-Rarn Lee 2 Materials 2014, 7 Materials 2014, 7 PLA/tapioca blends. The addition of MDI could improve the tensile strength of the blend with 60 wt% tapioca, from 19.8 to 42.6 MPa. In addition, because PLA lacked toughness, acetyl tributyl citrate (ATBC) was added as a plasticizer to improve the ductility of PLA. A significant decrease in the melting point and glass-transition temperature was observed on the basis of differential scanning calorimetry, which indicated that the PLA structure was not dense after ATBC was added. As such, the brittleness was improved, and the elongation at break was extended to several hundred percent. Therefore, mixing ATBC with PLA/tapioca/MDI blends did exhibit the effect of plasticization and biodegradation. The results also revealed that excessive plasticizer would cause the migration of ATBC and decrease the tensile properties. Keywords: biodegradable; poly(lactic acid) (PLA); tapioca; methylenediphenyl diisocyanate (MDI); acetyl tributyl citrate (ATBC) Chi-Hui Tsou 1,2,3,, Maw-Cherng Suen 4, Wei-Hua Yao 3, Jen-Taut Yeh 5, Chin-San Wu 6, Chih-Yuan Tsou 1, Shih-Hsuan Chiu 1, Jui-Chin Chen 3, Ruo Yao Wang 3, Shang-Ming Lin 3, Wei-Song Hung 2,, Manuel De Guzman 2, Chien-Chieh Hu 2 and Kueir-Rarn Lee 2 1 Department of Materials Science and Engineering, National Taiwan University of Science and Technology, No. 43, Sec. 4, Keelung Rd., Da’an Dist., Taipei 10607, Taiwan; E-Mails: keymykimo@gmail.com (C.-Y.T.); schiu@mail.ntust.edu.tw (S.-H.C.) 2 Research and Development Center for Membrane Technology, Department of Chemical Engineering, Chung Yuan University, Chung-Li 32023, Taiwan; E-Mails: maweidegz@gmail.com (M.D.G.); cchu@cycu.edu.tw (C.-C.H.); krlee@cycu.edu.tw (K.-R.L.) 3 Department of Materials and Textiles, Oriental Institute of Technology, Pan-Chiao 22064, Taiwan; E-Mails: angelayao@mail.oit.edu.tw (W.-H.Y.); fc011@mail.oit.edu.tw (J.-C.C.); steven5202001@yahoo.com.tw (R.Y.W.); fc013@mail.oit.edu.tw (S.-M.L.) 4 Department of Creative Fashion Design, Taoyuan Innovation Institute of Technology, Jhongli 32091, Taiwan; E-Mail: sun@tiit.edu.tw 5 Department of Chemical and Biochemical Engineering, Kao Yuan University, Kaohsiung County 82151, Taiwan; E-Mail: jyeh@mail.ntust.edu.tw 6 Faculty of Materials Science and Engineering, Hubei University, Wuhan 430062, China; E-Mail: cws1222@cc.kyu.edu.tw * Authors to whom correspondence should be addressed; E-Mails: mayko0301@hotmail.com (C.-H.T.); wshung@cycu.edu.tw (W.-S.H.); Tel.: +886-3-2654190 (C.-H.T. & W.-S.H.); Fax: +886-3-2654198 (C.-H.T. & W.-S.H.). Received: 9 June 2014; in revised form: 25 July 2014 / Accepted: 29 July 2014 / Published: 4 August 2014 Abstract: Granular tapioca was thermally blended with poly(lactic acid) (PLA). All blends were prepared using a plasti-corder and characterized for tensile properties, thermal properties and morphology. Scanning electron micrographs showed that phase separation occurred, leading to poor tensile properties. Therefore, methylenediphenyl diisocyanate (MDI) was used as an interfacial compatibilizer to improve the mechanical properties of 5618 2.1. Tensile Property The tensile strength (σf) and elongation at break (εf) of PLAxTapiocay and PLA90Tapioca10MDI are plotted in Figure 1. The σf of PLA is 48.3 MPa. After blending PLA with tapioca, PLAxtapiocay specimens revealed a substantial reduction in σf and εf. For example, the σf of PLAxTapiocay specimens was reduced from 48.3 to 17.5 MPa as the tapioca content increased from 0 to 60 wt%. When MDI was added, the σf of the PLAxtapiocay specimens was improved; the σf of PLA40Tapioca60MDI was 42.6 MPa, whereas that of PLA40Tapioca60 was 25.1 MPa. This improvement in σf is due to the increase in the PLAxTapiocay interfacial adhesion, as a result of the formation of urethane linkages between MDI and PLA, as well as those between MDI and tapioca, because MDI acts as a coupling agent [21]. The εf for all samples are between 1% and 4%; thus, their toughness is similar. Figure 1. Tensile strength vs. tapioca content for (▲) PLA/tapioca and (■) PLA/tapiocaMDI0.5; elongation at break vs. tapioca content for (△) PLA/tapioca and (□) PLA/tapiocayMDI0.5. The σf and εf of PLA and PLAxtapiocayMDI specimens as a function of ATBC are shown in Figures 2 and 3, respectively. With increasing plasticizer content, a common trend is shown by all series investigated: the σf decreases, whereas the εf increases. The σf of PLAxtapiocayMDI was reduced much more significantly after ATBC was added; the σf value of PLA50tapioca50MDI was reduced from 42.3 to 0.9 MPa, as the ATBC content was increased from 0 to 25 wt% (see Figure 2). This substantial reduction is due to the tapioca of PLAxtapiocayMDI that could not be plasticized by ATBC. In addition, the εf of PLA90tapioca10MDI, PLA80tapioca20MDI, PLA70tapioca30MDI, PLA60tapioca40MDI The σf and εf of PLA and PLAxtapiocayMDI specimens as a function of ATBC are shown in Figures 2 and 3, respectively. With increasing plasticizer content, a common trend is shown by all series investigated: the σf decreases, whereas the εf increases. The σf of PLAxtapiocayMDI was reduced much more significantly after ATBC was added; the σf value of PLA50tapioca50MDI was reduced from 42.3 to 0.9 MPa, as the ATBC content was increased from 0 to 25 wt% (see Figure 2). This substantial reduction is due to the tapioca of PLAxtapiocayMDI that could not be plasticized by ATBC. Materials 2014, 7 The result of this study could provide a database useful for the design and manufacture of biodegradable materials. 1. Introduction Poly(lactic acid) (PLA) resins are well-known biodegradable, linear aliphatic thermoplastics, which can be produced from renewable resources [1,2]. They are one of the most promising polymers of their family [3] and are highly accepted as biomedical materials, because of their biocompatibility and good mechanical properties [4,5]. However, their brittleness, slow crystallization and being easily hydrolyzed limit their usage in many applications. In fact, it is difficult to use them for film blowing or extrusion, unless their moisture content and processing conditions are carefully controlled. Moreover, their price competitiveness in the biodegradable plastic market is an essential attribute that cannot be ignored. The most effective approach to reduce the capital cost of PLA is to use fillers. Cost-effective reinforcements are organic renewable resources [6], flax [7–9], sisal [10], lyocell [11], short abaca [12], jute [13], bamboo [14], paper pulp [15,16], pineapple [17], Cordenka [18], microcrystalline cellulose [19], and kenaf [20]. Starch is attractive because of its low cost, renewability, biodegradability, low density and non-abrasiveness. A lot of studies on the blending of PLA/starch [21–30], such as wheat starch [21,24,30], corn starch [26,27,29,31] and cassava starch [23], have been researched. Tapioca was used as a filler in [32], because it is cheap, and fewer reports compared it with other starches. However, the poor interfacial adhesion between the filler and the polymer generally leads to composites with worse mechanical properties. Surface and bulk modifications of the filler and/or matrix are necessary to increase the interfacial compatibility between the hydrophilic filler and the hydrophobic PLA matrix. Some studies used methylenediphenyl diisocyanate (MDI) as a compatibilizer to improve the compatibility between PLA and starch [21,30] or between PLA and rice husk [33]. These biopolymers are successfully prepared with starch or rice husk blends using MDI as a coupling agent. Copolymerization or blending PLA with other polymers [34–43] or compounds (e.g., plasticizers) [44–49] was proven to be a feasible way to improve its processability in film products for extrusion and/or film blowing. In this study, tapioca was used as a filler to reduce the cost of PLA products; MDI was used as a coupling agent to enhance the interfacial compatibility between PLA and tapioca; and ATBC was used as a plasticizer to improve the processability, flexibility and ductility of glassy PLA/tapioca composites. 5619 2.1. Tensile Property In addition, the εf of PLA90tapioca10MDI, PLA80tapioca20MDI, PLA70tapioca30MDI, PLA60tapioca40MDI The σf and εf of PLA and PLAxtapiocayMDI specimens as a function of ATBC are shown in Figures 2 and 3, respectively. With increasing plasticizer content, a common trend is shown by all series investigated: the σf decreases, whereas the εf increases. The σf of PLAxtapiocayMDI was reduced much more significantly after ATBC was added; the σf value of PLA50tapioca50MDI was reduced from 42.3 to 0.9 MPa, as the ATBC content was increased from 0 to 25 wt% (see Figure 2). This substantial reduction is due to the tapioca of PLAxtapiocayMDI that could not be plasticized by ATBC. In addition, the εf of PLA90tapioca10MDI, PLA80tapioca20MDI, PLA70tapioca30MDI, PLA60tapioca40MDI Materials 2014, 7 5620 Materials 2014, 7 5621 Materials 2014, 7 Materials 2014, 7 Materials 2014, 7 Materials 2014, 7 and PLA50tapioca50MDI approaches the maximum value at 357.3%, 289.7%, 178.8%, 102.5% and 56.3%, respectively, as the ATBC content reaches an optimum value of 10 or 15 wt%. The εf of the PLAxtapiocayMDI specimens was reduced as the ATBC content increased from 10 or 15 wt%. These results clearly suggest that the relatively poor ductility of PLAxtapiocayMDI was improved after blending proper amounts of ATBC with the PLAxTapiocayMDI composites. Figure 2. Tensile strength vs. ATBC content for (■) PLA; (○) PLA90tapioca10MDI0.5; (□) PLA80tapioca20MDI0.5; (◇) PLA70tapioca30MDI0.5; (△) PLA60tapioca40MDI0.5; and (×) PLA50tapioca50MDI0.5. Figure 3. Elongation at break vs. ATBC content for (■) PLA; (○) PLA90tapioca10MDI0.5; (□) PLA80tapioca20MDI0.5; (◇) PLA70tapioca30MDI0.5; (△) PLA60tapioca40MDI0.5; and (×) PLA50tapioca50MDI0.5. Figure 3. Elongation at break vs. ATBC content for (■) PLA; (○) PLA90tapioca10MDI0.5; (□) PLA80tapioca20MDI0.5; (◇) PLA70tapioca30MDI0.5; (△) PLA60tapioca40MDI0.5; and (×) PLA50tapioca50MDI0.5. Materials 2014, 7 2.2. Fourier Transform Infra-Red Spectroscopy Figure 4 illustrates typical FTIR spectra of PLA, tapioca, MDI, PLAxtapiocay and PLAxtapiocayMDI specimens. Four characteristic absorption bands centered at 1750, 2950, 2995 and 3510 cm−1, corresponding to the motions of C=O bending, C-H aliphatic stretching, C-H aliphatic stretching (doublet) and C-O-O-H stretching vibrations, respectively, were found in the spectrum of PLA (see Figure 4a). The FTIR spectra of PLAxtapiocay specimens, indicated in Figure 4c, are very similar to those of PLA; the four main absorption bands centered at 1750, 2950, 2995 and 3510 cm−1 were also found in the spectra of PLAxtapiocay specimens. The absorption bands around 3000 to 3670 cm−1 were the O-H stretching vibration of tapioca. The FTIR spectra of PLAxtapiocay specimens are very similar to those of PLAxtapiocayMDI (see Figure 4d). However, the aforementioned 3000 to 3670 cm−1 absorption bands originally shown in the FTIR spectra of PLA were gradually replaced by a newly developed absorption band centered at 3315 cm−1, which corresponds to the motion of the amine (N-H) stretching vibration. The disappearance of the 3000 to 3670 cm−1 bending absorption bands and the appearance of 3315 and 1550 cm−1 (N-H) stretching absorption bands are attributed to the reaction of the hydroxyl (O-H) groups of tapioca molecules with the urethane (N=C=O) groups of MDI and/or to the reaction of the carboxylic acid (C-O-O-H) groups of PLA molecules with the urethane groups of MDI during the melt-blending of PLAxtapiocay specimens. The possible mechanism for PLA/tapioca/MDI composites is shown in Scheme 1. Figure 4. FTIR of: (a) PLA; (b) tapioca; (c) PLA/tapioca; (d) PLA/tapioca/MDI; and (e) MDI. gure 4. FTIR of: (a) PLA; (b) tapioca; (c) PLA/tapioca; (d) PLA/tapioca/MDI; and (e) M FTIR of: (a) PLA; (b) tapioca; (c) PLA/tapioca; (d) PLA/tapioca/MDI; and (e) MDI. Materials 2014, 7 Materials 2014, 7 5622 Scheme 1. Possible reactions for PLA/tapioca/MDI composites. 2.3. Morphology Analysis 2.3. Morphology Analysis [51], who analyzed PLA/triacetin (TAc) and PLA/ATBC composites, respectively, over a more limited composition range (0%–30% TAc and ATBC) A decreasing Tg trend with increasing ATBC content is also shown in Figure 6, which is related to the cold crystallization and melting phenomena. (g) (PLA70tapioca30MDI0.5)85ATBC15; (h) (PLA70tapioca30MDI0.5)80ATBC20; and (i) (PLA70tapioca30MDI0.5)75ATBC25. 2.4. Differential Scanning Calorimetry The thermal behavior of PLA and (PLA70tapioca30MDI)aATBCb specimens was investigated. Figure 6 shows the DSC curves of PLA, the (PLA70tapioca30MDI)aATBCb specimens and the plasticizer. The crystallization and melting enthalpies are identical, showing that PLA is totally amorphous after melt quenching. The DSC curves of PLA70tapioca30MDI show a single glass transition that decreases with increasing ATBC concentration (from 0 to 25 wt%). This result agrees with the data by Yeh et al. [50] and Baiardo et al. [51], who analyzed PLA/triacetin (TAc) and PLA/ATBC composites, respectively, over a more limited composition range (0%–30% TAc and ATBC). A decreasing Tg trend with increasing ATBC content is also shown in Figure 6, which is related to the cold crystallization and melting phenomena. 2.4. Differential Scanning Calorimetry The thermal behavior of PLA and (PLA70tapioca30MDI)aATBCb specimens was investigated. Figure 6 shows the DSC curves of PLA, the (PLA70tapioca30MDI)aATBCb specimens and the plasticizer. The crystallization and melting enthalpies are identical, showing that PLA is totally amorphous after melt quenching. The DSC curves of PLA70tapioca30MDI show a single glass transition that decreases with increasing ATBC concentration (from 0 to 25 wt%). This result agrees with the data by Yeh et al. [50] and Baiardo et al. [51], who analyzed PLA/triacetin (TAc) and PLA/ATBC composites, respectively, over a more limited composition range (0%–30% TAc and ATBC). A decreasing Tg trend with increasing ATBC content is also shown in Figure 6, which is related to the cold crystallization and melting phenomena. The thermal behavior of PLA and (PLA70tapioca30MDI)aATBCb specimens was investigated. Figure 6 shows the DSC curves of PLA, the (PLA70tapioca30MDI)aATBCb specimens and the plasticizer. The crystallization and melting enthalpies are identical, showing that PLA is totally amorphous after melt quenching. The DSC curves of PLA70tapioca30MDI show a single glass transition that decreases with increasing ATBC concentration (from 0 to 25 wt%). This result agrees with the data by Yeh et al. [50] and Baiardo et al. [51], who analyzed PLA/triacetin (TAc) and PLA/ATBC composites, respectively, over a more limited composition range (0%–30% TAc and ATBC). 2.3. Morphology Analysis 2.3. Morphology Analysis Typical SEM micrographs of (PLA70tapioca30MDI)aATBCb specimens are shown in Figure 5. As shown in Figure 5a, a relatively brittle and smooth surface morphology was found for PLA. Tapioca is shown in Figure 5b. After blending PLA with tapioca, intervals between PLA and tapioca granules appeared (see Figure 5c) These results are similar to some reports on PLA/wheat starch [21] and PLA/corn starch blends [27,29]. This morphology is typical of incompatible blends, resulting in a poor tensile property, which is consistent with the result in Figure 1. After MDI was added, the compatibility of the PLA70tapioca30 specimen was improved (see Figure 5d). It shows excellent compatible morphologies, without the interval and voids associated with poor interfacial adhesion. The better compatibility of PLA70tapioca30MDI was due to the reaction of the hydroxyl groups of tapioca with the urethane groups of MDI and the reaction of the carboxylic acid groups of PLA with the urethane groups of MDI. The SEM micrographs of PLA70tapioca30MDI as a function of the increasing ATBC content (from 5 to 25 wt%) are shown in Figure 5e–h. The surface of PLA70tapioca30MDI was still smooth and without intervals when the ATBC content was 5 wt%. Furthermore, two phases can be seen after the ATBC content reaches 10 wt%, which is the threshold limit value of high εf for (PLA70tapioca30MDI)aATBCb specimens (see Figure 3). With increasing ATBC content, more demarcated plastic-deformed PLA debris or fibrils were found on the surface of PLA70Tapioca30MDI (see Figure 5f–i). This was attributed to the increasing distance between PLA molecules, or the exudation of ATBC, and also to the deterioration of the interfacial adhesion between tapioca and PLA. Therefore, the σf of PLAxtapiocayMDI decreased significantly with increasing ATBC. 5623 Materials 2014, 7 Materials 2014, 7 (g) (PLA70tapioca30MDI0.5)85ATBC15; (h) (PLA70tapioca30MDI0.5)80ATBC20; and (i) (PLA70tapioca30MDI0.5)75ATBC25. 2.4. Differential Scanning Calorimetry The thermal behavior of PLA and (PLA70tapioca30MDI)aATBCb specimens was investigated Figure 6 shows the DSC curves of PLA, the (PLA70tapioca30MDI)aATBCb specimens and the plasticizer. The crystallization and melting enthalpies are identical, showing that PLA is totally amorphous after melt quenching. The DSC curves of PLA70tapioca30MDI show a single glas transition that decreases with increasing ATBC concentration (from 0 to 25 wt%). This result agrees with the data by Yeh et al. [50] and Baiardo et al. 2.5. Water Absorption 2.5. Water Absorption Figure 7 presents data on the water absorption of the PLAxATBCy specimens as a function of varying tapioca content. It shows a rising water absorption rate with increasing tapioca content at the same ATBC content. Water absorption rates were 0.58% to 18.57% when the tapioca content was from 0% to 50% at 25% ATBC content. This is due to the hydrophilic property of tapioca, causing the percentage of water absorption to increase. Furthermore, water absorption rates increased when ATBC was added; the water absorption rate of PLA60tapioca40 was 4.91% to 11.85% when the ATBC content was from 0% to 25%. ATBC is hydrophobic, but shows an interesting phenomenon of increasing moisture content. The trend of increasing water absorption is attributed to ATBC, which could enhance the free volume in PLA. This is evidenced by DSC analysis (see Figure 6): Tg decreased with increasing ATBC content. Therefore, the water molecule could be absorbed by PLAxtapiocay easily when the ATBC content increased the mobility of PLA molecules. The water adsorption for the lower tapioca content (10% to 30%) was first increasing and then decreasing. This might be due to slight exudation when the ATBC content is from 10% to 25%. It could be evidenced from the morphology analysis. The phase separation may be because of the exudation of ATBC; the phase separation worsens when the amount of ATBC was higher than 10% (See Figure 5f–i). However, when the tapioca content approached 40% and 50%, the ATBC might be absorbed by tapioca. Therefore, the exudation effect might not be apparent. On the basis of the results from the tensile property, Figure 7 presents data on the water absorption of the PLAxATBCy specimens as a function of varying tapioca content. It shows a rising water absorption rate with increasing tapioca content at the same ATBC content. Water absorption rates were 0.58% to 18.57% when the tapioca content was from 0% to 50% at 25% ATBC content. This is due to the hydrophilic property of tapioca, causing the percentage of water absorption to increase. Furthermore, water absorption rates increased when ATBC was added; the water absorption rate of PLA60tapioca40 was 4.91% to 11.85% when the ATBC content was from 0% to 25%. ATBC is hydrophobic, but shows an interesting phenomenon of increasing moisture content. The trend of increasing water absorption is attributed to ATBC, which could enhance the free volume in PLA. 2.3. Morphology Analysis A decreasing Tg trend with increasing ATBC content is also shown in Figure 6, which is related to the cold crystallization and melting phenomena. 5624 Materials 2014, 7 Figure 6. DSC for: (a) PLA; (b) PLA70tapioca30MDI; (c) (PLA70tapioca30MD)95ATBC5; (d) (PLA70tapioca30MDI)90ATBC10; (e) (PLA70tapioca30)85ATBC15; (f) (PLA70tapioca30MDI)aATBCb; and (g) (PLA70tapioca30MDI)75ATBC25. 2.5. Water Absorption Figure 6. DSC for: (a) PLA; (b) PLA70tapioca30MDI; (c) (PLA70tapioca30MD)95ATBC5; (d) (PLA70tapioca30MDI)90ATBC10; (e) (PLA70tapioca30)85ATBC15; (f) (PLA70tapioca30MDI)aATBCb; and (g) (PLA70tapioca30MDI)75ATBC25. Figure 6. DSC for: (a) PLA; (b) PLA70tapioca30MDI; (c) (PLA70tapioca30MD)95ATBC5; (d) (PLA70tapioca30MDI)90ATBC10; (e) (PLA70tapioca30)85ATBC15; (f) (PLA70tapioca30MDI)aATBCb; and (g) (PLA70tapioca30MDI)75ATBC25. 2.5. Water Absorption Figure 7 presents data on the water absorption of the PLAxATBCy specimens as a function of varying tapioca content. It shows a rising water absorption rate with increasing tapioca content at the same ATBC content. Water absorption rates were 0.58% to 18.57% when the tapioca content was from 0% to 50% at 25% ATBC content. This is due to the hydrophilic property of tapioca, causing the percentage of water absorption to increase. Furthermore, water absorption rates increased when ATBC was added; the water absorption rate of PLA60tapioca40 was 4.91% to 11.85% when the ATBC content was from 0% to 25%. ATBC is hydrophobic, but shows an interesting phenomenon of increasing moisture content. The trend of increasing water absorption is attributed to ATBC, which could enhance the free volume in PLA. This is evidenced by DSC analysis (see Figure 6): Tg decreased with increasing ATBC content. Therefore, the water molecule could be absorbed by PLAxtapiocay easily when the ATBC content increased the mobility of PLA molecules. The water adsorption for the lower tapioca content (10% to 30%) was first increasing and then decreasing. This might be due to slight exudation when the ATBC content is from 10% to 25%. It could be evidenced from the morphology analysis. The phase separation may be because of the exudation of ATBC; the phase separation worsens when the amount of ATBC was higher than 10% (See Figure 5f–i). However, when the tapioca content approached 40% and 50%, the ATBC might be absorbed by tapioca. Therefore, the exudation effect might not be apparent. On the basis of the results from the tensile property, Materials 2014, 7 5625 Materials 2014, 7 morphology analysis and water absorption, the miscibility limits of ATBC content are suggested to be 10% for PLAxtapiocayMDI0.5 composites. morphology analysis and water absorption, the miscibility limits of ATBC content are suggested to be 10% for PLAxtapiocayMDI0.5 composites. p gy y p , y gg 10% for PLAxtapiocayMDI0.5 composites. Figure 7. Water absorption vs. ATBC content for (●) PLA; (○) PLA90tapioca10MDI0.5; (□) PLA80tapioca20MDI0.5; (◇) PLA70tapioca30MDI0.5; (△) PLA60tapioca40MDI0.5; and (×) PLA50tapioca50MDI0.5. 2.6. Enzymatic Hydrolysis The weight loss of PLA70tapioca30MDI and (PLA70tapioca30MDI)aATBCb at varying enzymatic hydrolysis time is indicated in Figure 8. It shows a common result: the increasing percentage of weight loss for all series, as the hydrolysis time increases. The weight loss of PLA70tapioca30MDI increased significantly from 0.33% to 11.82%, as the ATBC content increased from 0 to 25 wt% after 120 h of hydrolysis time. Enzymes could attack the molecules of PLA easily after ATBC was blended with PLA70tapioca30MDI. Furthermore, the interfacial adhesion between PLA and tapioca might deteriorate when ATBC was added. The weight loss increased significantly when the ATBC content was higher than 5%. The migration of ATBC occurred from 10% content. This conjecture of exudation was evidenced by the morphological analysis and water absorption. Figure 7. Water absorption vs. ATBC content for (●) PLA; (○) PLA90tapioca10MDI0.5; (□) PLA80tapioca20MDI0.5; (◇) PLA70tapioca30MDI0.5; (△) PLA60tapioca40MDI0.5; and (×) PLA50tapioca50MDI0.5. Figure 7. Water absorption vs. ATBC content for (●) PLA; (○) PLA90tapioca10MDI0.5; (□) PLA80tapioca20MDI0.5; (◇) PLA70tapioca30MDI0.5; (△) PLA60tapioca40MDI0.5; and (×) PLA50tapioca50MDI0.5. 2.6. Enzymatic Hydrolysis 2.5. Water Absorption This is evidenced by DSC analysis (see Figure 6): Tg decreased with increasing ATBC content. Therefore, the water molecule could be absorbed by PLAxtapiocay easily when the ATBC content increased the mobility of PLA molecules. The water adsorption for the lower tapioca content (10% to 30%) was first increasing and then decreasing. This might be due to slight exudation when the ATBC content is from 10% to 25%. It could be evidenced from the morphology analysis. The phase separation may be because of the exudation of ATBC; the phase separation worsens when the amount of ATBC was higher than 10% (See Figure 5f–i). However, when the tapioca content approached 40% and 50%, the ATBC might be absorbed by tapioca. Therefore, the exudation effect might not be apparent. On the basis of the results from the tensile property, 2.6. Enzymatic Hydrolysis The weight loss of PLA70tapioca30MDI and (PLA70tapioca30MDI)aATBCb at varying enzymatic hydrolysis time is indicated in Figure 8. It shows a common result: the increasing percentage of weight loss for all series, as the hydrolysis time increases. The weight loss of PLA70tapioca30MDI increased significantly from 0.33% to 11.82%, as the ATBC content increased from 0 to 25 wt% after 120 h of hydrolysis time. Enzymes could attack the molecules of PLA easily after ATBC was blended with PLA70tapioca30MDI. Furthermore, the interfacial adhesion between PLA and tapioca might deteriorate when ATBC was added. The weight loss increased significantly when the ATBC content was higher than 5%. The migration of ATBC occurred from 10% content. This conjecture of exudation was evidenced by the morphological analysis and water absorption. Materials 2014, 7 5626 Figure 8. Weight loss for (●) PLA; (○) PLA70tapioca30MDI0.5; (□) (PLA70tapioca30MDI0.5)95ATBC5; (◇) (PLA70tapioca30MDI0.5)90ATBC10; (△) (PLA70tapioca30MDI0.5)85ATBC15; (×) (PLA70tapioca30MDI0.5)80ATBC20; and (+) (PLA70tapioca30MDI0.5)75ATBC25. 3. Experimental Section 3.1. Materials and Preparation PLA resin, with a trade name of Nature Green 4032D, was obtained from Cargill-Dow. Tapioca was purchased from United Global Agencies (Bangkok, Thailand). Before melt-blending, PLA and tapioca were dried in a vacuum oven at 80 °C for 8 h to remove the residual water. Acetyl tributyl citrate (ATBC-food grade) was supplied by Chou Feng Enterprise Co., Ltd. (Shulin, Taiwan). Dried components of PLA/starch at varying weight ratios were melt-blended using a Brabender. Three compounds were evaluated: PLAxTapiocay, PLAxTapiocayMDI and (PLAxTapiocayMDI)aATBCb. During each compounding process, the Brabender was operated at a temperature of 190 °C and a screw speed of 120 rpm for 3 min for all samples without ATBC and for an additional 2 min after adding ATBC. All prepared series were then hot-pressed at 190 °C and 10 MPa for 2 min and then cooled in air at about 25 °C. The compositions of all specimens are summarized in Table 1. Before hot-pressing, the specimens were dried in a vacuum oven at 80 °C for 12 h. Materials 2014, 7 Materials 2014, 7 5626 Figure 8. Weight loss for (●) PLA; (○) PLA70tapioca30MDI0.5; (□) (PLA70tapioca30MDI0.5)95ATBC5; (◇) (PLA70tapioca30MDI0.5)90ATBC10; (△) (PLA70tapioca30MDI0.5)85ATBC15; (×) (PLA70tapioca30MDI0.5)80ATBC20; and (+) (PLA70tapioca30MDI0.5)75ATBC25. 3.1. Materials and Preparation PLA resin, with a trade name of Nature Green 4032D, was obtained from Cargill-Dow. Tapioca was purchased from United Global Agencies (Bangkok, Thailand). Before melt-blending, PLA and tapioca were dried in a vacuum oven at 80 °C for 8 h to remove the residual water. Acetyl tributyl citrate (ATBC-food grade) was supplied by Chou Feng Enterprise Co., Ltd. (Shulin, Taiwan). Dried components of PLA/starch at varying weight ratios were melt-blended using a Brabender. Three compounds were evaluated: PLAxTapiocay, PLAxTapiocayMDI and (PLAxTapiocayMDI)aATBCb. During each compounding process, the Brabender was operated at a temperature of 190 °C and a screw speed of 120 rpm for 3 min for all samples without ATBC and for an additional 2 min after adding ATBC. All prepared series were then hot-pressed at 190 °C and 10 MPa for 2 min and then cooled in air at about 25 °C. The compositions of all specimens are summarized in Table 1. Before hot-pressing, the specimens were dried in a vacuum oven at 80 °C for 12 h. Table 1. Compositions of PLA, PLAxtapiocay, PLAxtapiocayMDI and (PLAxtapiocayMDI)aATBCb specimens. Sample PLA (%) Tapioca (%) ATBC (%) MDI (phr) PLA 100 0 0 0 PLA90tapioca10 90 10 0 0 PLA80tapioca20 80 20 0 0 PLA70tapioca30 70 30 0 0 1. Compositions of PLA, PLAxtapiocay, PLAxtapiocayMDI and tapiocayMDI)aATBCb specimens. Sample PLA (%) Tapioca (%) ATBC (%) MDI (phr) PLA 100 0 0 0 PLA90tapioca10 90 10 0 0 PLA80tapioca20 80 20 0 0 PLA70tapioca30 70 30 0 0 Materials 2014, 7 5627 Table 1. Cont. Table 1. Cont. 3.1. Materials and Preparation Sample PLA (%) Tapioca (%) ATBC (%) MDI (phr) PLA60tapioca40 60 40 0 0 PLA50tapioca50 50 50 0 0 PLA40tapioca60 0 60 0 0 PLA90tapioca10MDI 90 10 0 0.5 PLA80tapioca20MDI 80 20 0 0.5 PLA70tapioca30MDI 70 30 0 0.5 PLA60tapioca40MDI 60 40 0 0.5 PLA50tapioca50MDI 50 50 0 0.5 PLA60tapioca40MDI 40 60 0 0.5 (PLA90tapioca10MDI)95ATBC5 85.5 9.5 5 0.5 (PLA80tapioca20MDI)95ATBC5 76 19 5 0.5 (PLA70tapioca30MDI)95ATBC5 66.5 28.5 5 0.5 (PLA60tapioca40MDI)95ATBC5 57 38 5 0.5 (PLA50tapioca50MDI)95ATBC5 47.5 47.5 5 0.5 (PLA90tapioca10MDI)90ATBC10 81 9 10 0.5 (PLA80tapioca20MDI)90ATBC10 72 18 10 0.5 (PLA70tapioca30MDI)90ATBC10 63 27 10 0.5 (PLA60tapioca40MDI)90ATBC10 54 36 10 0.5 (PLA50tapioca50MDI)90ATBC10 45 45 10 0.5 (PLA90tapioca10MDI)85ATBC15 76.5 8.5 15 0.5 (PLA80tapioca20MDI)85ATBC15 68 17 15 0.5 (PLA70tapioca30MDI)85ATBC15 59.5 25.5 15 0.5 (PLA60tapioca40MDI)85ATBC15 51 34 15 0.5 (PLA50tapioca50MDI)85ATBC15 42.5 42.5 15 0.5 (PLA90tapioca10MDI)80ATBC20 72 8 20 0.5 (PLA80tapioca20MDI)80ATBC20 64 16 20 0.5 (PLA70tapioca30MDI)80ATBC20 56 24 20 0.5 (PLA60tapioca40MDI)80ATBC20 48 32 20 0.5 (PLA50tapioca50MDI)80ATBC20 40 40 20 0.5 (PLA90tapioca10MDI)75ATBC25 67.5 7.5 25 0.5 (PLA80tapioca20MDI)75ATBC25 60 15 25 0.5 (PLA70tapioca30MDI)75ATBC25 52.2 22.5 25 0.5 (PLA60tapioca40MDI)75ATBC25 45 30 25 0.5 (PLA50tapioca50MDI)75ATBC25 37.5 37.5 25 0.5 3.5. Differential Scanning Calorimetry The thermal properties of PLA composite resins were determined using a TA Q100 differential scanning calorimetry (DSC). All DSC scans were performed at a heating rate of 10 °C/min and under flowing nitrogen with a flow rate of 50 mL/min. The DSC was calibrated using pure indium. For Tg and Tm determination, samples weighing approximately 0.5 mg were placed in standard aluminum-sample pans. 3.3. Fourier Transform Infra-Red Spectroscopy FTIR measurements were performed on a PerkinElmer spectrometer (model Spectrum One, PerkinElmer Inc., Waltham, MA, USA). The spectra of the samples were obtained by averaging 15 scans, with a wavenumber range of 4000 to 650 cm−1 and a resolution of 2 cm−1. Materials 2014, 7 5628 Materials 2014, 7 3.6. Water Absorption Five specimens (10 mm × 10 mm × 0.5 mm) were used for the water absorption test. After conditioning in desiccators for three weeks, specimens were weighed. They were immersed in distilled water at room temperature for 24 h. Then, they were dabbed with tissue paper to remove the water from the surface. Water absorption was calculated using the following Equation (1): (1) (1) where W0 is the weight of the dry sample and W1 is the weight of the sample immersed in distilled water for 24 h. where W0 is the weight of the dry sample and W1 is the weight of the sample immersed in distilled water for 24 h. 3.4. Morphology Analysis The morphology of specimens, before and after the hydrolytic degradation, was observed by using a scanning electron microscope (model SU1510, Hitachi High-Technologies Corporation, Tokyo, Japan). Specimens of a 2 × 2 cm2 area were fixed on a sample holder using a conductive adhesive tape. They were coated with a thin layer of gold at 15 keV for 15 s to improve the image resolution and were then photographed at 3.00 K magnification and a low voltage of 2.1 kV. 3.2. Tensile Property The tensile properties of the hot-pressed PLA, PLAxTapiocay and (PLAxTapiocay)/ATBC specimens at 25 °C were determined using a tensile testing machine (model AG-10KNA, Shimadzu Corporation, Kyoto, Japan) with a crosshead speed of 50 mm/min. A 35-mm gauge length was used for each tensile experiment. Dog-bone-shaped specimens were prepared according to the ASTM D638 Type IV standard [52]. On the basis of the average tensile results of at least five tensile specimens, the values of tensile strength and elongation at break were obtained. Materials 2014, 7 Materials 2014, 7 Materials 2014, 7 Materials 2014, 7 5629 4. Conclusions The σf of PLAxtapiocayMDI specimens was significantly higher than that of PLAxtapiocay specimens. The εf of PLA and PLAxtapiocayMDI specimens approached the maximum value, as the ATBC content reached an optimum value of 10 and 15 wt%, respectively. The threshold limits of the εf were high when the ATBC content was 10 wt%. FTIR demonstrated the disappearance of the 3000 to 3670 cm−1 bending absorption band and the appearance of the 3315 and 1550 cm−1 NH stretching absorption band, which were attributed to the reaction of the OH groups of tapioca molecules with the N=C=O groups of MDI and/or to the reaction of the C-O-O-H groups of PLA molecules with the urethane groups of MDI during the melt-blending of PLAxtapiocay specimens. SEM micrographs revealed the intervals between PLA and tapioca. Voids from the matrix of PLAxtapiocay were significantly improved after MDI was added. Furthermore, two phases can be seen after the ATBC content reached 10 wt%. This is due to the exudation of ATBC; with increasing ATBC content, more demarcated plastic deformation was found on the surface of PLA70Tapioca30MDI. DSC curves of the PLA70tapioca30MDI specimen showed a single glass transition and cold crystallization that decreased as the ATBC content increased from 0 to 25 wt%. The increasing trend of water absorption with increasing ATBC content was attributed to the increasing free volume in PLA, causing the water molecules to be easily absorbed in the PLAxtapiocay specimens. Enzymatic hydrolysis tests indicated that the weight loss of PLA70tapioca30MDI increased significantly as the ATBC content increased. Author Contributions All authors contributed to this study. Chi-Hui Tsou and Wei-Song Hung designed the research and wrote this paper. Chi-Hui Tsou, Maw-Cherng Suen, and Chih-Yuan Tsou edited the paper and gave final approval of the version to be submitted. Chih-Yuan Tsou and Ruo Yao Wang did the analysis. Manuel De Guzman, Chien-Chieh Hu, and Kueir-Rarn Lee also contributed in analyzing data and in rewriting the revised manuscript. Jen-Taut Yeh, Wei-Hua Yao, Chin-San Wu, Shih-Hsuan Chiu, Jui-Chin Chen, Shang-Ming Lin, and Manuel De Guzman supervised the conduct of experiments and performed the theoretical analysis of the data. Acknowledgments The authors express their appreciation to Grabio Greentech Corporation, Ching-Yung Trading Co., Ltd., Fabric King Textile Co., Ltd., Ministry of Economic Affairs (100-EC-17-A-10-S1-186) and the National Science Council (NSC 102-2221-E-161-011, NSC 100-3113-E-033-001 and NSC 102-2221-E-003-065) for support of this work. Conflicts of Interest The authors declare no conflict of interest. 1. Handra, R.; Rustgi, R. Biodegradable polymers. Prog. Polym. Sci. 1998, 23, 1273–1335. 3.7. Enzymatic Hydrolysis The degradation of the enzymatic hydrolysis of specimens was evaluated at 27 °C using 50 mg starch enzyme in (0.025 mol Na2HPO4 + 0.025 mol KH2PO4) aqueous solution. Specimens with a dimension of 5 × 5 cm2 were tested for various days, washed with distilled water and dried completely in a vacuum oven at 70 °C for 8 h. On the basis of weight loss, the degree of degradation was determined using Equation (2): (2) (2) where W0 is the dry weight before degradation and Wt is the dry weight at time t. where W0 is the dry weight before degradation and Wt is the dry weight at time t. Materials 2014, 7 Materials 2014, 7 5630 2. Warwel, S.; Brüse, F.; Demes, C.; Kunz, M.; Rüsch gen Klaas, M. Polymers and surfactants on the basis of renewable resources. Chemosphere 2001, 43, 39–48. 3. Tsutsumi, N.; Kono, Y.; Oya, M.; Sakai, W.; Nagata, M. Recent development of biodegradable network polyesters obtained from renewable natureal resources. Clean Soil Air Water 2008, 36, 682–686. 4. Sinclair, R.G.J. The case for polylactic acid as a commodity packaging plastic. Macromol. Sci. Pure Appl. Chem. 1996, 33, 585–597. 5. Kricheldorf, H.R.; Kreiser-Saunders, I. Polylactides—Synthesis, characterization and medical application. Macromol. Symp. 1996, 103, 85–102. 6. Jang, W.Y.; Shin, B.Y.; Lee, T.J.; Narayan, R. Thermal properties and morphology of biodegradable PLA/starch compatibilized blends. J. Ind. Eng. Chem. 2007, 1, 457–464. 7. Oksman, K.; Skrifvars, M.; Selin, J.F. Natural fibres as reinforcement in polylactic acid (PLA) composites. Compos. Sci. 2003, 63, 1317–1324. 8. Shanks, R.A.; Hodzic, A.; Ridderhof, D. Composites of poly(lactic acid) with flax fibers modified by interstitial polymerization. J. Appl. Polym. Sci. 2006, 101, 3620–3629. 9. Bodros, E.; Pillin, I.; Montrelay, N.; Baley, C. Could biopolymers reinforced by randomly scattered flax fiber be used in structural applications. Compos. Sci. Technol. 2007, 67, 462–470. 10. Alvarez, V.A.; Ruscekaite, R.A.; Vázquez, A. Mechanical properties and water absorption behavior of composites made from a biodegradable matrix and alkaline-treated sisal fibers. J. Compos. Mater. 2003, 37, 1575–1588. 11. Lee, S.Y.; Kang, I.A.; Doh, G.H.; Yoon, H.G.; Park, B.D.; Wu, Q. Thermal and mechanical properties of wood flour/talc-filled polylactic acid composites: Effect of filler content and coupling treatment. J. Thermoplast. Compos. Mater. 2008, 21, 209–223. 12. Shibata, M.; Ozawa, K.; Teramoto, N.; Yosomiya, R.; Takeishi, H. Biocomposites made from short abaca fiber and biodegradable polyesters. Macromol. Mater. Eng. 2003, 288, 35–43. 13. Plackett, D.; Andersen, T.L.; Pedersen, W.B.; Nielsen, L. Biodegradable composites based on L-polylactide and jute fibres. Compos. Sci. Technol. 2003, 63, 1287–1296. 14. Shibata, M.; Oyamada, S.; Kobayashi, S.I.; Yaginuma, D. Mechanical properties and biodegradability of green composites based on biodegradable polyesters and lyocell fabric. J. Appl. Polym. Sci. 2004, 92, 3857–3863. 15. Huda, M.S.; Drzal, L.T.; Misra, M.; Mohanty, A.K. Wood-fiber-reinforced poly(lactic acid) composites: Evaluation of the physicomechanical and morphological properties. J. Appl. Polym. Sci. 2006, 102, 4856–4869. 16. Huda, M.S.; Drzal, L.T.; Misra, M.; Mohanty, A.K.; Williams, K.; Mielewski, D.F. A study onbiocomposites from recycled newspaper fiber and poly(lactic acid). Ind. Eng. Chem. Res. 2005, 44, 5593–5601. 17. References 1. Handra, R.; Rustgi, R. Biodegradable polymers. Prog. Polym. Sci. 1998, 23, 1273–1335. Materials 2014, 7 Materials 2014, 7 5631 20. Avella, M.; Buzarovska, A.; Errico, M.E. Gennaro gentile and grozdanov aeco-challenges of bio-based polymer composites. Materials 2009, 2, 911–925. 21. Wang, H.; Sun, X.Z.; Seib, P. Strengthening blends of poly(lactic acid) and starch with methylenediphenyl diisocyanate. J. Appl. Polym. Sci. 2001, 82, 1761–1767. 22. Ohkita, T.; Lee, S.H. Effect of aliphatic isocyanates (HDI and LDI) as a coupling agent on the properties of eco-composite from biodegradable polymers and corn starch. J. Adhes. Sci. Technol. 2004, 18, 905–924. 23. Teixeira, E.M.; Da Roz, A.L.; Carvalho, A.J.F.; Curvelo, A.A.S. The effect of glycerol/sugar/water and sugar/water mixtures on the plasticization of thermoplastic cassava starch. Carbohydr. Polym. 2007, 69, 619–624. 24. Rodriguez-Gonzalez, F.J.; Ramsay, B.A.; Favis, B.D. Rheological and thermal properties of thermoplastic starch with high glycerol content. Carbohydr. Polym. 2004, 58, 139–147. 25. Ma, X.F.; Yu, J.G.; Wan, J.J. Urea and ethanolamine as a mixed plasticizer for thermoplastic starch. Carbohydr. Polym. 2006, 64, 267–273. 26. Shi, R.; Zhang, Z.Z.; Liu, Q.Y.; Han, Y.M.; Zhang, L.Q.; Chen, D.F. Characterization of citric acid/glycerol co-plasticized thermoplastic starch prepared by melt blending. Carbohydr. Polym. 2007, 69, 748–755. 27. Jang, J.W.Y.; Shin, B.Y. Thermal properties and morphology of biodegradable PLA/starch Compatibilized Blends. Eng. Chem. 2007, 13, 457–464. 28. Wang, J.W.; Zhai, W.T.; Zheng, W.G. Poly(ethylene glycol) grafted starch introducing a novel interphase in poly(lactic acid)/poly(ethylene glycol)/starch ternary composite. J. Polym. Environ. 2012, 2, 528–539. 29. Xiong, Z.; Yang, Y.; Feng, J.X.; Zhang, X.; Zhang, C.Z.; Tang, Z.B. Preparation and characterization of poly(lactic acid)/starch composites toughened with epoxidized soybean oil. Carbohydr. Polym. 2013, 92, 810–816. 30. Acioli-Moura, R.; Sun, X.S. Thermal degradation and physical aging of poly(lactic acid) and its blends with starch ricardo. Polym. Eng. Sci. 2008, 48, 829–836. 31. Xiong, Z.; Zhang, L.S.; Ma, S.Q.; Yang, Y.; Zhang, C.Z.; Tang, Z.B.; Zhu, J. Effect of castor oil enrichment layer produced by reaction on the properties of PLA/HDI-g-starch blends. Carbohydr. Polym. 2013, 94, 235–243. 32. Poramacom, N.; Ungsuratana, A.O.; Ungsuratana, P.; Supavititpattana, P. Cassava production, prices and related policy in Thailand. Am. Int. J. Contemp. Res. 2013, 3, 43–51. 33. Tsou, C.H.; Hung, W.S.; Chen, J.C.; Huang, C.Y.; Wu, C.S.; Chiu, S.H.; Tsou, C.Y.; Chen, J.C.; Chu, C.K.; Hu, C.C.; et al. New composition of maleic-anhydride-grafted poly(lactic acid)/rice husk with methylenediphenyl diisocyanate. Mater. Sci. 2014, in press. 34. Zhao, Y.L.; Cai, Q.; Shuai, X.T.; Bei, J.Z.; Chen, C.F.; Xi, F. 35. Kim, E.S.; Kim, B.C.; Kim, S.H. Structural effect of linear and star-shaped poly(L-lactic acid) on physical properties. J. Polym. Sci. Part B Polym. Phys. 2004, 42, 939–946. Materials 2014, 7 Liu, W.; Misra, M.; Askeland, P.; Drzal, L.; Mohanty, A.K. Green composites from soy based plastic and pineapple leaf fiber: Fabrication and properties evaluation. Polymer 2005, 46, 2710–2721. 18. Ardente, F.; Beccali, M.; Cellura, M.; Mistretta, M. Building energy performance: A LCA case study of kenaf-fibres insulation board. Energy Build. 2008, 40, 1–10. 19. Mathew, A.P.; Oksman, K.; Sain, M. Mechanical properties of biodegradable composites from poly lactic acid (PLA) and microcrystalline cellulose (MCC). J. Appl. Polym. Sci. 2005, 97, 10–20. Materials 2014, 7 Materials 2014, 7 5632 36. Liu, H.; Chen, F.; Liu, B.; Estep, G.; Zhang, J. Super toughened poly(lactic acid) ternary blends by simultaneous dynamic vulcanization and interfacial compatibilization. Macromolecules 2010, 43, 6058–6066. 36. Liu, H.; Chen, F.; Liu, B.; Estep, G.; Zhang, J. Super toughened poly(lactic acid) ternary blends by simultaneous dynamic vulcanization and interfacial compatibilization. Macromolecules 2010, 43, 6058–6066. 37. Jiang, L.; Wolcott, M.P.; Zhang, J. Study of biodegradable polylactide/poly(butylene adipate-co- terephthalate) blends. Biomacromolecules 2006, 7, 199–207. 38. Williams, G.I.; Wool, R.P. Composites from natural fibers and soy oil resins. Wool. Appl. Compos. Mater. 2000, 7, 421–432. 39. Natural Fibre: Plastics and Composites; Wallenberger, F.T., Weston, N.E., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 2004. 40. Martin, P.; Maquet, C.; Legras, R.; Bailly, C.; Leemans, L.; van Gurp, M. Conjugated effects of the compatibilization and the dynamic vulcanization on the phase inversion behavior in poly(butylene terephthalate)/epoxide-containing rubber reactive polymer blends. Polymer 2004, 45, 5111–5125. 41. Martin, P.; Maquet, R.C.; Legras, C.; Bailly, L.; van Gurp Leemans, M. Particle-in-particle morphology in reactively compatibilized poly(butylene terephthalate)/epoxide-containing rubber blends. Polymer 2004, 45, 3277–3284. 42. Lim, J.S.; Park, K.I.; Chung, G.S.; Kim, J.H. Effect of composition ratio on the thermal and physical properties of semicrystalline PLA/PHB-HHx composites. Mater. Sci. Eng. 2013, 33, 2131–2137. 43. Gerard, T.; Budtova, T. Morphology and molten-state rheology of polylactide and polyhydroxyalkanoate blends. Eur. Polym. J. 2002, 48, 1110–1117. 44. Labrecque, L.V.; Dave, R.A.; Gross, R.A. Citrate esters as plasticizers for poly(lactic acid). J. Appl. Polym. Sci.1997, 66, 1507–1513. 45. Kulinski, Z.; Piorkowska, E.; Gadzinowska, K. Plasticization of poly(L-lactide) with poly(propylene glycol). Biomacromolecules 2006, 7, 2128–2135. 46. Ljungberg, N.; Wesslen, B. Preparation and properties of plasticized poly(lactic acid) films. Biomacromolecules 2005, 6, 1789–1796. 47. Ljungberg, N.; Andersson, T.; Wesslen, B. Film extrusion and film weldability of poly(lactic acid) plasticized with triacetine and tributyl citrate. J. Appl. Polym. Sci. 2003, 88, 3239–3247. 48. Martin, O.; Averous, L. Poly(lactic acid): Plasticization and properties of biodegradable multiphase systems. Polymer 2001, 42, 6209–6219. 49. Jacobsen, S.; Fritz, H.G. Plasticizing polylactide-the effect of different plasticizers on the mechanical properties. Polym. Eng. Sci. 1999, 39, 1303–1310. 50. Yeh, J.T.; Huang, C.Y.; Chai, W.L.; Chen, K.N. Plasticized properties of poly (lactic acid) and triacetine blends. J. Appl. Polym. Sci. 2009, 112, 2757–2763. 51. Baiardo, M.; Frisoni, G.; Scandola, M.; Michel, R.; Lips, D.; Ruffieux, K.; Wintermantel, E. Thermal and mechanical properties of plasticized poly(L-lactic acid). J. Materials 2014, 7 Synthesis and thermal properties of novel star-shaped poly(L-lactide)s with starburst PAMAM–OH dendrimer macroinitiator. Polymer 2002, 43, 5819–5825. 35. Kim, E.S.; Kim, B.C.; Kim, S.H. Structural effect of linear and star-shaped poly(L-lactic acid) on physical properties. J. Polym. Sci. Part B Polym. Phys. 2004, 42, 939–946. Materials 2014, 7 Appl. Polym. Sci. 2003, 90, 1731–1738. 52. Standard Test Method for Tensile Properties of Plastics. Available online: http://www.astm.org/ Standards/D638.htm (accessed on 31 July 2014). © 2014 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 license (http://creativecommons.org/licenses/by/3.0/).
https://openalex.org/W3027401261	https://link.springer.com/content/pdf/10.1007/s10461-020-02929-8.pdf	English	null	Patient-Reported Outcomes in ATLAS and FLAIR Participants on Long-Acting Regimens of Cabotegravir and Rilpivirine Over 48 Weeks	AIDS and behavior	2,020	cc-by	9,579	Abstract The phase 3 ATLAS and FLAIR studies demonstrated that maintenance with Long-Acting (LA) intramuscular cabotegravir and rilpivirine is non-inferior in efficacy to current antiretroviral (CAR) oral therapy. Both studies utilized Patient-Reported Outcome instruments to measure treatment satisfaction (HIVTSQ) and acceptance (ACCEPT general domain), health status (SF-12), injection tolerability/acceptance (PIN), and treatment preference. In pooled analyses, LA-treated patients (n = 591) demonstrated greater mean improvements from baseline than the CAR group (n = 591) in treatment satisfaction (Week 44, + 3.9 vs. +0.5 HIVTSQs-points; p < 0.001) and acceptance (Week 48, +8.8 vs. +2.0 ACCEPT-points; p < 0.001). The acceptability of injection site reactions (PIN) significantly improved from week 5 (2.10 points) to week 48 (1.62 points; p < 0.001). In both studies, ≥ 97% of LA group participants with recorded data preferred LA treatment compared with prior oral therapy. These results further support the potential of a monthly injectable option for people living with HIV seeking an alternative to daily oral treatment. Keywords Patient-reported outcomes · Antiretroviral therapy · Cabotegravir · Rilpivirine · Long-acting treatment AIDS and Behavior (2020) 24:3533–3544 https://doi.org/10.1007/s10461-020-02929-8 AIDS and Behavior (2020) 24:3533–3544 https://doi.org/10.1007/s10461-020-02929-8 ORIGINAL PAPER Patient‑Reported Outcomes in ATLAS and FLAIR Participants on Long‑Acting Regimens of Cabotegravir and Rilpivirine Over 48 Weeks Miranda Murray1 · Antonio Antela2 · Anthony Mills3 · Jenny Huang4 · Hans Jäger5,6 · Enrique Bernal7 · Johan Lombaard8 · Harold Katner9 · Sharon Walmsley10 · Marie‑Aude Khuong‑Josses11 · Krischan Hudson12 · David Dorey4 · Sandy Griffith12 · William Spreen12 · Simon Vanveggel13 · Mark Shaefer12 · David Margolis12 · Vasiliki Chounta14 Miranda Murray1 · Antonio Antela2 · Anthony Mills3 · Jenny Huang4 · Hans Jäger5,6 · Enrique Bernal7 · Johan Lombaard8 · Harold Katner9 · Sharon Walmsley10 · Marie‑Aude Khuong‑Josses11 · Krischan Hudson12 · David Dorey4 · Sandy Griffith12 · William Spreen12 · Simon Vanveggel13 · Mark Shaefer12 · David Margolis12 · Vasiliki Chounta14 Published online: 23 May 2020 © The Author(s) 2020 * Vasiliki Chounta vasiliki.x.chounta@viivhealthcare.com Study Design ATLAS and FLAIR (Fig. 1) are ongoing, phase 3, rand- omized, open-label, parallel-group studies comparing the efficacy and safety of monthly intramuscular (IM) CAB+RPV LA (400 mg CAB+600 mg RPV) vs. continu- ation of an oral cART regimen—the current antiretroviral regimen (CAR) group—consisting of either a pre-existing stable daily oral therapy of at least 6 months’ prior duration (ATLAS) or a defined oral induction treatment comprising a single-tablet coformulation of dolutegravir, abacavir, and lamivudine (FLAIR). Participant recruitment and study designs have been fully described in the primary clinical manuscripts [12, 13]. LA injectable formulations have been developed for cabotegravir (CAB), an integrase strand transfer inhibitor (INSTI), and rilpivirine (RPV), a non-nucleoside reverse transcriptase inhibitor (NNRTI) [10, 11]. It has been shown in both individual and pooled analyses from two pivotal phase 3 studies in treatment-experienced patients (ATLAS; NCT02951052) and in previously treatment- naïve patients (FLAIR; NCT02938520) that monthly dos- ing of CAB+RPV LA is non-inferior to daily oral ART for maintaining HIV suppression [12–14]. In addition, other than injection-site reactions (ISRs), which declined over time, the overall incidence of adverse events was compa- rable between the oral and LA treatment groups [12–14]. Similarly, in the phase 2b LATTE-2 study (NCT02120352), CAB+RPV LA maintenance every 1 or 2 months resulted in similar rates of virologic suppression to continuing on the oral induction regimen of oral CAB with nucleoside analogues through 96 weeks [15]. In qualitative interviews to assess their preference for LA injections over oral medications, LATTE-2 participants in Spain and the US cited both the convenience of LA injections vs. daily pills, and psychosocial benefits of LA treatment such as confidentiality, the lack of a daily reminder of living with HIV, and the reduced risk of stigma associated with dis- ease disclosure as reasons supporting their preference for an LA therapy [16]. LA injectable formulations have been developed for cabotegravir (CAB), an integrase strand transfer inhibitor (INSTI), and rilpivirine (RPV), a non-nucleoside reverse transcriptase inhibitor (NNRTI) [10, 11]. It has been shown in both individual and pooled analyses from two pivotal phase 3 studies in treatment-experienced patients (ATLAS; NCT02951052) and in previously treatment- naïve patients (FLAIR; NCT02938520) that monthly dos- ing of CAB+RPV LA is non-inferior to daily oral ART for maintaining HIV suppression [12–14]. Assessments and Endpoints PRO instruments (Table 1) were included at specific time- points in each study to assess health status, treatment satis- faction, acceptance and preference, and the tolerability and acceptability of injections. These instruments were selected based on qualitative interviews and PRO data from patients enrolled on the phase 2b LATTE-2 study [16]. The PRO instruments used in ATLAS and FLAIR included: Patient-reported outcomes (PROs) are important for the holistic assessment of new medicines and innova- tive treatment options. In ATLAS, PROs were assessed in participants who had been virologically suppressed (HIV-1 RNA < 50 c/mL) on oral regimens for a median of 4 years, allowing participants to compare their expe- riences of LA treatment against prior oral therapies. In contrast, the assessment of PROs in FLAIR presented an opportunity to assess switching to the LA treatment vs. remaining on first-line oral treatment in patients who were treatment-naïve prior to study enrollment. Both studies assessed patient satisfaction with, and acceptance of, the oral or LA treatment received; the acceptability and toler- ability of CAB+RPV LA injections, and overall patient health status. Treatment preference and reason for wish- ing to switch from oral to LA therapy were assessed as exploratory endpoints. Herein, we present both pooled and individual PRO data from the ATLAS and FLAIR studies. Methods associated with daily cART impact many PLWHIV which in turn has led to significant interest in antiretroviral ther- apy (ART) with less frequent dosing [3–9]. As a result, a clinical development program has been dedicated to intro- ducing a long-acting (LA) alternative to daily oral cART, providing an additional treatment option that may improve treatment adherence and satisfaction for PLWHIV. Introduction Combination antiretroviral therapy (cART) has dramati- cally reduced mortality and improved the quality of life of people living with HIV (PLWHIV) [1, 2], but HIV treat- ment currently requires a lifelong commitment to daily oral therapy. Emotional and adherence-related challenges Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10461-020-02929-8) contains supplementary material, which is available to authorized users. 7 Hospital General Universitario Reina Sofía, Murcia, Spain 8 Josha Research, Bloemfontein, South Africa 9 Mercer University Medical School, Macon, GA, USA 10 University Health Network, Toronto, ON, Canada 11 Hôpital Delafontaine, Saint‑Denis, France 12 ViiV Healthcare, Research Triangle Park, NC, USA 13 Janssen Research & Development, Beerse, Belgium 14 ViiV Healthcare, 980 Great West Road, Brentford TW8 9GS, Middlesex, UK 7 Hospital General Universitario Reina Sofía, Murcia, Spain 8 Josha Research, Bloemfontein, South Africa 9 Mercer University Medical School, Macon, GA, USA 10 University Health Network, Toronto, ON, Canada 11 Hôpital Delafontaine, Saint‑Denis, France 12 ViiV Healthcare, Research Triangle Park, NC, USA 13 Janssen Research & Development, Beerse, Belgium 14 ViiV Healthcare, 980 Great West Road, Brentford TW8 9GS, Middlesex, UK * Vasiliki Chounta vasiliki.x.chounta@viivhealthcare.com 1 Health Analytics and Outcomes Ltd, London, UK 2 Hospital Clínico Universitario, Santiago de Compostela, Spain 3 Southern California Men’s Medical Group, West Hollywood, CA, USA 4 GlaxoSmithKline, Mississauga, ON, Canada 5 MUC Research GmbH, Munich, Germany 6 MVZ Karlsplatz, HIV Research and Clinical Care Centre, Munich, Germany :(0123 1 23456789) 3 AIDS and Behavior (2020) 24:3533–3544 3534 Study Design In addition, other than injection-site reactions (ISRs), which declined over time, the overall incidence of adverse events was compa- rable between the oral and LA treatment groups [12–14]. Both studies were conducted in accordance with the prin- ciples founded in the Declaration of Helsinki and with Good Clinical Practice. All participants provided written informed consent, and the protocol was approved by an institutional review board or ethics committee of each study site. The authors can attest for adherence to the study protocol and for the accuracy and completeness of the data and analyses. g p Similarly, in the phase 2b LATTE-2 study (NCT02120352), CAB+RPV LA maintenance every 1 or 2 months resulted in similar rates of virologic suppression to continuing on the oral induction regimen of oral CAB with nucleoside analogues through 96 weeks [15]. In qualitative interviews to assess their preference for LA injections over oral medications, LATTE-2 participants in Spain and the US cited both the convenience of LA injections vs. daily pills, and psychosocial benefits of LA treatment such as confidentiality, the lack of a daily reminder of living with HIV, and the reduced risk of stigma associated with dis- ease disclosure as reasons supporting their preference for an LA therapy [16]. Treatment Satisfaction: HIVTSQ Treatment satisfaction was assessed using a recent adapta- tion of the validated 10-item HIV Treatment Satisfaction Treatment satisfaction was assessed using a recent adapta- tion of the validated 10-item HIV Treatment Satisfaction Questionnaire (HIVTSQ) [17, 18], which included two addi- tional items to account for LA dosing [19, 20], specifically: Questionnaire (HIVTSQ) [17, 18], which included two addi- tional items to account for LA dosing [19, 20], specifically: 11. How easy or difficult have you been finding your treat- ment to be recently?i 12. How satisfied are you with the amount of discomfort or pain involved with your present form of treatment? Extensive psychometric analyses on the 12-item HIVTSQ suggest that these two additional items do not reduce the overall validity of the questionnaire [19, 20]. Item 11 con- tributes to the structure of the scale, improving the outcomes 1 3 3535 AIDS and Behavior (2020) 24:3533–3544 Fig. 1 The ATLAS [12] and FLAIR [13] study design. Eligible individuals were randomly assigned (1:1) to continue their cur- rent antiretroviral regimen (CAR arm) or switch to the long-acting regimen (LA arm). Those assigned to the LA arm initially received 4 weeks of oral CAB+RPV QD, then transitioned to the injectable regimen. aUninterrupted ART for 6 months and VL < 50 c/mL at screening, 2 × VL < 50 c/mL for ≤ 12 months. bINSTI-based regi- men capped at 40% of enrollment; Triumeq excluded from study. cOptional switch to CAB+RPV LA at week 52 for those on CAR. dParticipants who withdraw/complete CAB+RPV LA must com- plete 52 weeks of follow-up. eParticipants received an initial loading dose of CAB LA (600 mg) and RPV LA (900 mg) at week 4b. From week 8 onwards, participants received CAB LA (400 mg)+RPV LA (600 mg) injections every 4 weeks. fNNRTI RAMs but not K103N were exclusionary. gOf the 631 participants who entered the induc- tion phase, two withdrew prior to receiving study drug. DTG plus two alternative non-ABC NRTIs was permitted if participant was intolerant or HLA-B5701-positive (n = 30 as last regimen during induction: n = 2 discontinued during induction, n = 14 randomized to CAB+RPV LA, n = 14 randomized to DTG/ABC/3TC arm and con- tinued on DTG plus two alternative non-ABC NRTIs in the mainte- nance phase). hParticipants who withdraw/complete CAB+RPV LA enter 52-week long-term follow-up. Treatment Satisfaction: HIVTSQ 3TC lamivudine, ABC abacavir, ART antiretroviral therapy, CAB cabotegravir, CAR current antiretro- viral regimen, DTG dolutegravir, IM intramuscular, INSTI integrase strand transfer inhibitor, HBsAg hepatitis B surface antigen, LA long- acting, NNRTI non-nucleoside reverse transcriptase inhibitor, NRTI nucleoside reverse transcriptase inhibitor, PI protease inhibitor, RAM resistance-associated mutation, RPV rilpivirine, VL viral load Fig. 1 The ATLAS [12] and FLAIR [13] study design. Eligible individuals were randomly assigned (1:1) to continue their cur- rent antiretroviral regimen (CAR arm) or switch to the long-acting regimen (LA arm). Those assigned to the LA arm initially received 4 weeks of oral CAB+RPV QD, then transitioned to the injectable regimen. aUninterrupted ART for 6 months and VL < 50 c/mL at screening, 2 × VL < 50 c/mL for ≤ 12 months. bINSTI-based regi- men capped at 40% of enrollment; Triumeq excluded from study. cOptional switch to CAB+RPV LA at week 52 for those on CAR. dParticipants who withdraw/complete CAB+RPV LA must com- plete 52 weeks of follow-up. eParticipants received an initial loading dose of CAB LA (600 mg) and RPV LA (900 mg) at week 4b. From week 8 onwards, participants received CAB LA (400 mg)+RPV LA (600 mg) injections every 4 weeks. fNNRTI RAMs but not K103N were exclusionary. gOf the 631 participants who entered the induc- tion phase, two withdrew prior to receiving study drug. DTG plus two alternative non-ABC NRTIs was permitted if participant was intolerant or HLA-B5701-positive (n = 30 as last regimen during induction: n = 2 discontinued during induction, n = 14 randomized to CAB+RPV LA, n = 14 randomized to DTG/ABC/3TC arm and con- tinued on DTG plus two alternative non-ABC NRTIs in the mainte- nance phase). hParticipants who withdraw/complete CAB+RPV LA enter 52-week long-term follow-up. 3TC lamivudine, ABC abacavir, ART antiretroviral therapy, CAB cabotegravir, CAR current antiretro- viral regimen, DTG dolutegravir, IM intramuscular, INSTI integrase strand transfer inhibitor, HBsAg hepatitis B surface antigen, LA long- acting, NNRTI non-nucleoside reverse transcriptase inhibitor, NRTI nucleoside reverse transcriptase inhibitor, PI protease inhibitor, RAM resistance-associated mutation, RPV rilpivirine, VL viral load group in both studies, while the HIVTSQc was applied only at week 48 to both treatment groups in FLAIR and to the LA group only in ATLAS. of confirmatory factor analysis, and preserves the option of calculating the total score with items 1 through 11. Treatment Satisfaction: HIVTSQ Item 12 can be reported individually and is not included in the total score [19–21]. Two versions exist of the HIVTSQ: the status version (HIVTSQs), which was the first to be developed, and the subsequent change version (HIVTSQc) developed to mitigate ceiling effects common in treatment satisfaction measures [18]. The HIVTSQs asked patients to rank their response on a 6-point Likert scale, from 6 (very satisfied) to 0 (very dissatisfied), while the HIVTSQc asked patients to rank their response from 3 (much more satisfied now) to − 3 (much less satisfied now) [18]. On both versions, the scores are added together, to provide the total summary score. The HIVTSQs was assessed at maintenance baseline (MBL) and weeks 4, 24, and 44 in the LA treatment group and the CAR of confirmatory factor analysis, and preserves the option of calculating the total score with items 1 through 11. Item 12 can be reported individually and is not included in the total score [19–21]. Two versions exist of the HIVTSQ: the status version (HIVTSQs), which was the first to be developed, and the subsequent change version (HIVTSQc) developed to mitigate ceiling effects common in treatment satisfaction measures [18]. The HIVTSQs asked patients to rank their response on a 6-point Likert scale, from 6 (very satisfied) to 0 (very dissatisfied), while the HIVTSQc asked patients to rank their response from 3 (much more satisfied now) to − 3 (much less satisfied now) [18]. On both versions, the scores are added together, to provide the total summary score. The HIVTSQs was assessed at maintenance baseline (MBL) and weeks 4, 24, and 44 in the LA treatment group and the CAR HAT-QoLb Do you agree with the following statement: “My medica- tion has more advantages than disadvantages”? Given the advantages and disadvantages of your medi- cation, do you consider it to be an acceptable solution? Are you convinced that in the long term, it is worth taking your medications? Do you agree with the following statement: “My medica- tion has more advantages than disadvantages”? Given the advantages and disadvantages of your medi- cation, do you consider it to be an acceptable solution? Are you convinced that in the long term, it is worth taking your medications? from the earlier Vaccinees’ Perception of Injection (VAPI) questionnaire [24], while retaining the same scoring sys- tem. The questionnaire contains 21 items in total and con- sists of four dimensions: “acceptance of ISRs”, “bother of ISRs”, “sleep”, and “leg movement”, and five individually reported items related to anxiety before and after the injec- tion, willingness to receive an HIV injectable treatment at the following visit, pain during injection, and satisfaction with the mode of treatment administration. Participants were asked to score their responses from 1 to 5, where scores of 1 through 3 refer to, in order, “totally acceptable”, “very acceptable”, and “moderately acceptable”; a score of 4 is “a little acceptable”, and 5 is “not at all acceptable”. To avoid multiplicity issues, hypothesis testing was preplanned only for the “acceptance of ISRs” dimension of the PIN. Responses were rated on a 5-point Likert scale with a score of 5 representing “I don’t know” for all three questions, and scores of 1 through 4 representing increasing levels of agreement or acceptance from “Totally disagree/Not at all acceptable/Not at all convinced” through to “Totally agree/ Totally acceptable/Totally convinced”. Item scores were grouped together to form an aggregate that was linearly transformed to range from 0 to 100, with a higher score suggesting greater acceptance [23]. Treatment Acceptance: ACCEPT Questionnaire Participants in both treatment groups completed the General Acceptance domain of the Chronic Treatment Acceptance (ACCEPT) questionnaire [22], a generic medication accept- ance instrument validated for chronic conditions that was administered at MBL and weeks 8, 24, and 48. This domain consists of three questions: 1 3 AIDS and Behavior (2020) 24:3533–3544 3536 Table 1 The PRO instruments conducted in ATLAS and FLAIR ACCEPT Chronic Treatment Acceptance questionnaire, CAB cabotegravir, HAT-QoL HIV/AIDS-targeted quality of life, HIVTSQs/c HIV Treat- ment Satisfaction Questionnaire (status version)/(change version), LA long-acting, PIN Perception of Injection questionnaire, PRO patient- reported outcome, RPV rilpivirine, SF-12 12-Item Short Form Health Survey a HIVTSQc only administered to participants receiving LA therapy b No meaningful differences between arms were reported with the three dimensions included in the phase 3 studies. Results are not discussed here c Consistent with the PIN, numerical reduction in post-injection pain was reported in the Numeric Rating Scale over time. No significance testing was preplanned for this measure. Results are not discussed here PRO instrument Assessment Timepoints measured Pooled analy- sis HIVTSQs/ca Patient satisfaction with HIV treatment Status version: maintenance baseline, week 4, week 24, week 44 Change version: week 48 ✔ ACCEPT Patient acceptance of treatment Maintenance baseline, week 8, week 24, week 48 ✔ PIN questionnaire Perception of pain and injection site reac- tions Week 5, week 41, week 48 ✔ SF-12 General health status and degree of mental health distress Maintenance baseline, week 24, week 48 ✖ Preference of HIV treatment (single ques- tion) Patient preference for CAB + RPV LA vs. their current oral therapy Week 48 ✖ Reason for switch (single question) Patient reasoning for switching to LA therapy from oral therapy for ATLAS study only Week 52 ✖ HAT-QoLb Overall function and wellbeing. Only 3 out of 9 dimensions were assessed Maintenance baseline, week 24, week 48 ✖ Numeric Rating Scalec Intensity of post-injection pain Week 4, week 5, week 40, week 41 ✖ Reason for Switch Additionally, in the ATLAS study only, a single-question “Reason for switch” questionnaire with six response options was administered to all participants (including those contin- uing on oral CAR) on day 1 to explore the reasons why par- ticipants wished to enter an LA trial. A similar questionnaire was also administered at week 52 to participants in the oral CAR arm who had opted to switch to CAB+RPV LA after the primary analysis at week 48, under the study protocol. Injection Acceptability and Tolerability: PIN Questionnaire General health was assessed in both studies using the generic validated 12-Item Short Form Health Survey (SF- 12) questionnaire [25–28]. The SF-12 includes the same eight domains as the older SF-36 questionnaire [29], but with fewer questions, making it more practical, especially for larger and more complex populations [30]. The physical The acceptability and tolerability of monthly injections and ISRs at early (week 5) and later stages of treatment (weeks 41 and 48) was assessed in the LA treatment groups of both studies using the Perception of Injection (PIN) questionnaire. This instrument was adapted for gluteal IM administration 1 3 3537 AIDS and Behavior (2020) 24:3533–3544 exploratory endpoints (preference question and reason for switch) proportions are reported for observed cases without imputation, statistical modeling, or testing. Missing data for non-exploratory PROs were imputed using a last-observa- tion-carried-forward approach including measures assessed at time of withdrawal. component score (PCS) and mental component score (MCS) of the SF-12 are norm-based and range from 0 to 100, with higher scores indicating better health. In the US population, the mean SF-12 score is 50 with a standard deviation of 10 [31]. SF-12 was administered at MBL, week 24, and week 48 to participants on both treatment arms. Results For both studies, a single-item preference question was developed to assess participants’ preference for CAB+RPV LA compared with the oral cART received prior to randomi- zation. This was administered, at week 48, to participants in the LA arm only. The question reads: “For the past 44 weeks you have received Long Acting injectable HIV medication every month. Today we would like you to compare your expe- rience on the Long Acting injections with the oral medica- tion you received during the induction phase of the study. Which therapy do you prefer?” Participants had the option of selecting either daily oral treatment or monthly injections. Baseline Characteristics The pooled intention-to-treat-exposed (ITT-E) population of all randomized participants who received at least one dose of study medication in either ATLAS or FLAIR consisted of 1182 individuals: 591 in each treatment group (LA or CAR). MBL characteristics were generally similar between groups in each study and were also similar between the two studies [12, 13], although ATLAS had a numerically higher percent- age of participants in the CAR group aged over 50 years compared with the LA group (31% vs. 21%, respectively) [12]. More than 20% of patients enrolled in each study were female, exceeding the recruitment goals for both. Of note, participants enrolled in ATLAS had been on previous cART for a median of 4 years (range 1–21) [12], while participants in FLAIR had no cART experience prior to the induction phase of the study [13]. ACCEPT of the standard deviation at baseline [32, 33]. Changes from MBL in ATLAS were greater for LA dosing across all indi- vidual items in the HIVTSQs, particularly for items related to overall regimen satisfaction; the ease, convenience, and flexibility of treatment; and satisfaction at the prospect of continuing current treatment (Supplementary Fig. 1).f Mean general acceptance domain scores were high and similar in both studies between the LA and CAR treatment groups at MBL. Pooled mean (SD) MBL values were 80.5 (24.8) for the LA arm and 78.8 (25.3) for the CAR arm, out of a maximum 100 points. In ATLAS, the mean (SD) baseline score was 75.9 (26.5) in the LA group, and 74.7 (26.1) in the CAR group, while in FLAIR these MBL val- ues were 86.0 (21.3) and 83.4 (23.7), respectively.i Mean general acceptance domain scores were high and similar in both studies between the LA and CAR treatment groups at MBL. Pooled mean (SD) MBL values were 80.5 (24.8) for the LA arm and 78.8 (25.3) for the CAR arm, out of a maximum 100 points. In ATLAS, the mean (SD) baseline score was 75.9 (26.5) in the LA group, and 74.7 (26.1) in the CAR group, while in FLAIR these MBL val- ues were 86.0 (21.3) and 83.4 (23.7), respectively.i A smaller LA treatment effect for HIVTSQs total score was reported in the FLAIR study. A statistically significant greater improvement from MBL of 2.2 (95% CI 1.0–3.4) points in the adjusted mean HIVTSQs total score favoring the LA group vs. CAR was observed at week 24 that was not maintained at week 44 (Fig. 2). Significantly greater (p < 0.001) improvement in the adjusted mean general acceptance domain score change from MBL was noted for LA treatment vs. oral cART in pooled data at weeks 24 and 48 (Fig. 3). Consist- ent with HIVTSQs data, this improvement was driven largely by ATLAS, in which increases of 16–18% over MBL were seen on LA treatment at weeks 24 and 48 (week 24 adjusted mean change 12.3 [95% CI 9.9–14.8] points; week 48 adjusted mean change 13.7 [95% CI 11.2–16.3] points) compared with lesser changes in the CAR group (week 24 adjusted mean change 5.5 [95% CI 3.0–8.0] points; week 48 adjusted mean change 3.0 [95% CI 0.4–5.6] points). Statistical Analysis Mean HIVTSQs total score values at MBL were high and similar between the oral and LA treatment groups in both ATLAS and FLAIR, with higher mean scores in FLAIR. Out of a maximum of 66 points, mean MBL values in the pooled dataset were 57.1 (SD 8.4) for the CAR group and 57.1 (8.6) for the LA group; while individually these values were, in ATLAS, 55.4 (8.7) and 55.3 (9.1), respectively, and in FLAIR, 59.1 (7.6) and 59.3 (7.4), respectively. Descriptive statistics summarize questionnaire scores for each timepoint. Statistical comparisons between treatment groups in change from MBL for prespecified endpoints was performed with an ANCOVA model adjusting for covariates selected a priori: MBL score, sex at birth, age, race (white, non-white), third agent class (integrase inhibitors, protease inhibitors, NNRTI) for ATLAS only, and induction baseline HIV RNA for FLAIR only. P values and 95% CI for the treatment difference between groups were reported. Within- group comparisons in change over time for the “Acceptance of ISRs” dimension of the PIN was based on the Wilcoxon signed-rank test. The significance threshold was set at 0.05. In addition to individual study analyses, pooled analyses combining data from both ATLAS and FLAIR were per- formed post hoc for instruments assessing treatment satis- faction, treatment acceptance, and the tolerability of injec- tions and ISRs, adjusting for baseline score, sex at birth, age (< 50, ≥ 50 years) and race (white, non-white). For the Pooled data at weeks 24 and 44 showed a statistically significant greater improvement in treatment satisfaction from MBL on LA treatment compared with the CAR group (Fig. 2). This difference was mainly driven by ATLAS data, where LA-treated participants showed a large and stable 5.4 (95% CI 4.2–6.6) to 5.7 (95% CI 4.4–7.0)-point treatment difference in the adjusted mean change from MBL in HIVTSQs total score vs. the CAR group at both timepoints that meets the minimum clinically important difference threshold using the distribution-based approach, with the mean difference between the two groups exceeding one half 1 3 AIDS and Behavior (2020) 24:3533–3544 3538 Fig. 2 Change from main- tenance baseline HIVTSQs total score through week 44. Adjusted mean change from maintenance baseline is estimated from an ANCOVA model. Covariates are: ATLAS: baseline score, sex at birth, age (< 50 vs. ≥ 50 years), race, and third agent class, FLAIR: maintenance baseline score (day 1), sex at birth, age (< 50 vs. Statistical Analysis ≥ 50 years), race, and induction baseline (week –20) HIV-1 RNA (< 105 vs. ≥ 105 c/mL) Pooled: maintenance baseline score, sex at birth, age (< 50 vs. ≥ 50 years), and race. CAB cabotegravir, CAR current antiretroviral treatment (oral), CI confidence interval, HIVTSQs HIV Treatment Satisfaction Questionnaire (status version), LA long-acting, RPV rilpivirine, SD standard deviation ACCEPT The treatment difference in FLAIR Inter-group comparative data for the HIVTSQ change instrument in FLAIR, allowed for a direct comparison of LA maintenance with the oral induction regimen at week 48, with mitigation of ceiling effects caused by high MBL satis- faction. Here, a statistically significant difference (p < 0.001) in mean HIVTSQc total score was observed in favor of the LA arm, with an LA adjusted mean of 29.6 (SE 0.49) and a CAR adjusted mean of 25.2 (SE 0.48) giving an adjusted mean treatment difference of 4.1 (95% CI 2.8–5.5). 1 3 3539 AIDS and Behavior (2020) 24:3533–3544 was smaller and non-significant, partly due to the similarly high MBL values noted also in the HIVTSQs. PIN According to the “Acceptance of ISRs” dimension of the PIN, most participants in both studies reported that pain and ISRs were “very acceptable” or “totally accept- able” when questioned a week after their first injections of CAB+RPV LA (week 5). In pooled data, the mean (SD) score for the acceptance domain at week 5 was 2.10 (1.04), with favorable scores also reported for the other domains and individual PIN items after the first injec- tion (Supplementary Fig. 2). A statistically significant (p < 0.001) mean improvement from week 5 in the accept- ance domain was observed in pooled data at weeks 41 (mean 1.67 [SD 0.86]) and 48 (1.62 [0.81]) indicating improved acceptability of ISRs over time, alongside high initial acceptance rates (Fig. 4). For the two items gen- erating the “Acceptance of ISRs” dimension, 90% and 86% of participants in ATLAS and FLAIR, respectively, reported that their ISRs were either “totally accept- 84% of participants, respectively, reported that the level of pain experienced was either “totally acceptable” or “very acceptable” at week 48. Consistent results were Fig. 3 Change from mainte- nance baseline General Accept- ance domain scores through week 48. Adjusted mean change from maintenance baseline is estimated from an ANCOVA model. Covariates are: ATLAS: treatment, baseline score, sex at birth, age (< 50 vs. ≥ 50 years), race, and third agent class, FLAIR: maintenance baseline score (day 1), sex at birth, age (< 50 vs. ≥ 50 years), race, and induction baseline (week –20) HIV-1 RNA (< 105 vs. ≥ 105 c/mL) Pooled: maintenance baseline score, sex at birth, age (< 50 vs. ≥ 50 years), and race. ACCEPT 4 Summary of PIN “Acceptability of ISRs” scores through week 48. Week 48 was compared with the 1st visit (week 5) based on Wil- coxon signed-rank test, respectively. P values are derived for ‘Accept- ance’ only and not adjusted for multiple testing. ISR injection site reaction, PIN perception of injection, SD standard deviation was smaller and non-significant, partly due to the similarly high MBL values noted also in the HIVTSQs. was smaller and non-significant, partly due to the similarly high MBL values noted also in the HIVTSQs. PIN According to the “Acceptance of ISRs” dimension of the PIN, most participants in both studies reported that pain and ISRs were “very acceptable” or “totally accept- able” when questioned a week after their first injections of CAB+RPV LA (week 5). In pooled data, the mean (SD) score for the acceptance domain at week 5 was 2.10 (1.04), with favorable scores also reported for the other domains and individual PIN items after the first injec- tion (Supplementary Fig. 2). A statistically significant (p < 0.001) mean improvement from week 5 in the accept- ance domain was observed in pooled data at weeks 41 (mean 1.67 [SD 0.86]) and 48 (1.62 [0.81]) indicating improved acceptability of ISRs over time, alongside high initial acceptance rates (Fig. 4). For the two items gen- erating the “Acceptance of ISRs” dimension, 90% and 86% of participants in ATLAS and FLAIR, respectively, reported that their ISRs were either “totally accept- able” or “very acceptable” at week 48, while 86% and According to the “Acceptance of ISRs” dimension of the PIN, most participants in both studies reported that pain and ISRs were “very acceptable” or “totally accept- able” when questioned a week after their first injections of CAB+RPV LA (week 5). In pooled data, the mean (SD) score for the acceptance domain at week 5 was 2.10 (1.04), with favorable scores also reported for the other domains and individual PIN items after the first injec- tion (Supplementary Fig. 2). A statistically significant (p < 0.001) mean improvement from week 5 in the accept- ance domain was observed in pooled data at weeks 41 (mean 1.67 [SD 0.86]) and 48 (1.62 [0.81]) indicating improved acceptability of ISRs over time, alongside high initial acceptance rates (Fig. 4). ACCEPT ACCEPT Chronic Treatment Acceptance questionnaire, CAB cabotegravir, CAR current antiretroviral treatment (oral), CI confidence interval, LA long-acting, RPV rilpivirine, SD standard deviation Fig. 4 Summary of PIN “Acceptability of ISRs” scores through week 48. Week 48 was compared with the 1st visit (week 5) based on Wil- coxon signed-rank test, respectively. P values are derived for ‘Accept- ance’ only and not adjusted for multiple testing. ISR injection site reaction, PIN perception of injection, SD standard deviation AIDS and Behavior (2020) 24:3533–3544 3539 Fig. 3 Change from mainte- nance baseline General Accept- ance domain scores through week 48. Adjusted mean change from maintenance baseline is estimated from an ANCOVA model. Covariates are: ATLAS: treatment, baseline score, sex at birth, age (< 50 vs. ≥ 50 years), race, and third agent class, FLAIR: maintenance baseline score (day 1), sex at birth, age (< 50 vs. ≥ 50 years), race, and induction baseline (week –20) HIV-1 RNA (< 105 vs. ≥ 105 c/mL) Pooled: maintenance baseline score, sex at birth, age (< 50 vs. ≥ 50 years), and race. ACCEPT Chronic Treatment Acceptance questionnaire, CAB cabotegravir, CAR current antiretroviral treatment (oral), CI confidence interval, LA long-acting, RPV rilpivirine, SD standard deviation was smaller and non-significant, partly due to the similarly high MBL values noted also in the HIVTSQs. Fig. 3 Change from mainte- nance baseline General Accept- ance domain scores through week 48. Adjusted mean change from maintenance baseline is estimated from an ANCOVA model. Covariates are: ATLAS: treatment, baseline score, sex at birth, age (< 50 vs. ≥ 50 years), race, and third agent class, FLAIR: maintenance baseline score (day 1), sex at birth, age (< 50 vs. ≥ 50 years), race, and induction baseline (week –20) HIV-1 RNA (< 105 vs. ≥ 105 c/mL) Pooled: maintenance baseline score, sex at birth, age (< 50 vs. ≥ 50 years), and race. ACCEPT Chronic Treatment Acceptance questionnaire, CAB cabotegravir, CAR current antiretroviral treatment (oral), CI confidence interval, LA long-acting, RPV rilpivirine, SD standard deviation score (day 1), sex at birth, age (< 50 vs. ≥ 50 years), race, and induction baseline (week –20) HIV-1 RNA (< 105 vs. ≥ 105 c/mL) Pooled: maintenance baseline score, sex at birth, age (< 50 vs. ≥ 50 years), and race. ACCEPT Chronic Treatment Acceptance questionnaire, CAB cabotegravir, CAR current antiretroviral treatment (oral), CI confidence interval, LA long-acting, RPV rilpivirine, SD standard deviation Fig. ACCEPT For the two items gen- erating the “Acceptance of ISRs” dimension, 90% and 86% of participants in ATLAS and FLAIR, respectively, reported that their ISRs were either “totally accept- able” or “very acceptable” at week 48, while 86% and Fig. 4 Summary of PIN “Acceptability of ISRs” scores through week 48. Week 48 was compared with the 1st visit (week 5) based on Wil- coxon signed-rank test, respectively. P values are derived for ‘Accept- ance’ only and not adjusted for multiple testing. ISR injection site reaction, PIN perception of injection, SD standard deviation 84% of participants, respectively, reported that the level of pain experienced was either “totally acceptable” or “very acceptable” at week 48. Consistent results were observed in all remaining domains and individual item 1 3 1 3 AIDS and Behavior (2020) 24:3533–3544 3540 Preference Question (Exploratory) scores, although hypothesis testing was not undertaken for other components of the PIN to avoid multiplicity. In the ITT-E population, 86% (266/308) of LA arm par- ticipants in ATLAS and 91% (257/283) of LA arm partici- pants in FLAIR rated the LA treatment as their preferred option over daily oral therapy after 48 weeks of treatment. Only 2% (7/308) in ATLAS and 1% (2/283) in FLAIR preferred daily oral treatment, with the remaining 11% (35/308) and 8% (24/283), respectively, classified as miss- ing data. A post hoc observed analysis of participants with response data at week 48 showed that almost all respond- ers preferred the LA regimen over CAR: 97% (266/273) in ATLAS and 99% (257/259) in FLAIR. No participant refused to respond to the preference question. SF‑12 No LA vs. CAR difference was observed in either ATLAS or FLAIR for the SF-12 MCS or PCS at MBL or later. In each study, MBL scores were above the US national average of 50 [31] and no significant change from MBL was observed or expected in either domain over 48 weeks. ATLAS patients receiving LA therapy had a mean adjusted treatment differ- ence of 0.64 (95% CI, − 0.64, 1.91; p = 0.327) in the MCS domain and 0.70 (95% CI, − 0.11, 1.51; p = 0.092) in the PCS domain at 48 weeks. In FLAIR, patients receiving LA therapy had a mean adjusted treatment difference of 1.10 (95% CI [− 0.25, 2.45]; p = 0.109) in the MCS domain and − 0.17 (95% CI [− 0.99, 0.66]; p = 0.689) in the PCS domain at 48 weeks (Fig. 5). Reason for Switch in ATLAS (Exploratory) At day 1 (randomization), ATLAS participants indicated “I am interested in research of new therapies” (82%; 505/616) and “My clinician asked me to participate” (25%; 151/616) as the top two reasons for wanting to enter the study and receive an LA regimen. At week 52, those opting to switch from continued oral CAR to open- label CAB+RPV LA in the extension phase indicated that 1 3 Fig. 5 The SF-12 component scores adjusted treatment difference through 48 weeks. Adjusted mean is the estimated mean change from maintenance baseline score by visit in each treatment calculated from an ANCOVA model including the covariates, which are: ATLAS: baseline score, sex at birth, age, race (white, non-white), and third agent class (integrase inhibitors, protease inhibitors, NNRTI), FLAIR: maintenance baseline (day 1) score, induction baseline (week –20) HIV-1 RNA (< 100,000 vs. 100,000 c/mL), sex at birth, age (< 50 vs. ≥ 50 years), and race (white vs. non-white). CI confidence interval, SF-12 12-Item Short Form Health Survey Fig. 5 The SF-12 component scores adjusted treatment difference through 48 weeks. Adjusted mean is the estimated mean change from maintenance baseline score by visit in each treatment calculated from an ANCOVA model including the covariates, which are: ATLAS: baseline score, sex at birth, age, race (white, non-white), and third agent class (integrase inhibitors, protease inhibitors, NNRTI), FLAIR: maintenance baseline (day 1) score, induction baseline (week –20) HIV-1 RNA (< 100,000 vs. 100,000 c/mL), sex at birth, age (< 50 vs. ≥ 50 years), and race (white vs. non-white). CI confidence interval, SF-12 12-Item Short Form Health Survey Discussion Monthly CAB+RPV LA injections have demonstrated similar efficacy vs. daily oral ART in the pivotal phase 3 clinical trials ATLAS and FLAIR [12–14]. Although ISRs were common, they declined over time [12–14] and LA participants reported significantly higher levels of treatment satisfaction and preference for the LA regimen over previous daily oral ART, with respect to baseline, after almost 1 year of follow-up. Discontinuations due to ISRs were very uncommon (1%; 6/489), supporting these findings. In terms of general health outcomes, as measured by the SF-12 instrument, neither study showed statistically relevant changes from baseline in the physical or mental component subdomains for either oral or LA treatment. This is consist- ent with both the generally healthy baseline status of the two patient populations, and the noninferior efficacy of LA vs. oral treatment shown in both studies. The data also suggest that monthly injection therapy did not significantly alter par- ticipants’ general perceptions of their overall health status or functioning compared with oral therapy. i Treatment satisfaction with LA therapy, as assessed by the HIVTSQs instrument, reached similarly high scores in both studies, highlighting very high levels of satisfaction with LA treatment irrespective of prior ART experience. Differences in the change from MBL in treatment satisfac- tion were observed between ATLAS and FLAIR which could be explained by a difference in baseline satisfaction between the two studies. Changes from MBL in scores for the individual items within the HIVTSQs were higher in the LA group in ATLAS, showing the extent to which LA treatment contributes to improvement across almost all aspects of treatment satisfaction compared with oral therapy. Changes from baseline in FLAIR were generally smaller, particularly in the LA group, due to the ceiling effects caused by very high MBL scores. This observa- tion is consistent with the assumption that the previously treatment-naïve patient group in FLAIR, who were viro- logically suppressed prior to LA maintenance by a short course of oral treatment with a well-tolerated, single-tab- let dolutegravir-based modern regimen, would tend to be inherently more satisfied with their experience of their first oral treatment compared with those in ATLAS, who had already spent a number of years on oral therapies and were better able to compare treatment modalities. 1 1 3 3 3541 AIDS and Behavior (2020) 24:3533–3544 “Interest in the convenience of a monthly injectable treat- ment” (53%; 164/308) and “Discretion associated with a monthly injectable treatment” (32%; 98/308) were the top two reasons for choosing LA treatment. over many years in ATLAS appeared to show a stronger predisposition in favor of LA therapy. The results of the PIN questionnaire, assessing patient acceptability of injections, showed very similar attitudes towards IM administration between the two studies. Despite the common occurrence of ISRs, most participants indicated that the level of pain and ISRs was “very accept- able” 1 week after the initial LA injections, and scores improved significantly by 48 weeks of treatment. These trends are consistent with both the low discontinuation rate for ISRs and a declining incidence of ISRs across the maintenance phase of both studies [12, 13], similar to the time-dependent declines in ISRs seen with chronic LA parenteral treatment for other conditions, including infu- sions of ocrelizumab [34], rituximab [35], or ofatumumab [36] for multiple sclerosis, or IM injections of paliperi- done [37] for treatment of schizophrenia. Compliance with Ethical Standards In addition, although treatment satisfaction has been shown to be positively correlated with adherence [38–40], the very high adherence rates in both studies do not allow for demonstration of potential adherence benefits with an LA treatment, with the additional caveat that adherence in a clinical trial may not always reflect that in the routine clinic. In ATLAS and FLAIR, 98% of LA dosing visits took place within the 7-day dosing window. Also, although pill counts were not provided, no protocol deviation was marked for patients in the CAR groups in terms of adherence to oral medication, signifying that total number of days with daily oral treatment interruptions did not exceed 10% per patient [41]. To address these evidence gaps, an ongoing clinical trial of CAB LA+RPV LA vs. oral treatment (LATITUDE; NCT03635788) is currently recruiting a more difficult-to- treat population with adherence challenges and a wider range of clinical, behavioral and demographic characteris- tics, where satisfaction with treatment and adherence with the LA regimen might be more pronounced.f Conflict of interest Vasiliki Chounta, Krischan Hudson, Sandy Grif- fith, William Spreen, Mark Shaefer and David Margolis are employ- ees of ViiV Healthcare and own company stocks of GlaxoSmithKline (GSK). Jenny Huang and David Dorey are employees of GSK and own company stocks of GSK. Anthony Mills has received honoraria for consulting for the following companies: Merck, ViiV Healthcare, Janssen, and Gilead Sciences. The Men’s Health Foundation receives research support for clinical trials for which Anthony Mills is an inves- tigator. Hans Jäger has received lecture sponsorship or has served on advisory boards for the following companies: AbbVie, Gilead, GSK, Janssen, MSD Sharp & Dohme, TAD and ViiV Healthcare. Sharon Walmsley has served on advisory boards, speaking engagements, meetings, symposiums, and clinical studies for the following compa- nies: ViiV Healthcare, GSK, Merck, Janssen, and Gilead Sciences. Simon Vanveggel is an employee and shareholder of Janssen, Pharma- ceutical Companies of Johnson & Johnson. All other authors declare that they have no conflicts of interest. Ethical Approval All procedures performed in studies involving human participants were in accordance with the ethical standards of a national, regional, or investigational center ethics committee, or institutional review board, and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Discussion Also of note, even though both studies exceeded their female recruitment tar- gets, which were set with high thresholds, this number was still low compared with the female proportion in the overall HIV population. While subgroup analyses may help provide additional clarity, for the more difficult-to-recruit groups it is likely that large observational cohorts will provide more robust future data on patient experience with LA treat- ment, including data on how well these trial results reflect the routine clinic situation. Also, the effect of longer (more than 48 weeks) LA treatment on PROs will be explored in forthcoming 96-week data from FLAIR, and from the results of the ATLAS-2M trial (NCT03299049) of 8-weekly vs. 4-weekly LA treatment, which included a high proportion of patients who rolled over from ATLAS after the 48-week primary analysis. In summary, these PRO analyses of participants in the FLAIR and ATLAS studies provide a patient-oriented per- spective of the LA regimen. The results indicate a high degree of satisfaction, acceptance, tolerability, and prefer- ence for the LA regimen in multiple dimensions, supporting the therapeutic potential of monthly injectable LA therapy. Acknowledgements The authors thank all study participants and their families, the ATLAS and FLAIR clinical investigators and their staff, and study team members at ViiV Healthcare, GlaxoSmithKline, and Janssen. The PIN questionnaire is a modified version of the VAPI © Sanofi Pasteur, 2009 All Rights Reserved. VAPI contact information and permission to use: Mapi Research Trust, Lyon, France. Email: PROinformation@mapi-trust.org—internet: www.mapi-trust.org. Financial support for these studies was provided by ViiV Healthcare and Janssen. Editorial assistance with the preparation of the manu- script was provided by Nicole Ogbonnaya and Nick Fitch, PhD, of ArticulateScience (London, UK) with funding from ViiV Healthcare. Discussion The results of the preference question were striking in both studies: 97% (ATLAS) and 99% (FLAIR) of responding participants preferred the LA regimen over prior oral ther- apy at week 48. Although willingness to participate in these studies assumes a predisposition to at least consider inject- able therapy, the continued preference for the LA regimen is reassuring, suggesting that the LA regimen met partici- pants’ expectations despite the challenges of monthly clinic visits and the prevalence of ISRs. Missing assessments, which contribute to the difference between ITT-E and per- responder analysis, are attributed to malfunctioning of PRO devices used for capturing data, scheduling of visits that fall within the dosing window but outside of the data collection window for PROs, and study withdrawals prior to week 48. Specific factors driving the individual preferences in ATLAS may be multifaceted and vary between patients; however, convenience (53% of responders) and discretion (32%) were reported as the primary reasons for switching to LA therapy in the ATLAS study extension among those who had remained on oral treatment during the comparative treatment period. Similarly, treatment acceptance scores for participants receiving CAB+RPV LA also reached the same levels at weeks 24 and 48 in both ATLAS and FLAIR, consist- ent with what was previously observed with the HIVTSQ. The lack of a statistically significant change from MBL in FLAIR was again mostly driven by high initial treat- ment acceptance and suggests that these newly treated patients are likely to find monthly LA treatment and daily oral treatment with a modern and well-established single- tablet regimen similarly acceptable. By contrast, those with more extensive experience of daily oral therapies Cognizance must be made of the limitations of these data in terms of the generalizability of the ATLAS and FLAIR study datasets to important sociodemographic groups chronically underrepresented in all HIV randomized tri- als—for whom the experience of LA vs. oral treatment may 1 AIDS and Behavior (2020) 24:3533–3544 3542 who simply prefer the convenience and flexibility associated with an LA treatment schedule. be different either in degree or in kind. Relevant sociode- mographic factors such as employment, socioeconomic stratum, access to care, disability status, and drug use were not statistically captured in these studies. Compliance with Ethical Standards While treatment differences for any individual PRO in an open-label trial may be subject to selection bias, there was a clear preference to continue with LA treatment in both the FLAIR and ATLAS patient groups randomized to receive it. In ATLAS, there was a clear advantage for LA treatment satisfaction and acceptance among patients who had previously received oral cART. Real-world uptake of LA treatment would in fact be driven by patient choice. The data suggest that availability of an alternative to daily oral treatment option will address the needs of a number of PLWHIV, including those who have challenges with remain- ing adherent to daily pills despite their efforts, medical con- ditions interfering with oral administration, those who wish to reduce the daily reminder of their HIV status, or those References 19. Romaine J, Murray M, Bradley C. Psychometric evaluation of the revised HIV treatment satisfaction questionnaire (HIVTSQ). Value Health. 2016;19(7):A420. 1. May M, Gompels M, Sabin C. Life expectancy of HIV-1-positive individuals approaches normal conditional on response to antiret- roviral therapy: UK collaborative HIV cohort study. J Int AIDS Soc. 2012;15(Suppl 4):18078. 20. Romaine J, Murray M, Bradley C. Investigating the responsiveness to change of the HIV Treatment Satisfaction Questionnaire change version (HIVTSQc) in overcoming ceiling effects in the HIV Treatment Satisfaction Questionnaire status version (HIVTSQs). ISPOR Europe. Copenhagen, Denmark, 2019 [abstract PIH62].fi pp 2. Bor J, Herbst AJ, Newell ML, Barnighausen T. Increases in adult life expectancy in rural South Africa: valuing the scale-up of HIV treatment. Science. 2013;339(6122):961–5. OR Europe. Copenhagen, Denmark, 2019 [abstract PIH62]. 21. Murray M, Dorey D, Griffith S, et al. Satisfaction, tolerability, and acceptability of cabotegravir (CAB) + rilpivirine (RPV) long- acting therapy: LATTE-2 results. International AIDS Conference. Durban, South Africa, 2016 [poster THPEB052]. 3. Ortego C, Huedo-Medina TB, Llorca J, et al. Adherence to highly active antiretroviral therapy (HAART): a meta-analysis. AIDS Behav. 2011;15(7):1381–96.f Durban, South Africa, 2016 [poster THPEB052]. 22. Marant C, Longin J, Gauchoux R, et al. Long-term treatment acceptance: what is it, and how can it be assessed? Patient. 2012;5(4):239–49. 4. Gardner EM, Sharma S, Peng G, et al. Differential adherence to combination antiretroviral therapy is associated with virological failure with resistance. AIDS. 2008;22(1):75–82. 23. Lambert J, Chekroun M, Gilet H, Acquadro C, Arnould B. Assess- ing patients’ acceptance of their medication to reveal unmet needs: results from a large multi-diseases study using a patient online community. Health Qual Life Outcomes. 2018;16(1):134. 5. Bangsberg DR, Perry S, Charlebois ED, et al. Non-adherence to highly active antiretroviral therapy predicts progression to AIDS. AIDS. 2001;15(9):1181–3. 6. Weld ED, Rana MS, Dallas RH, et al. Interest of youth living with HIV in long-acting antiretrovirals. J Acquir Immune Defic Syndr. 2019;80(2):190–7. 24. Chevat C, Viala-Danten M, Dias-Barbosa C, Nguyen VH. Devel- opment and psychometric validation of a self-administered ques- tionnaire assessing the acceptance of influenza vaccination: the Vaccinees’ Perception of Injection (VAPI) questionnaire. Health Qual Life Outcomes. 2009;7:21. 7. Williams J, Sayles HR, Meza JL, et al. Long-acting par- enteral nanoformulated antiretroviral therapy: interest and attitudes of HIV-infected patients. Nanomedicine (Lond). 2013;8(11):1807–13. 25. Ware J Jr, Kosinski M, Keller SD. References A 12-Item Short-Form Health Survey: construction of scales and preliminary tests of reliability and validity. Med Care. 1996;34(3):220–33. 8. Iacob SA, Iacob DG, Jugulete G. Improving the adherence to antiretroviral therapy, a difficult but essential task for a success- ful HIV treatment–clinical points of view and practical considera- tions. Front Pharmacol. 2017;8:831. 26. Failde I, Medina P, Ramírez C, Arana R. Assessing health-related quality of life among coronary patients: SF-36 vs SF-12. Public Health. 2009;123(9):615–7. 9. Been SK, Schadé A, Bassant N, Kastelijns M, Pogány K, Verbon A. Anxiety, depression and treatment adherence among HIV- infected migrants. AIDS Care. 2019;31(8):979–87. 27. Webster KE, Feller JA. Comparison of the short form-12 (SF-12) health status questionnaire with the SF-36 in patients with knee osteoarthritis who have replacement surgery. Knee Surg Sports Traumatol Arthrosc. 2016;24(8):2620–6. g 10. Trezza C, Ford SL, Spreen W, Pan R, Piscitelli S. Formulation and pharmacology of long-acting cabotegravir. Curr Opin HIV AIDS. 2015;10(4):239–45. 28. Wukich DK, Sambenedetto TL, Mota NM, Suder NC, Rosario BL. Correlation of SF-36 and SF-12 component scores in patients with diabetic foot disease. J Foot Ankle Surg. 2016;55(4):693–6. 11. Edurant (rilpivirine) prescribing information. 2018. Janssen Ther- apeutics. https://www.janssenlabels.com/package-insert/product- monograph/prescribing-information/EDURANT-pi.pdf. Accessed March 25 2020. 29. Ware JE Jr, Sherbourne CD. The MOS 36-item short-form health survey (SF-36). I. Conceptual framework and item selection. Med Care. 1992;30(6):473–83. 12. Swindells S, Andrade-Villanueva J-F, Richmond GJ, et al. Long- acting cabotegravir and rilpivirine for maintenance of HIV-1 sup- pression. N Engl J Med. 2020;382(12):1112–23. 30. Huo T, Guo Y, Shenkman E, Muller K. Assessing the reliability of the Short Form 12 (SF-12) Health survey in adults with mental health conditions: a report from the wellness incentive and naviga- tion (WIN) study. Health Qual Life Outcomes. 2018;16(1):34. 13. Orkin C, Arasteh K, Hernández-Mora MG, et al. Long-acting cabotegravir and rilpivirine after oral induction for HIV-1 infec- tion. N Engl J Med. 2020;382(12):1124–35. 31. King JT Jr, Horowitz MB, Kassam AB, Yonas H, Roberts MS. The short form-12 and the measurement of health status in patients with cerebral aneurysms: performance, validity, and reliability. J Neurosurg. 2005;102(3):489–94. 14. Overton E, Orkin C, Swindells S, et al. Monthly long-acting cabo- tegravir and rilpivirine is noninferior to oral ART as maintenance therapy for HIV-1 infection: Week 48 pooled analysis from the Phase 3 ATLAS and FLAIR studies. IAS Conference on HIV Science. Mexico City, Mexico, 2019 [poster MOPEB257]. 32. Informed Consent Informed consent was obtained from all individual participants included in the study. Informed Consent Informed consent was obtained from all individual participants included in the study. Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, 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/. 1 3 3 AIDS and Behavior (2020) 24:3533–3544 3543 References Rai SK, Yazdany J, Fortin PR, Aviña-Zubieta JA. Approaches for estimating minimal clinically important differences in systemic lupus erythematosus. Arthritis Res Ther. 2015;17(1):143. 15. Margolis DA, Gonzalez-Garcia J, Stellbrink H-J, et al. Long- acting intramuscular cabotegravir and rilpivirine in adults with HIV-1 infection (LATTE-2): 96-week results of a ran- domised, open-label, phase 2b, non-inferiority trial. Lancet. 2017;390(10101):1499–510. 33. Ousmen A, Touraine C, Deliu N, et al. Distribution- and anchor- based methods to determine the minimally important difference on patient-reported outcome questionnaires in oncology: a structured review. Health Qual Life Outcomes. 2018;16(1):228. 34. Mayer L, Kappos L, Racke MK, et al. Ocrelizumab infusion experience in patients with relapsing and primary progressive multiple sclerosis: results from the phase 3 randomized OPERA I, OPERA II, and ORATORIO studies. Mult Scler Relat Disord. 2019;30:236–43. 16. Kerrigan D, Mantsios A, Gorgolas M, et al. Experiences with long acting injectable ART: a qualitative study among PLHIV partici- pating in a phase II study of cabotegravir + rilpivirine (LATTE-2) in the United States and Spain. PLoS ONE. 2018;13(1):e0190487. 17. Woodcock A, Bradley C. Validation of the HIV treatment satisfac- tion questionnaire (HIVTSQ). Qual Life Res. 2001;10(6):517–31. 35. Hawker K, O’Connor P, Freedman MS, et al. Rituximab in patients with primary progressive multiple sclerosis: results of a randomized double-blind placebo-controlled multicenter trial. Ann Neurol. 2009;66(4):460–71. 18. Woodcock A, Bradley C. Validation of the revised 10-item HIV Treatment Satisfaction Questionnaire status version and new change version. Value Health. 2006;9(5):320–33. 1 3 3544 AIDS and Behavior (2020) 24:3533–3544 39th ESCP European symposium on clinical pharmacy & 13th SFPC congress: clinical pharmacy at the front line of innova- tions. 21–23 October 2010, Lyon, France. Int J Clin Pharm. 2011;33:285–467. 36. Sorensen PS, Lisby S, Grove R, et al. Safety and efficacy of ofatu- mumab in relapsing-remitting multiple sclerosis: a phase 2 study. Neurology. 2014;82(7):573–81.fi 39th ESCP European symposium on clinical pharmacy & 13th SFPC congress: clinical pharmacy at the front line of innova- tions. 21–23 October 2010, Lyon, France. Int J Clin Pharm. 2011;33:285–467. 39th ESCP European symposium on clinical pharmacy & 13th SFPC congress: clinical pharmacy at the front line of innova- tions. 21–23 October 2010, Lyon, France. Int J Clin Pharm. 2011;33:285–467. gy 37. Savitz AJ, Xu H, Gopal S, et al. Efficacy and safety of paliperidone palmitate 3-month formulation for patients with schizophrenia: a randomized, multicenter, double-blind, noninferiority study. Int J Neuropsychopharmacol. 2016;19(7):pyw018.i 41. 41. References Teichner P, Cutrell A, D’Amico R, et al. Patient adherence to long- acting injectable cabotegravir + rilpivirine through 48 weeks of maintenance therapy in the Phase 3 ATLAS and FLAIR studies. Open Forum Infect Dis. 2019;6(Suppl 2):S20. 38. Boretzki J, Wolf E, Wiese C, et al. Highly specific reasons for nonadherence to antiretroviral therapy: results from the German adherence study. Patient Prefer Adherence. 2017;11:1897–906. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 39. Jordan J, Cahn P, Goebel F, Matheron S, Bradley C, Woodcock A. Abacavir compared to protease inhibitors as part of HAART regimens for treatment of HIV infection: patient satisfac- tion and implications for adherence. AIDS Patient Care STDs. 2005;19(1):9–18. 40. Delestras S, Roustit M, Mazet R, et al. Patient satisfaction with medication as an outcome for clinical pharmacists. Presented at 1 3 3
https://openalex.org/W1974552527	https://www.scielo.br/j/rbgo/a/DxFXx7n4grfKnTtjGrF79hg/?lang=pt&format=pdf	Portuguese	null	Relação entre a Classificação Clínica de White e a Histopatologia das Placentas de Gestantes Diabéticas	Revista brasileira de ginecologia e obstetrícia	2,000	cc-by	7,029	RESUMO Objetivo: analisar a relação entre a classificação clínica de White e as alterações histopatológicas de placentas de gestantes diabéticas, comparando, de forma qualitativa, as alterações histopatológicas de placentas de gestantes não-diabéticas e diabéticas gestacionais (classes A e A/B), clínicas de curta duração (classes B e C) e clínicas com vasculopatia (classes D a FRH), no termo e no pré-termo, e de acordo com a qualidade do controle glicêmico na gestação. Pacientes e Métodos: foram colhidas amostras de placentas de todas as gestantes diabéticas, atendidas entre 1991 e 1996 na Maternidade do Hospital das Clínicas da Faculdade de Medicina de Botucatu, coradas pela técnica de hematoxilina-eosina e submetidas a exame histopatológico. A qualidade do controle glicêmico foi analisada pela média glicêmica da gestação e classificada em adequada e inadequada, com limite de 120 mg/dl. A idade da gestação foi individualizada em termo e pré-termo. Objetivo: analisar a relação entre a classificação clínica de White e as alterações histopatológicas de placentas de gestantes diabéticas, comparando, de forma qualitativa, as alterações histopatológicas de placentas de gestantes não-diabéticas e diabéticas gestacionais (classes A e A/B), clínicas de curta duração (classes B e C) e clínicas com vasculopatia (classes D a FRH), no termo e no pré-termo, e de acordo com a qualidade do controle glicêmico na gestação. Pacientes e Métodos: foram colhidas amostras de placentas de todas as gestantes diabéticas, atendidas entre 1991 e 1996 na Maternidade do Hospital das Clínicas da Faculdade de Medicina de Botucatu, coradas pela técnica de hematoxilina-eosina e submetidas a exame histopatológico. A qualidade do controle glicêmico foi analisada pela média glicêmica da gestação e classificada em adequada e inadequada, com limite de 120 mg/dl. A idade da gestação foi individualizada em termo e pré-termo. p Resultados: observou-se que 42 recém-nascidos (43,3%) eram de termo e o restante, de pré- termo (56,7%). O índice de prematuridade foi maior nas diabéticas clínicas (classes B e C; D a FRH). Algumas alterações histopatológicas só foram encontradas em placentas de gestantes diabéticas: degeneração cistóide, edema corial, edema da íntima, dismaturidade, hiperplasia das células de Hofbauer, vilite, células fantasmas, dois vasos no cordão umbilical e endarterite. Conclusões: as alterações histopatológicas de placentas de gestantes com diabete gestacional (classes A e A/B), clínico de curta duração (classes B e C) e clínico com vasculopatia (classes D a FRH) foram semelhantes às das não-diabéticas e, portanto, independeram da classificação clínica de White. 22 (7): 401-411, 2000 RBGO 22 (7): 401-411, 2000 RBGO Trabalhos Originais RESUMO As alterações histopatológicas de placentas de gestantes diabéticas não se relacionaram com a idade gestacional ao nascimento e com a qualidade do controle glicêmico materno. A comparação entre as alterações histopatológicas e a elevada proporção de recém- nascidos pré-termo nas diabéticas clínicas, classes D a FRH, sugerem amadurecimento placentário precoce nas diabéticas clínicas com vasculopatia. PALAVRAS-CHAVE: Diabetes mellitus. Placenta. Prematuridade. The Relationship between White’s Classification and the Histopathological Changes in the Placentas of Diabetic Pregnant Women The Relationship between White’s Classification and the Histopathological Changes in the Placentas of Diabetic Pregnant Women Iracema de Mattos Paranhos Calderon1, César Pereira Lima2,3, Marilza Vieira Cunha Rudge1 Gilberto De Napoli2, Emílio Antonio Jeckel Neto4, Maria Delgi Ramos1 Introdução rios foram realizados na tentativa de explicar a fisiopatologia do comprometimento fetal no diabete. O problema é que as alterações encon- tradas não são características das placentas das diabéticas1-6. As alterações macroscópicas de- pendem da severidade do diabete materno, pois a análise da literatura7-9 mostra correlação en- tre a classificação de White10 e os critérios mor- fológicos da placenta11. Entretanto, são discor- dantes os resultados dos trabalhos correlacio- nando a gravidade do diabete materno, pela 1 Faculdade de Medicina de Botucatu - SP 2 Fundação Faculdade Federal de Ciências Médicas de Porto Alegre - RS 3 Faculdade de Medicina da PUC-RS / Porto Alegre - RS 4 Instituto de Geriatria e Gerontologia da PUC-RS / Porto Alegre - RS Correspondência: Iracema de Mattos Paranhos Calderon Rua Atílio Losi, 226 - Jardim Paraíso 18610-260 – Botucatu – SP Inúmeros estudos morfológicos placentá- Pacientes e Métodos É um estudo baseado na análise histopa- tológica, prospectivo e cego, de placentas prove- nientes de gestantes normais e diabéticas, acom- panhadas no Serviço de Diabete e Gravidez da Disciplina de Obstetrícia da Faculdade de Medi- cina de Botucatu - UNESP. A análise histopato- lógica foi realizada no Departamento de Patolo- gia da Fundação Faculdade Federal de Ciências Médicas de Porto Alegre. Foram colhidas amos- tras de placentas de todas as gestantes diabéti- cas (n = 91) atendidas no período de 1991 a 1996 e de seis gestantes não diabéticas. Nas gestantes diabéticas sem vasculopatia, as placentas são maiores, mais pesadas, com au- mento do número de células, provavelmente de- vido ao aumento na velocidade de divisão celu- lar, que ocorre antes da 36a semana. Quando a angiopatia é grave, podem ser encontrados infartos a partir de 32-34 semanas12-14. Foram incluídas no estudo as placentas de pacientes com diagnóstico de diabete clínico, das classes B a FRH, e de diabete gestacional, das classes A e A/B10. O grupo não-diabético foi com- posto por placentas de pacientes com rastreamento positivo para diabete sem confir- mação diagnóstica (grupo IA)18. No diabete existe imaturidade estrutural e funcional da placenta em relação à idade gesta- cional, que inclui tamanho aumentado do vilo terminal, aparência edematosa, diminuição da vascularização e da formação da membrana vásculo-sincicial, citotrofoblasto residual e au- mento das células de Hofbauer15. O significado deste citotrofoblasto metabolicamente ativo no final da gestação é controverso: não está claro se representa retardo na maturação do órgão ou regressão a um estadio celular mais primitivo, induzido por alteração degenerativa do sinciciotrofoblasto. As gestantes e suas placentas foram clas- sificadas segundo White10 e agrupadas, de acor- do com Gabbe19, em diabéticas gestacionais (clas- ses A e A/B), diabéticas clínicas de curta dura- ção (classes B e C) e diabéticas clínicas com vasculopatia (classes D a FRH). A qualidade do controle glicêmico materno foi analisada pela média glicêmica da gestação (MG), calculada pelo somatório de todas as gli- cemias plasmáticas avaliadas nos perfis glicêmicos de 24 horas, dividido pelo total de dosagens realizadas. O controle glicêmico foi con- siderado adequado quando a MG foi igual ou in- ferior a 120 mg/dl e inadequado quando superi- or a este limite20. Inúmeros estudos morfológicos placentá- 1 Faculdade de Medicina de Botucatu - SP 2 Fundação Faculdade Federal de Ciências Médicas de Porto Alegre - RS 3 Faculdade de Medicina da PUC-RS / Porto Alegre - RS 4 Instituto de Geriatria e Gerontologia da PUC-RS / Porto Alegre - RS Correspondência: Iracema de Mattos Paranhos Calderon Rua Atílio Losi, 226 - Jardim Paraíso 18610-260 – Botucatu – SP Inúmeros estudos morfológicos placentá- RBGO - v. 22, nº 7, 2000 401 Calderon et al Histopatologia da placenta em diabéticas Histopatologia da placenta em diabéticas classificação de White 10, com a patologia placentária. Isso pode ser decorrente da heterogeneidade dos fatores maternos e fetais, que resultariam em alterações da estrutura ou fun- ção placentárias. Whitsett e Brownscheidle2 des- tacaram que lesões placentárias semelhantes po- dem ocorrer em gravidez normal, e sugeriram que as diferenças deveriam ser mais quantitativas do que qualitativas. corial. Dois vasos: ausência de uma artéria no cor- dão umbilical. Edema da íntima: túnica íntima dos vasos es- pessada. Endarterite: oclusão vascular, geralmente por proliferação endotelial ou conjuntiva, conseqüen- te a processos inflamatórios vasculares ou na vi- zinhança de vasos. Hemorragia intersticial: extravasamento de sangue para o tecido extravascular. g p Congestão: vasos repletos de sangue. Infarto subcorial: hemorragia em área sub- corial. Membrana duplicada: espessamento de membrana basal pelo alargamento do espaço sub-celular. Edema vilositário: identificado pelo espaça- mento das células, pelo acúmulo excessivo de líquido extravascular e extracelular distendendo o vilo, com distribuição irregular. Hemorragia de cordão: presença de hemácias na geléia de Wharton. Tais alterações foram agrupadas pelo pa- tologista, de acordo com a etiopatogenia, em: Fibrose do vilo ou intervilosa: caracterizada pelo aumento de colágeno no interior do vilo ou como organização de áreas de infarto. lesões circulatórias: degeneração cistóide, edema da íntima, hemorragia intersticial, conges- tão e infarto subcorial. Calcificação: achados corados pela HE em azul escuro, sem tecido placentário identificável em seu interior, comuns em áreas mortas ou zo- nas nas quais o metabolismo está diminuído. Ocorrem como manifestações do processo distrófico. É um tecido degenerado onde o cálcio se precipita. lesões degenerativas: edema vilositário, fibrose do vilo ou intervilosa, calcificação e de- generação focal hialina. lesões proliferativas: dismaturidade, hiperpla- sia das células de Hofbauer, corioangiose e nós sinciciais. Degeneração focal hialina: é identificada pela presença de massas homogêneas, amorfas e eosinofílicas, de uma substância fortemente refringente, tingível com corantes ácidos. A patogenia não está totalmente clara. Discute-se a possibilidade de precipitação, por fibrinólise parcial ou imperfeita, ou absorção das proteínas do colágeno nos processos inflamatórios ou al- terações circulatórias como conseqüência da acidose cística. lesões inflamatórias: vilite e amnionite focal. outras lesões: incluindo “células fantasmas” ou restos celulares, dois vasos, endarterite, membrana duplicada e hemorragia de cordão (Fi- guras 1 a 5). O exame histopatológico do material foi cego por parte do patologista, que não tinha co- nhecimento prévio da classe diabética e da MG da paciente. Na análise individual das placentas, foram atribuídos escores para a presença (1) ou au- sência (0) das alterações histopatológicas. O somatório destes escores, dentro das classes de gestantes diabéticas, relacionado à idade gesta- cional (termo e pré-termo) e à qualidade do con- trole glicêmico (adequado e inadequado), foi usa- do para calcular o escore total. Dismaturidade: vilos com características de imaturidade entremeados com vilos madu- ros. Pacientes e Métodos Após o parto, as placentas fo- ram classificadas como sendo de gestação ter- mo e pré-termo, com limite de idade gestacio- nal, respectivamente, superior e inferior a 37 semanas completas. al-Okail & al-Attas16 não conseguiram correlacionar as alterações estruturais placen- tárias em pacientes com diabete de longa dura- ção e gestacional mal controlado, comparadas às não diabéticas. Jones e Fox15,17 demonstraram, por estudos ultra-estruturais e histoquímicos, que as alterações morfológicas placentárias do diabete gestacional são semelhantes às descri- tas para as insulino-dependentes, porém em menor quantidade. Essas alterações, mais quan- titativas do que qualitativas, entre as diabéticas gestacionais e clínicas, reforçam a hipótese de que a doença materna pode produzir uma gama de alterações no meio micrometabólico da pla- centa2. Imediatamente após a dequitação, foi co- lhido um fragmento central do cotilédone, no lo- cal da inserção do cordão umbilical na placenta, desde a face materna até a face fetal, e um frag- mento proximal do cordão umbilical. Ambos fo- ram fixados em formol e confeccionadas as lâmi- nas, coradas pela técnica de hematoxilina-eosina (HE). O objetivo deste trabalho é analisar a in- fluência da classificação clínica de White sobre as alterações histopatológicas de placentas de gestantes diabéticas, comparando, de forma qua- litativa, as alterações histopatológicas de placen- tas de gestantes não-diabéticas e diabéticas gestacionais (classes A e A/B), clínicas de curta duração (classes B e C) e clínicas com vasculopatia (classes D a FRH), no termo e no pré-termo, e de acordo com a qualidade do con- trole glicêmico na gestação. Num estudo piloto, a análise de 20 placen- tas de gestantes diabéticas identificou 22 alte- rações histopatológicas, descritas a seguir: Degeneração cistóide: presença de lacunas acelulares, contendo material fluido, proteináceo, encontradas no cório e, eventualmente, no vilo. Edema corial: aumento de líquido ao nível RBGO - v. 22, nº 7, 2000 402 Calderon et al Calderon et al Calderon et al Histopatologia da placenta em diabéticas Histopatologia da placenta em diabéticas corial. Hiperplasia das células de Hofbauer: aumento das células de Hofbauer ao nível dos vilos. Corioangiose: caracterizada por aumento do número dos vasos vilositários em extensão vari- ável do disco placentário, ao nível dos cotilédones. Pode ser compensatória, decorren- te de insuficiente aporte sanguíneo. O índice (%) de alteração placentária (IAP) foi calculado pela seguinte fórmula: IAP (%)= Es- core total . 22 . n . 100; onde 22 é número de altera- ções histopatológicas observadas nas placentas de gestantes diabéticas (estudo piloto) e n é o número de placentas estudadas por grupo. Nós sinciciais: denominação para a especiali- zação do trofoblasto que se caracteriza por aglo- merado nuclear com projeções periféricas dos vilos. Fazem parte da estrutura da placenta e têm coloração escura. É índice de maturação placentar e tem diminuição das células do citotrofoblasto. A ocorrência de alterações histopatológicas foi analisada pelo teste do χ2, empregando-se, quando necessário, o teste exato de Fisher. As médias de glicemia materna e peso do recém- nascido foram comparadas pelo teste “t” de Student. Adotou-se 5% como nível de significân- cia estatística (p<0,05). Vilite: presença de exsudato inflamatório comprometendo os vilos. Amnionite focal: exsudato inflamatório ao ní- vel do âmnio. Este trabalho foi aprovado pelo Comitê de Ética em Pesquisa da Faculdade de Medicina de Botucatu. “Células fantasmas” ou restos celulares: cé- lulas mortas, com desaparecimento do núcleo, restando apenas o contorno celular. RBGO - v. 22, nº 7, 2000 403 Calderon et al Histopatologia da placenta em diabéticas Figura 1 - Alterações circulatórias: A) degeneração cistóide: lacunas acelulares, com material fluido, proteináceo, encontradas no cório e, eventualmente, no vilo (HE lupa); B) edema corial: aumento de líquido ao nível corial (HE 40X); C) edema da íntima: túnica íntima dos vasos espessada (HE 10X); D) hemorragia intersticial: extravasamento de sangue para o tecido extra-vascular (HE 20X); E) congestão: vasos repletos de sangue (HE 20X); F) infarto sub-corial: hemorragia em área sub-corial (HE lupa). corial. A B C D E A B C D C D C D Figura 2 - Alterações degenerativas: A) edema vilositário: identificado pelo espaçamento das células, pelo acúmulo excessivo de líquido extravascular e extracelular distendendo o vilo, com distribuição irregular (HE 20X); B e C) fibrose do vilo (HE 10X) ou intervilosa (HE 20X): caracterizada pelo aumento de colágeno no interior do vilo ou como organização de áreas de infarto; D) calcificação: achados corados pela HE em azul-escuro, sem tecido placentário identificável em seu interior, comuns em áreas mortas ou zonas nas quais o metabolismo está diminuído. Ocorrem como manifestações do processo distrófico. É um tecido degenerado onde o cálcio se precipita (HE 20X); E) degeneração focal hialina: é identificada pela presença de massas homogêneas, amorfas e eosinofílicas, de uma substância fortemente refringente, tingível com corantes ácidos. A patogenia não está totalmente clara. Discute-se a possibilidade de precipitação, por fibrinólise parcial ou imperfeita, ou absorção das proteínas do colágeno nos processos inflamatórios ou alterações circulatórias como conseqüência da acidose cística (HE 40X). E E Figura 2 - Alterações degenerativas: A) edema vilositário: identificado pelo espaçamento das células, pelo acúmulo excessivo de líquido extravascular e extracelular distendendo o vilo, com distribuição irregular (HE 20X); B e C) fibrose do vilo (HE 10X) ou intervilosa (HE 20X): caracterizada pelo aumento de colágeno no interior do vilo ou como organização de áreas de infarto; D) calcificação: achados corados pela HE em azul-escuro, sem tecido placentário identificável em seu interior, comuns em áreas mortas ou zonas nas quais o metabolismo está diminuído. Ocorrem como manifestações do processo distrófico. É um tecido degenerado onde o cálcio se precipita (HE 20X); E) degeneração focal hialina: é identificada pela presença de massas homogêneas, amorfas e eosinofílicas, de uma substância fortemente refringente, tingível com corantes ácidos. A patogenia não está totalmente clara. Discute-se a possibilidade de precipitação, por fibrinólise parcial ou imperfeita, ou absorção das proteínas do colágeno nos processos inflamatórios ou alterações circulatórias como conseqüência da acidose cística (HE 40X). corial. A B C D E F Histopatologia da placenta em diabéticas Calderon et al Calderon et al Calderon et al A B C D Figura 1 - Alterações circulatórias: A) degeneração cistóide: lacunas acelulares, com material fluido, proteináceo, encontradas no cório e, eventualmente, no vilo (HE lupa); B) edema corial: aumento de líquido ao nível corial (HE 40X); C) edema da íntima: túnica íntima dos vasos espessada (HE 10X); D) hemorragia intersticial: extravasamento de sangue para o tecido extra-vascular (HE 20X); E) congestão: vasos repletos de sangue (HE 20X); F) infarto sub-corial: hemorragia em área sub-corial (HE lupa). E F Figura 1 - Alterações circulatórias: A) degeneração cistóide: lacunas acelulares, com material fluido, proteináceo, encontradas no cório e, eventualmente, no vilo (HE lupa); B) edema corial: aumento de líquido ao nível corial (HE 40X); C) edema da íntima: túnica íntima dos vasos espessada (HE 10X); D) hemorragia intersticial: extravasamento de sangue para o tecido extra-vascular (HE 20X); E) congestão: vasos repletos de sangue (HE 20X); F) infarto sub-corial: hemorragia em área sub-corial (HE lupa). RBGO - v. 22, nº 7, 2000 RBGO - v. 22, nº 7, 2000 404 Calderon et al Histopatologia da placenta em diabéticas Histopatologia da placenta em diabéticas A B C D A B B Figura 2 - Alterações degenerativas: A) edema vilositário: identificado pelo espaçamento das células, pelo acúmulo excessivo de líquido extravascular e extracelular distendendo o vilo, com distribuição irregular (HE 20X); B e C) fibrose do vilo (HE 10X) ou intervilosa (HE 20X): caracterizada pelo aumento de colágeno no interior do vilo ou como organização de áreas de infarto; D) calcificação: achados corados pela HE em azul-escuro, sem tecido placentário identificável em seu interior, comuns em áreas mortas ou zonas nas quais o metabolismo está diminuído. Ocorrem como manifestações do processo distrófico. É um tecido degenerado onde o cálcio se precipita (HE 20X); E) degeneração focal hialina: é identificada pela presença de massas homogêneas, amorfas e eosinofílicas, de uma substância fortemente refringente, tingível com corantes ácidos. A patogenia não está totalmente clara. Discute-se a possibilidade de precipitação, por fibrinólise parcial ou imperfeita, ou absorção das proteínas do colágeno nos processos inflamatórios ou alterações circulatórias como conseqüência da acidose cística (HE 40X). corial. Figura 2 - Alterações degenerativas: A) edema vilositário: identificado pelo espaçamento das células, pelo acúmulo excessivo de líquido extravascular e extracelular distendendo o vilo, com distribuição irregular (HE 20X); B e C) fibrose do vilo (HE 10X) ou intervilosa (HE 20X): caracterizada pelo aumento de colágeno no interior do vilo ou como organização de áreas de infarto; D) calcificação: achados corados pela HE em azul-escuro, sem tecido placentário identificável em seu interior, comuns em áreas mortas ou zonas nas quais o metabolismo está diminuído. Ocorrem como manifestações do processo distrófico. É um tecido degenerado onde o cálcio se precipita (HE 20X); E) degeneração focal hialina: é identificada pela presença de massas homogêneas, amorfas e eosinofílicas, de uma substância fortemente refringente, tingível com corantes ácidos. A patogenia não está totalmente clara. Discute-se a possibilidade de precipitação, por fibrinólise parcial ou imperfeita, ou absorção das proteínas do colágeno nos processos inflamatórios ou alterações circulatórias como conseqüência da acidose cística (HE 40X). RBGO - v. 22, nº 7, 2000 405 Histopatologia da placenta em diabéticas Calderon et al Calderon et al Calderon et al A B C D Figura 3 - Alterações proliferativas: A) dismaturidade: vilos com características de imaturidade entremeados com vilos maduros (HE 40X); B) hiperplasia das células de Hofbauer: aumento das células de Hofbauer ao nível dos vilos (HE 20X); C) corioangiose: caracterizada por aumento do número dos vasos vilositários em extensão variável do disco placentar, ao nível dos cotilédones. Pode ser compensatória, decorrente de insuficiente aporte sanguíneo (HE 40X); D) nós sinciciais: denominação para a especialização do trofoblasto que se caracteriza por aglomerado nuclear com projeções periféricas dos vilos. Fazem parte da estrutura da placenta e têm coloração escura. É índice de maturação placentar e tem diminuição das células do citotrofoblasto (HE 20X). Calderon et al Histopatologia da placenta em diabéticas A B B C D Figura 3 - Alterações proliferativas: A) dismaturidade: vilos com características de imaturidade entremeados com vilos maduros (HE 40X); B) hiperplasia das células de Hofbauer: aumento das células de Hofbauer ao nível dos vilos (HE 20X); C) corioangiose: caracterizada por aumento do número dos vasos vilositários em extensão variável do disco placentar, ao nível dos cotilédones. Pode ser compensatória, decorrente de insuficiente aporte sanguíneo (HE 40X); D) nós sinciciais: denominação para a especialização do trofoblasto que se caracteriza por aglomerado nuclear com projeções periféricas dos vilos. corial. Fazem parte da estrutura da placenta e têm coloração escura. É índice de maturação placentar e tem diminuição das células do citotrofoblasto (HE 20X). Figura 3 - Alterações proliferativas: A) dismaturidade: vilos com características de imaturidade entremeados com vilos maduros (HE 40X); B) hiperplasia das células de Hofbauer: aumento das células de Hofbauer ao nível dos vilos (HE 20X); C) corioangiose: caracterizada por aumento do número dos vasos vilositários em extensão variável do disco placentar, ao nível dos cotilédones. Pode ser compensatória, decorrente de insuficiente aporte sanguíneo (HE 40X); D) nós sinciciais: denominação para a especialização do trofoblasto que se caracteriza por aglomerado nuclear com projeções periféricas dos vilos. Fazem parte da estrutura da placenta e têm coloração escura. É índice de maturação placentar e tem diminuição das células do citotrofoblasto (HE 20X). A B Figura 4 - Alterações inflamatórias: A) vilite: presença de exsudato inflamatório comprometendo os vilos (HE 40X); B) amnionite focal: exsudato inflamatório ao nível do âmnio (HE 20X). ite: presença de exsudato inflamatório comprometendo os vilos (HE 40X); B) amnionite focal: exsudato inflamatório ao nível do âmnio (HE 20X). Figura 4 - Alterações inflamatórias: A) vilite: presença de exsudato inflamatório comprometendo os vilos (HE 40X); B) amnionite focal: exsudato inflamatório ao nível do âmnio (HE 20X). RBGO - v. 22, nº 7, 2000 RBGO - v. 22, nº 7, 2000 406 Histopatologia da placenta em diabéticas Histopatologia da placenta em diabéticas Calderon et al Figura 5 - Outra alterações: A) dois vasos: ausência de uma artéria no cordão umbilical (HE lupa); B) endarterite: oclusão vascular, geralmente por proliferação endotelial ou conjuntiva, conseqüente a processos inflamatórios vasculares ou na vizinhança de vasos (HE 20X); C) membrana duplicada: espessamento de membrana basal pelo alargamento do espaço sub-celular (HE 40X); D) hemorragia de cordão: presença de hemácias na geléia de Wharton (HE lupa). A B C D B A B A Figura 5 - Outra alterações: A) dois vasos: ausência de uma artéria no cordão umbilical (HE lupa); B) endarterite: oclusão vascular, geralmente por proliferação endotelial ou conjuntiva, conseqüente a processos inflamatórios vasculares ou na vizinhança de vasos (HE 20X); C) membrana duplicada: espessamento de membrana basal pelo alargamento do espaço sub-celular (HE 40X); D) hemorragia de cordão: presença de hemácias na geléia de Wharton (HE lupa). Resultados A membrana Tabela 1 - Características maternas e dos recém-nascidos (RN) e média glicêmica da gestação (MG) de não-diabéticas e diabéticas gestacionais (classes A e A/B), clínicas de curta duração (classes B e C) e clínicas com vasculopatia (classes D a FRH). Características Maternas MG (mg/dl) () Recém-nascidos Peso médio (g) () RN termo () RN pré-termo () Total de casos Não-diabéticas 79,1 3250 6 (100,0%) 0 6 A e A/B 100,7 3417 24 (66,7%) 12 (33,3%) 36 B e C 119,2 3229 11 (25,6%) 32 (72,1%) 43 D a FRH 113,0 2458 1 (8,3%) 11 (91,7%) 12 Diabéticas (classes) () Significância estatística (p<0,05): MG: (Não-diabéticas < A e A/B) < B e C = D a FRH; Peso RN: D a FRH < demais; RN termo: Não-diabéticas = A e A/B > B e C = D a FRH; RN pré-termo: A e A/B < B e C = D a FRH. ém-nascidos (RN) e média glicêmica da gestação (MG) de não-diabéticas e diabéticas gestacionais (classes A e A/B), clínicas de curta duração (classes B e RH) ( )Significância estatística (p<0,05): MG: (Não-diabéticas < A e A/B) < B e C = D a FRH; Peso RN: D a FRH < demais; RN termo: Não-diabéticas = A e A/B > B e C = D a FRH; RN pré-termo: A e A/B < B e C = D a FRH. vasculopatia (classes D a FRH). Resultados ção de recém-nascidos, relacionando peso e ida- de gestacional, não se observou diferença entre as proporções de adequados, pequenos e gran- des, sendo a maioria deles de peso adequado para a idade gestacional (AIG). Observou-se que 42 recém-nascidos (43,3%) eram de termo e o restante, de pré-termo (56,7%). O índice de pre- maturidade foi maior nas diabéticas clínicas (clas- ses B e C; D a FRH) (Tabela 1). Foram analisadas 97 gestações, classifica- das de acordo com White10 e agrupadas segun- do Gabbe19: não-diabéticas (n = 6), diabéticas gestacionais (classes A e A/B - n = 36), diabéti- cas clínicas de curta duração (classes B e C - n = 43) e diabéticas clínicas com vasculopatia (clas- ses D a FRH - n = 12). A idade materna foi seme- lhante entre os grupos, variando entre 26 e 29 anos completos. As pacientes diabéticas gestacionais apresentaram MG superior às não- diabéticas e inferior às das diabéticas clínicas. Entre as diabéticas clínicas, o tempo de evolu- ção e a vasculopatia não interferiram nos valo- res da MG. Algumas alterações histopatológicas só fo- ram encontradas em placentas de gestantes di- abéticas: degeneração cistóide, edema corial, edema da íntima, dismaturidade, hiperplasia das células de Hofbauer, vilite, células fantasmas, dois vasos no cordão umbilical e endarterite (Ta- bela 2). Outras, puderam ser observadas tanto em placentas de gestantes diabéticas como não- diabéticas, mas apenas algumas mostraram dife- rença significativa. Quando se considerou a clas- sificação clínica de White10 e o agrupamento de Gabbe19, o infarto subcorial foi mais freqüente em placentas de gestantes normais, comparadas com Os menores pesos de recém-nascidos fo- ram observados nas classes D a FRH. Nas de- mais classes de gestantes diabéticas, os recém- nascidos tiveram média de peso equivalente aos dos filhos de mães não-diabéticas. Na classifica- RBGO - v. 22, nº 7, 2000 407 Histopatologia da placenta em diabéticas Calderon et al duplicada foi mais observada em placentas de gestantes não-diabéticas quando comparadas com as diabéticas gestacionais (A e A/B) e clíni- cas de curta duração (B e C) (Tabela 2). placentas de diabéticas gestacionais (classes A e A/B). A fibrose intervilosa diferenciou as placen- tas de diabéticas gestacionais e clínicas de curta duração (classes A e A/B > B e C). Calderon et al Calderon et al Histopatologia da placenta em diabéticas glicêmico (MG adequada e inadequada). O limi- te de MG de 120 mg/dl não diferenciou as alte- rações histopatológicas placentárias nas classes de gestantes diabéticas (Tabela 3). Nas classes de gestantes avaliadas, o IAP foi semelhante em todas as placentas e não dife- renciou as de termo e pré-termo. O mesmo se observou em relação à qualidade do controle es placentárias (IAP), agrupadas conforme a etiopatogenia, em gestantes não-diabéticas e diabéticas, relacionadas à idade gestacional e à qualidade Tabela 3 - Freqüência (%), escore total e índice de alterações placentárias (IAP), agrupadas conforme a etiopatogenia, em gestantes não-diabéticas e diabéticas, relacionadas à idade gestacional e à qualidade do controle glicêmico: adequado e inadequado. Alterações Histopatológicas Circulatórias Degenerativas Proliferativas Inflamatórias Outras Escore total IAP (%) () Não-diabéticas Termo n = 6 5 (19,3) 13 (50,0) 2 (7,7) 1 (3,8) 5 (19,2) 26 19,7 Termo n = 36 29 (18,7) 98 (63,2) 14 (9,0) 4 (2,6) 10 (6,5) 155 19,6 Pré-Termo n = 55 42 (20,7) 125 (61,6) 13 (6,4) 6 (3,0) 17 (8,3) 203 16,8 MG adeq n = 68 55 (19,8) 171 (61,7) 22 (8,0) 7 (2,5) 22 (8,0) 277 18,5 MG inadeq n = 23 16 (19,8) 52 (64,2) 5 (6,2) 3 (3,6) 5 (6,2) 81 16,0 Diabéticas Diabéticas () ns (p>0,05) ela 3 - Freqüência (%), escore total e índice de alterações placentárias (IAP), agrupadas conforme a etiopatogenia, em gestantes não-diabéticas e diabéticas, relac ontrole glicêmico: adequado e inadequado. tológicas em placentas de gestantes não-diabé- ticas e diabéticas, a fibrose intervilosa foi mais freqüente em diabéticas gestacionais do que em diabéticas clínicas de curta evolução. Isto pode estar relacionado ao diagnóstico, que é mais tar- dio no diabete gestacional e, em conseqüência, o início do tratamento é retardado. Este aspecto clínico torna-se importante, pois a fibrose leva à hipoxia fetal, complicação associada a índices baixos de Apgar e aumento da morbimortalidade neonatal25, evidenciando a importância do diag- nóstico e tratamento precoces do diabete gesta- cional. Resultados Alterações Histopatológicas Circulatórias Degeneração cistóide Edema corial Edema da íntima Hemorragia intersticial Congestão Infarto sub-corial () Degenerativas Edema vilositário Fibrose do vilo Fibrose intervilosa () Calcificação Degeneração focal hialina Proliferativas Dismaturidade Hiperplasia Hofbauer Corioangiose Nós sinciciais Inflamatórias Vilite Amnionite focal Outras Células fantasmas Dois vasos Endarterite Membrana duplicada () Hemorragia do cordão Total de casos Não-Diabéticas 1 (16,7) 2 (33,3) 2 (33,3) 1 (16,7) 1 (16,7) 3 (50,0) 4 (66,7) 4 (66,7) 1 (16,7) 1 (16,7) 1 (16,7) 2 (33,3) 3 (50,0) 6 A e A/B 2 (5,6) 3 (8,3) 1 (2,8) 21 (58,3) 1 (2,8) 8 (22,2) 5 (13,9) 32 (88,9) 17 (47,2) 33 (91,7) 6 (16,7) 2 (5,6) 3 (8,3) 5 (13,9) 5 (13,9) 7 (19,4) 36 B e C 1 (2,3) 1 (2,3) 1 (2,3) 2 (4,7) 28 (65,1) 2 (4,6) 8 (18,6) 5 (11,7) 30 (69,8) 22 (51,7) 34 (79,1) 2 (4,6) 1 (2,3) 5 (1,7) 4 (9,3) 1 (2,3) 3 (7,0) 1 (2,3) 2 (4,6) 2 (4,6) 5 (11,7) 43 D a FRH 1 (8,3) 1 (8,3) 6 (50,0) 5 (41,7) 1 (8,3) 9 (75,0) 4 (33,3) 10 (83,3) 3 (25,0) 1 (8,3) 1 (8,3) 1 (8,3) 1 (8,3) 1 (8,3) 2 (16,7) 12 (*) Significância estatística (p<0,05): Infarto subcorial: Não-diabéticas > A e A/B; Fibrose intervilosa: A e A/B > B e C; Membrana duplicada: Não-diabéticas > A e A/B = B e C Classes de diabéticas RBGO - v. 22, nº 7, 2000 408 Calderon et al Discussão Na opinião de Benirschke e Kaufmann27, ne- nhuma das alterações é específica para o diabete materno, refletindo apenas a concentração de glicose disponível e os ajustes fetais para a ofer- ta intermitente e excessiva deste metabólito. Os resultados aqui encontrados reforçam tal opinião - a histopatologia placentária não tem caracte- rísticas específicas do diabete e não se relaciona com a gravidade do quadro clínico materno. Con- cluímos que as alterações histopatológicas de placentas de gestantes com diabete gestacional (classes A e A/B), clínico de curta duração (clas- ses B e C) e clínico com vasculopatia (classes E a FRH) foram semelhantes às das não-diabéticas e, portanto, independeram da classificação clí- nica de White. As alterações histopatológicas de placentas de gestantes diabéticas não se relaci- onaram com a idade gestacional e com a quali- dade do controle glicêmico materno. Isto também aconteceu neste trabalho, em que a faixa média de glicemia materna não foi muito diferente entre os grupos e não atingiu 120 mg/ dl. Na opinião de Benirschke e Kaufmann27, ne- nhuma das alterações é específica para o diabete materno, refletindo apenas a concentração de glicose disponível e os ajustes fetais para a ofer- ta intermitente e excessiva deste metabólito. Os resultados aqui encontrados reforçam tal opinião - a histopatologia placentária não tem caracte- rísticas específicas do diabete e não se relaciona com a gravidade do quadro clínico materno. Con- cluímos que as alterações histopatológicas de placentas de gestantes com diabete gestacional (classes A e A/B), clínico de curta duração (clas- ses B e C) e clínico com vasculopatia (classes E a FRH) foram semelhantes às das não-diabéticas e, portanto, independeram da classificação clí- nica de White. As alterações histopatológicas de placentas de gestantes diabéticas não se relaci- onaram com a idade gestacional e com a quali- dade do controle glicêmico materno. Isto também aconteceu neste trabalho, em que a faixa média de glicemia materna não foi muito diferente entre os grupos e não atingiu 120 mg/ dl. Na opinião de Benirschke e Kaufmann27, ne- nhuma das alterações é específica para o diabete materno, refletindo apenas a concentração de glicose disponível e os ajustes fetais para a ofer- ta intermitente e excessiva deste metabólito. Discussão Os resultados aqui encontrados reforçam tal opinião O IAP traduziu o percentual das caracte- rísticas histopatológicas nas placentas de ges- tantes não-diabéticas e diabéticas, no termo e pré-termo e no controle glicêmico materno ade- quado e inadequado. Apesar do cuidado na di- ferenciação destas variáveis confundidoras, o IAP não diferenciou as placentas resultantes de di- ferentes condições clínicas, idade gestacional ou qualidade de tratamento. al-Okail & al-Attas16 encontraram altera- ções mais acentuadas nas placentas das diabé- ticas gestacionais, comparadas às das clínicas de longa evolução. Entretanto, não conseguiram correlacionar as alterações estruturais placen- tárias provenientes de pacientes com diabete de longa duração com as de diabete gestacional mal controlado. Teasdale26 mostrou que diferentes alterações estruturais placentárias provêm de pacientes com a mesma gravidade clínica da do- ença. Fox22 e Haust24 já chamavam atenção para o cuidado que deve ser tomado no exame das placentas, ou seja, comparar placentas de mes- ma idade gestacional. Whitsett e Brownscheidle2 aventaram a hipótese de que a dismaturidade representaria retardo na maturação ou regres- são a um estadio celular mais primitivo. Mesmo aceitando que a imaturidade estrutural e funci- onal da placenta é alteração que pode ser ob- servada em gestações complicadas pelo diabete em idades gestacionais mais avançadas, não foi possível diferenciar as alterações histopatológi- cas, no termo e pré-termo, entre diabéticas e não- diabéticas. É interessante a concordância dos achados histopatológicos do grupo controle e do grupo com diabete clínico com vasculopatia, uma vez que as placentas do grupo controle são todas de termo, ao contrário daquelas de diabé- ticas clínicas com vasculopatia, nas quais 91,7% são pré-termo. Pode-se inferir que as placentas resultantes de gestações das classes D a FRH no pré-termo são semelhantes às das gestantes normais no termo. Este achado sugere senescência precoce das placentas no diabete materno com vasculopatia. A comparação entre as alterações histopa- tológicas e a elevada proporção de recém-nasci- dos pré-termo nas diabéticas clínicas, classes D a FRH, sugerem amadurecimento placentário precoce nas diabéticas clínicas com vasculopatia. Discussão Vários estudos demonstraram que as pla- centas de diabéticas não têm achados histopa- tológicos patognomônicos3-6,21. Outros trabalhos relataram elevada incidência de anomalias nas placentas, porém sem definir achado caracterís- tico22. Vogel23 descreveu a placopatia diabética, caracterizada por vilo embriônico persistente, dismaturidade, desordens de ramificação do vilo e corioangiose. Fox22 encontrou anomalias na maturação do vilo, endarterite obliterativa intravilosa, espessamento da característica mem- brana basal do trofoblasto e necrose fibrinóide do vilo corial. As lesões degenerativas e circulatórias fo- ram as mais comuns, tanto nas placentas das diabéticas como das não-diabéticas. Os estudos pioneiros sobre placentas de gestantes diabéti- cas já caracterizavam a pletora, que se relaciona tanto à congestão e hemorragias, como à hipóxia tecidual, levando à corioangiose25. As alterações degenerativas dos vilos - edema, fibrose, calcificação e degeneração hialina - foram inter- pretadas por esses autores como imaturidade do órgão. Entretanto, estudos posteriores admitem a dismaturidade da placenta no diabete, associ- ada à corioangiose, alterações vilositárias e au- mento dos nós sinciciais6,23,24, características ob- servadas, também, em placentas de gestantes não-diabéticas. Das 22 alterações histopatológicas avalia- das neste trabalho, nove foram encontradas ape- nas nas placentas de gestantes diabéticas. A dismaturidade esteve presente em 11 de 91 (12,1%) e a endarterite, em cerca de 9% das pla- centas de gestantes diabéticas. Essas alterações foram consideradas, por outros autores, como lesões freqüentes em placentas de gestantes di- abéticas20. As placentas das gestantes diabéticas mos- traram características histopatológicas semelhan- tes às das não-diabéticas, sendo que algumas características que apresentaram diferença es- tatística não foram suficientes para separar as placentas das gestantes não-diabéticas das dia- béticas gestacionais ou clínicas, dificuldade sa- lientada, também, por Haust24. As lesões proliferativas também não foram exclusivas do diabete materno. A corioangiose e o aumento dos nós sinciciais, conseqüentes à hipoxia tecidual, podem estar presentes na hi- pertensão, na doença hemolítica perinatal e em tabagistas crônicas, o que explicaria o encontro Chama a atenção que, a despeito das se- melhanças entre a incidência de lesões histopa- RBGO - v. 22, nº 7, 2000 409 Calderon et al Histopatologia da placenta em diabéticas Calderon et al dessas alterações histopatológicas nas placen- tas de gestantes não-diabéticas. Isto também aconteceu neste trabalho, em que a faixa média de glicemia materna não foi muito diferente entre os grupos e não atingiu 120 mg/ dl. SUMMARY Purpose: to analyze the relationship between White’s classification and the histopathological, changes occurring in the placentas of diabetic pregnant women, performing a qualitative comparison of histopathological changes in the placentas of nondiabetic pregnant women with those in diabetic ones (classes A and A/B), clinical, short duration (classes B and C), and clinical with vasculopathy (classes D to FRH), studying the influence of the quality of glycemic control and of gestational age on placental changes in the three groups of diabetic pregnant women. p g Patients and methods: specimens of placentas were collected from all diabetic pregnant women seen between 1991 and 1996 in the Maternity Section of the Hospital das Clínicas, Faculdade de Medicina de Botucatu, stained using the hematoxylin-eosin technique, and submitted to a histopathological examination. The quality of glycemic control was analyzed by the glycemia average of gestation and classified as adequate or inadequate, with a limit of 120 mg/dl. Gestational age was individualized as term and preterm. É necessário que se analise com precau- ção os trabalhos que mostram resultados histo- patológicos diferentes nas placentas de gestan- tes diabéticas. Nos últimos tempos, houve mu- dança na abordagem do diabete materno e, con- seqüentemente, nos resultados perinatais. O rí- gido controle da glicemia materna, princípio bá- sico do tratamento atual no diabete, previne as complicações decorrentes da hiperglicemia nos meios materno, fetal, neonatal e placentário20. p Results: forty-two newborns (43.3%) were born at term and the remaining were preterm (56.7%). The prematurity rate was higher for women with clinical diabetes (classes B and C; D to FRH). Some histopathological alterations were observed only in placentas from diabetic pregnant RBGO - v. 22, nº 7, 2000 RBGO - v. 22, nº 7, 2000 410 Histopatologia da placenta em diabéticas Calderon et al 9. Vogel M, Kloss K. Diabetes in der Schwangerschaft: neue morphologische Befunde Plazenta und Fetus in perinataler Medizin bd. 6.7. Dtsch. Kongress für Perinatale Medizin; Berlin 1974. women: cystoid degeneration, chorial edema, intima edema, dysmaturity, Hofbauer cell hyperplasia, villitis, ghost cells, two vessels in the umbilical cord, and endarteritis. 10.White P. Classification of obstetric diabetes. Am J Obstet Gynecol 1978; 130:228-30. Referências 17.Jones CJ, Fox H. Placental changes in gestational diabetes. An ultrastructural study. Obstet Gynecol 1976; 48:274–80. 1. Oh W. Neonatal care and long-term outcome in infants of diabetic mothers. In: Merkatz IR, Adam PAJ. The Diabetic Pregnancy: a perinatal perspective. 1st ed. New York: Grune & Stratton; 1979. p.195-205. 18.Rudge, MVC. Diabetes e gestação. Femina 1988; 16:125–40. 19.Gabbe SG. Management of diabetes mellitus in pregnancy. Am J Obstet Gynecol 1985; 153:824–8. 2. Whitsett JA, Brownscheidle CM. Aspects of placental structure and function in maternal diabetes. In: Merkatz IR, Adam AJ. The Diabetic Pregnancy: a perinatal perspective. New York: Grune & Stratton; 1979. p.123–43. 20.Rudge MVC, Calderon IMP, Ramos MD, Abbade JF, Rugolo LMSS. Perinatal outcome of pregnancies complicated by diabetes and by maternal daily hyperglycemia not related to diabetes - A retrospective 10 years analysis. Gynecol Obstet Invest - in press. 3. Driscoll SG. The pathology of pregnancy complicated by diabetes mellitus. Med Clin North Am 1965;49:1053-67. 21.Zacks SI, Blazar AS. Chorionic villi in normal pregnancy, pre-eclamptic toxemia, erythroblastosis, and diabetes mellitus: a light and electron microcoscope study. Obstet Gynecol 1963; 22:149–53. 4. Benirschke K, Driscoll SG. The pathology of the human placenta. 1st ed. Berlin: Springer-Verlag; 1967. p.512. 22.Fox H. Pathology of the placenta in maternal diabetes mellitus. Obstet Gynecol 1969; 34:792-8. 5. Jácomo KH, Benedetti WL, Sala MA, Alvarez H. Pathology of the trophoblast and fetal vessels of the placenta in maternal diabetes mellitus. Acta Diabetol Lat 1976; 13:216-35. 23.Vogel M. Plakopathia diabetica. Entwicklungsstörungen der Plazenta bei Diabetes mellitus der Mutter. Virchows Arch Pathol Anat Physiol Klin Med 1967; 343:51–63. 6. Singer DB. The placenta in pregnancies complicated by diabetes mellitus. Perspect Pediatr Pathol 1984; 8:199–212. 24.Haust D. Maternal diabetes mellitus effects on the fetus and placenta. In: Naeye RL, Kissane JM, Kaufman N, editors. Perinatal Diseases. 1st ed. Baltimore: Williams & Wilkins; 1981. p.201-77. 7. Emmrich P, Gödel E. Morphologie der Plazenta bei mütterlichem Diabetes mellitus. Ergebnisse morphologischer Untersuchungen. Zentralbl Allg Pathol 1972;116:56–63. 25.Naeye RL. Disorders of the placenta, fetus and neonate: diagnosis and clinical significance. 1st ed. St. Louis: Mosby Year Book; 1992. p.375. 8. Emmrich P. Morphologie der Plazenta bei mütterlichem Diabetes mellitus. I. Vorschlag einer einheitlichen morphologischen Plazentadiagnostik unter Berücksichtigung internistischer, geburtshilflicher und pädiatrischer Gesichtspunkte sowie Literaturübersicht. Zentralbl Gynäkol 1972; 94:881-7. 26.Teasdale F. Histomorphometry of the human placenta in class B diabetes mellitus. Placenta 1983; 4:1–12. 27.Benirschke K, Kaufmann P. SUMMARY Conclusions: histopathological changes in the placentas of pregnant women with gestational diabetes (classes A and A/B), clinical, short duration (classes B and C), and clinical with vasculopathy (classes D to FRH) were similar to those in the nondiabetic ones, and, therefore, were independent of White’s clinical classification. The histopathological changes in the placentas of pregnant women with gestational diabetes (classes A and A and B), clinical, short duration (classes B and C), and clinical with vasculopathy (classes D to FRH) were not related to gestational age at birth and to the quality of glycemic control of the mother. The comparison between histopathological changes and the increased number of preterm newborns in clinical diabetes, class D to FRH, suggest early placental ageing in clinical diabetes patients. 11.Semmler K, Emmrich P. Morphologie der Plazenta in Relation zur Glykämielage in der Schwangerschaft beim Diabetes mellitus. Z Geburtshilfe Perinatol 1989; 193:124–8. 12.Benirschke K, Brown WH. Vascular anomaly of umbilical cord: Absence of one umbilical artery in umbilical cords of normal and abnormal fetuses. Obstet Gynecol 1955; 6:399–404. 13.Benirschke K. A review of the pathologic anatomy of the human placenta. Am J Obstet Gynecol 1962; 84:1595–622. 14.Driscoll SG. Placental manifestations of malformation and infection. In: Gruenwald P, editor. The Placenta and its Maternal Supply Line: effects of insufficiency on the fetus. 1st ed. Baltimore: University Park; 1975. p.248. 15.Jones CJ, Fox H. An ultrastructural and ultrahistochemical study of the placenta of the diabetic woman. J Pathol 1976; 119:91–9. KEY WORDS: Diabetes. Placenta. Prematurity. 16.al-Okail MS, al-Attas OS. Histological changes in placental syncytiotrophoblasts of poorly controlled gestational diabetic patients. Endocr J 1994; 41:355–60. Referências Pathology of the human placenta. 3rd ed. New York: Springer- Verlag; 1995. p.871. RBGO - v. 22, nº 7, 2000 411
https://openalex.org/W2556458297	https://metodickividici.ff.uns.ac.rs/index.php/MV/article/download/687/693	Bosnian	null	PRENOSNE GREŠKE KOD IZVORNIH GOVORNIKA BOSANSKOG/HRVATSKOG/SRPSKOG JEZIKA TOKOM PISANE PRODUKCIJE NA NJEMAČKOM KAO STRANOM JEZIKU	Metodički vidici/Metodički vidici	1,970	cc-by-sa	5,513	UDC [811.112.2:811.163.4]’255 UDC [811.112.2:811.163.4]’255 UDC [811.112.2:811.163.4]’255 Belma S. Prndelj Šator Univerzitet „Džemal Bijedić” u Mostaru Odsjek za njemački jezik i književnost Belma.Prndelj@unmo.ba Sažetak U ovladavanju njemačkim kao stranim jezikom neizbježna su od- stupanja koja se nerijetko javljaju kao posljedica transfera iz dominantnog jezičkog sistema, a to je maternji jezik kao J1. Ovaj rad se bavi odstupanji- ma ili greškama koje nastaju kao rezultat preklapanja bosanskog/hrvatskog/ srpskog jezika kao J1 i njemačkog jezika kao J2. Za navedenu analizu po- služili su pismeni radovi studenata Fakulteta humanističkih radova koji uče njemački jezik kao strani jezik, a njihov maternji jezik je bosanski/hrvatski/ srpski jezik – službeni jezici u BiH. Vrši se komparacija prenosnih grešaka u završnim radovima studenata nakon prve i druge godine učenja njemač- kog kao stranog jezika. Odstupanja se dijele na: pravopisna, gramatička i leksička. Neke greške nestaju ili se smanjuju na drugoj godini učenja, dok se neka odstupanja prenose iz prve na drugu godinu učenja. Ključne riječi: prenosne greške, bosanski/hrvatski/srpski jezik kao J1, njemački kao J2, dominantni jezički sistem 1. UVOD Istraživanje je potaknuto iskustvom podučavanja njemačkog kao stranog jezika te pilot istraživanjem provedenim među grupom od 16 polaznika (izvornih govornika bosanskog/hrvatskog/srpskog jezika) kursa njemačkog jezika u Kultur- nom centru „Kralj Fahd” u Mostaru. Kod navedenih polaznika je uočeno da najče- šće griješe prilikom upotrebe određenog/neodređenog člana, pisanja imenica itd. S obzirom na to da je autorica ovog rada po vokaciji germanistica, a ma- ternji jezik joj je bosanski (s tim da se bavila kontrastiranjem njemačkog i sa bo- 231 Методички Видици 4 Prndelj Šator: PRENOSNE GREŠKE KOD IZVORNIH GOVORNIKA BOSANSKOG/.. sanskim/hrvatskim/srpskim jezikom), u ovom istraživanju će se analizirati greške koje se pojavljuju kod ispitanika, izvornih govornika bosanskog/hrvatskog/srp- skog jezika, koji na Fakultetu humanističkih nauka Univerziteta „Džemal Bije- dić” u Mostaru uče njemački kao strani jezik. Autorica se koristila u radu kontrastivnim pristupom koji se sastoji u iden- tificiranju grešaka koje nastaju kao rezultat interferencije između bosanskog/hr- vatskog/srpskog kao maternjeg jezika i njemačkog kao stranog jezika. Ovakvim pristupom se htjela skrenuti pažnja na negativan prenos, kao i na činjenicu da bi se zbog navedenog trebalo u nastavi više insistirati na uvježbavanju određenog područja u njemačkom jeziku, pri čemu bi se izbjegle greške i odstupanja koja nastaju u jezičkoj produkciji koju studenti čine. Zbog razlika u gramatičkim i leksičkim sistemima dva jezika, maternji jezik može imati negativan utjecaj na napredak u stranom jeziku. Ta se pojava naziva interferencija maternjeg jezika ili negativan jezički transfer. Uočava se analizom grešaka koje se pojavljuju u govoru ili prilikom pisanja kod onih koji uče strani jezik (Petrović 1997: 114). Nakon učenja dolazi do supostojanja uporednih jezičkih kodova, čime na- staje tzv. međujezičko polje, polje učenja jezika (Pavličević-Fanić 2006: 1). Me- đujezik koji proizvodi učenik drugog ili stranog jezika je sistem koji ima pravila i odlikuje se odstupanjima kao normalnom pojavom u procesu učenja i ovladava- nja drugim jezikom. Odstupanja se definiraju kao otklon od norme jezika koji se uči. Mogu biti prenosna i razvojna. Razvojna se javljaju kao dio procesa učenja i nisu rezultat transfera iz prvog jezika, a prenosna nastaju transferom i utjecajem prvog jezika na drugi (Gulešić-Machata, Udier 2008:20). Pojavljuju se na svim jezičkim razinama: fonetskoj, fonološkoj, morfološkoj, sintaktičkoj, semantičkoj, pragmatičkoj i kulturološkoj (Jelaska 2005: 101). Zastupljenost prenosnih odstu- panja zavisi o stepenu znanja onog koji uči. Na početnom nivou ih ima mnogo, dok se kasnije ta odstupanja smanjuju, a razvojna odstupanja su u porastu (Macan i Kolaković 2008: 35). 1.3. Vrste prenosnih odstupanja iz b/h/s kao J1 u njemački kao J2 Ponekad je teško odrediti o kojoj vrsti odstupanja se radi jer se pojedini pri- mjeri mogu tumačiti na više načina, ili pak uključuju više od jedne razine. U ovom radu je napravljena podjela na tri osnovne vrste: pravopisna odstupanja (uključu- jući i fonološka), gramatička (obuhvataju morfološka i sintaktička) i leksička, u kojima su razdvojena tvorbena i semantička. МЕТОДИЧКИ ВИДИЦИ vima su se analizirala najučestalija prenosna odstupanja iz b/h/s kao J1 u njemački kao J2. Građa je podijeljena na dva dijela. U prvom dijelu su analizirani radovi studenata koji polažu Njemački jezik I i II (prva godina učenja, 120 sati ukupno) i onih koji polažu Njemački jezik III (druga godina učenja, 60 sati ukupno). Bilo je 70 radova sa prve i 70 radova sa druge godine. 1.2. Pretpostavke Na osnovu prijašnjih istraživanja koja su se bavila problematikom ovog rada prilikom učenja drugih jezika može se pretpostaviti da će tip kao i zastuplje- nost određenog odstupanja tokom učenja njemačkog jezika zavisiti od stepena znanja studenata. Očekivala su se odstupanja na svim jezičkim razinama, i kod studenata koji su učili Njemački jezik I i II, i kod onih koji su pohađali nastavu iz Njemačkog jezika III. Međutim, pretpostavljalo se da će se navedena odstupanja razlikovati po karakteristikama i zastupljenosti. Želi se, također, pokazati i ukazati na najčešća prenosna odstupanja kod studenata b/h/s J1 te kako se ona mijenjaju ili ne mijenjaju sa povećanjem znanja njemačkog jezika. Poređenje međujezika i dominantnog jezičkog sistema vrlo je važno za ra- zumijevanje i otklanjanje odstupanja pri učenju njemačkog J2. Rezultati ovog rada moći će doprinijeti boljoj i kvalitetnijoj nastavi njemač- kog kao stranog jezika. Nastavnik će biti upućen koja odstupanja može očekivati te će na taj način moći uložiti više vremena i truda kako bi na vrijeme spriječio ukorijenjivanje određenih odstupanja. 1.1. Građa i ispitanici U ovom radu su kao izvor za analizu korišteni testovi, diktati i završni pi- smeni radovi studenata Odsjeka za engleski jezik i književnost, Odsjeka za bo- sanski/hrvatski/srpski jezik i književnost, Odsjeka za historiju/istoriju/povijest i Odsjeka za komunikologiju Fakulteta humanističkih nauka Univerziteta „Džemal Bijedić” u Mostaru koji u sastavu svog plana i programa studiranja imaju nje- mački jezik kao izborni predmet. Prikupljeni su u akademskoj 2012/2013. godini. Ispitanicima je glavni jezik, tj. J1, bio bosanski/hrvatski/srpski. Na njihovim rado- 232 Методички Видици 4 МЕТОДИЧКИ ВИДИЦИ 2.1. Pravopisna odstupanja kod studenata prve godine učenja U radovima studenata koji su učili njemački jezik jednu akademsku godinu česta su pravopisna (ortografska) odstupanja, tj. prenošenje pravopisnih obilježja iz b/h/s J1 u njemački J2. Pisanje imenica malim početnim slovom – U njemačkom jeziku se imenice pišu velikim početnim slovom, što nije slučaj u J1 pa nastaju vrlo često odstupanja kao u (1). p p , j j p j p j kao u (1). (1) i. er ist 38 jahre alt (umj. Jahre) ii. und liest eine zeitung (umj. Zeitung) iii. finden Sie lampen (umj. Lampen) Zamjena grafema – kao u (2). (2) i. und spricht Francözisch (umj. Französisch) (z se u njemačkom jeziku izgovara kao c, a s na početku riječi i sloga kao z, što nije slučaj u J1) ii. Geschlecht: veiblich (umj. weiblich) (w se u njemačkom jeziku izgovara kao v, što nije slučaj u J1. Abeceda J1 ne sadrži grafem w) iii. aber auch fiele günstige Angebote (umj. viele) (v se u njemačkom jeziku u većini slučajeva izgovara kao f, osim kod nekih riječi stranog porijekla Violine, Vokativ itd). iv. und begrist die Nachbarin (umj. begrüßt) (J2 poznaje preglašene samoglasnike ä, ö, i ü, te slovo ß, što nije slučaj u J1. Govornici J1 navedene grafeme realiziraju u pisanoj produkciji grafemima e, i i s.) Izostavljanje grafema – kao u (3). (3) i. und begrüßt di Nachbarin (umj. die) (Diftong ie se u J2 izgovara kao dugo i. Izvorni govornici J1 ne poznaju na- vedeni diftong, te ga produciraju u pismenoj formi onako kako se izgovara, kao i.) ii. abe keine Küche (umj. aber) (r se na kraju sloga u J2 obično ne izgovara, što nije slučaj u J1. Ono se če- sto „vokalizira” u J2, posebno iza dugog vokala/samoglasnika i kod nenaglašenih nastavaka. Takvo r se realizira kao [ɐ].) iii. im Kaufhaus gibt es fast ales (umj. alles) (Kratki izgovor samoglasnika pojavljuje se kod udvojenog suglasnika koji slijedi iza samoglasnika u J2, dok se u bosanskom/hrvatskom/srpskom jeziku ud- vojeni suglasnici samo pišu u superlativu pridjeva na j, npr. najjači, i u nekim složenicama radi lakšeg razumijevanja, npr. vannastavni.) ( ) (1) i. er ist 38 jahre alt (umj. Jahre) ii. und liest eine zeitung (umj. Zeitung) iii. finden Sie lampen (umj. Lampen) Zamjena grafema – kao u (2). (2) i. und spricht Francözisch (umj. Französisch) (z se u njemačkom jeziku izgovara kao c, a s na početku riječi i sloga kao (umj. 2.1. Pravopisna odstupanja kod studenata prve godine učenja Jahre) (umj. Zeitung) (umj. Lampen) ii. und liest eine zeitung iii. finden Sie lampen Zamjena grafema – kao u (2). (2) i. und spricht Francözisch (z se u njemačkom jeziku izgovara kao c, a s na početku riječi i slo z, što nije slučaj u J1) ii. Geschlecht: veiblich (umj. weiblich) (w se u njemačkom jeziku izgovara kao v, što nije slučaj u J1. Abeceda J1 z, što nije slučaj u J1) z, što nije slučaj u J1) ii. Geschlecht: veiblich (w se u njemačkom jezik ii. Geschlecht: veiblich ne sadrži grafem w) iii. aber auch fiele günstige Angebote (umj. viele) (v se u njemačkom jeziku u većini slučajeva izgovara kao f, osim kod nekih ne sadrži grafem w) iii. aber auch fiele günstige Angebote (umj. viele) (v se u njemačkom jeziku u većini slučajeva izgovara kao f, osim kod nekih riječi stranog porijekla Violine Vokativ itd) v se u njemačkom jeziku u većini slučajeva izgovara kao f, osim kod nekih (v se u njemačkom jeziku u većini slučajeva izgovara kao f, osim kod nekih riječi stranog porijekla Violine, Vokativ itd). iv. und begrist die Nachbarin (umj. begrüßt) (J2 poznaje preglašene samoglasnike ä, ö, i ü, te slovo ß, što nije slučaj u J1. Govornici J1 navedene grafeme realiziraju u pisanoj produkciji grafemima e, i i s.) Izostavljanje grafema – kao u (3). (3) i. und begrüßt di Nachbarin (umj. die) (Diftong ie se u J2 izgovara kao dugo i. Izvorni govornici J1 ne poznaju na- vedeni diftong, te ga produciraju u pismenoj formi onako kako se izgovara, kao i.) ii. abe keine Küche (umj. aber) (r se na kraju sloga u J2 obično ne izgovara, što nije slučaj u J1. Ono se če- sto „vokalizira” u J2, posebno iza dugog vokala/samoglasnika i kod nenaglašenih nastavaka. Takvo r se realizira kao [ɐ].) iii. im Kaufhaus gibt es fast ales (umj. alles) (Kratki izgovor samoglasnika pojavljuje se kod udvojenog suglasnika koji slijedi iza samoglasnika u J2, dok se u bosanskom/hrvatskom/srpskom jeziku ud- vojeni suglasnici samo pišu u superlativu pridjeva na j, npr. najjači, i u nekim složenicama radi lakšeg razumijevanja, npr. vannastavni.) riječi stranog porijekla Violine, Vokativ itd). (J2 poznaje preglašene samoglasnike ä, ö, i ü, te slovo ß, što nije slučaj u J1. Govornici J1 navedene grafeme realiziraju u pisanoj produkciji grafemima e, i i s.) Izostavljanje grafema – kao u (3). (3) i. und begrüßt di Nachbarin (umj. 2. PRAVOPISNA ODSTUPANJA Pravopisna odstupanja se odnose samo na pisani jezik. Međutim, rezultati ukazuju na to da pravopisna odstupanja mogu biti povezana sa izgovornim, pri- mjerice pri obezvučenju šumnika, što je jedan od razloga uključivanja i fonološ- kih odstupanja u ovu skupinu. 233 Методички Видици 4 МЕТОДИЧКИ ВИДИЦИ Obezvučavanje ili obezvučenje kao u (4). Ova glasovna promjena (u b/h/s jeziku se koristi termin „obezvučavanje ili obezvučenje suglasnika na kraju sloga/ riječi”, a u njemačkom jeziku „Auslautverhärtung”) je česta kod studenata prve godine učenja, dok izvorni govornici njemačkog jezika koji uče hrvatski jezik (u prvoj godini učenja) zamjenjuju bezvučne šumnika zvučnim (npr. Orlandov stub, umj. stup; imam ped kornjača, umj. pet) (Macan i Kolaković 2008: 38). j p; p j , j p ) ( (4) i. er hat keinen Hert ( ii h t k i St ( j St (umj. Herd) (umj. Staubsauger) (umj. Herd) ii. er hat keinen Staupsauger ii. er hat keinen Staupsauger 2.1. Pravopisna odstupanja kod studenata druge godine učenja I studenti koji njemački jezik uče drugu godinu prenose pravopisne karak teristike iz J1 u J2, mada su kod studenata druge godine ove greške mnogo rjeđe Pisanje imenica malim početnim slovom – U njemačkom jeziku se imenice pišu velikim početnim slovom, što nije slučaj u J1. Ovakvih odstupanja je vrlo malo kod studenata druge godine učenja, ali se još uvijek pojavljuju kao u (5). (5) i. Deutschland ist ein großes land (umj. Land) ii. hast du hunger? (umj. Hunger) (5) i. Deutschland ist ein großes land (umj. Lan ii. hast du hunger? (umj. Hunge ii. hast du hunger? (Pretpostavlja se da su studenti pisali riječ Hunger „glad” malim početnim slovom, razmišljajući na J1. U J1 se ne postavlja pitanje, kao u J2, „imaš li glad?” nego „jesi li gladan”, dakle koristi se pridjev koji se u oba jezika piše malim po- četnim slovom.) (Pretpostavlja se da su studenti pisali riječ Hunger „glad” malim početnim slovom, razmišljajući na J1. U J1 se ne postavlja pitanje, kao u J2, „imaš li glad?” nego „jesi li gladan”, dakle koristi se pridjev koji se u oba jezika piše malim po- četnim slovom.) Izostavljanje grafema – kao u (6). Ovakva odstupanja su vrlo rijetka kod studenata druge godine učenja. Uglavnom je to slučaj kod udvojenog suglasnika koji slijedi iza samoglasnika. (6) i. vor einem Jahr konte er noch kein Wort Deutsch (umj. konnt (6) i. vor einem Jahr konte er noch kein Wort Deutsch (umj. konnte) ii. warum hatest du keine Zeit (umj. hattest) ii. warum hatest du keine Zeit Obezvučavanje ili obezvučenje – kao u (7). Ovakvih odstupanja je kod studenata druge godine učenja manje. (7) i. wir sint im Kino (7) i. wir sint im Kino ii. obwohl du wenig Gelt hast (7) i. wir sint im Kino (umj. sind) ii. obwohl du wenig Gelt hast (umj. Geld) ii. obwohl du wenig Gelt hast Upotreba zareza – Odstupanje koje se tek pojavljuje na ovom stepenu pro- izlazi iz većeg obima poznavanja jezika. Studenti druge godine učenja već koriste složene rečenice koje prije nisu poznavali. Tako se pojavljuje prenos pravila iz J1 o nepisanju zareza ispred svake zavisne rečenice kao u (8), iako se u njemačkom jeziku u takvim primjerima stavlja zarez. Obrnuto odstupanje se pojavljuje kod izvornih govornika njemačkog jezika koji uče hrvatski jezik (npr. Ona zna, da me uvijek može nazvati; Definicija prijateljstva je teška, jer je to apstraktan pojam. 2.1. Pravopisna odstupanja kod studenata prve godine učenja die) (Diftong ie se u J2 izgovara kao dugo i. Izvorni govornici J1 ne poznaju na- vedeni diftong, te ga produciraju u pismenoj formi onako kako se izgovara, kao i.) ii. abe keine Küche (umj. aber) (r se na kraju sloga u J2 obično ne izgovara, što nije slučaj u J1. Ono se če- sto „vokalizira” u J2, posebno iza dugog vokala/samoglasnika i kod nenaglašenih nastavaka. Takvo r se realizira kao [ɐ].) iii. im Kaufhaus gibt es fast ales (umj. alles) (Kratki izgovor samoglasnika pojavljuje se kod udvojenog suglasnika koji slijedi iza samoglasnika u J2, dok se u bosanskom/hrvatskom/srpskom jeziku ud- vojeni suglasnici samo pišu u superlativu pridjeva na j, npr. najjači, i u nekim složenicama radi lakšeg razumijevanja npr vannastavni ) (Kratki izgovor samoglasnika pojavljuje se kod udvojenog suglasnika koji slijedi iza samoglasnika u J2, dok se u bosanskom/hrvatskom/srpskom jeziku ud- vojeni suglasnici samo pišu u superlativu pridjeva na j, npr. najjači, i u nekim složenicama radi lakšeg razumijevanja, npr. vannastavni.) 234 Методички Видици 4 МЕТОДИЧКИ ВИДИЦИ 3. GRAMATIČKA ODSTUPANJA Gramatička odstupanja su rezultat prenošenja gramatičkih obilježja iz J1 u J2. Tu se misli na: red riječi u rečenici (prostoj i složenoj), upotrebu određenog i neodređenog člana, upotrebu roda, konjugaciju, padež, upotrebu prijedloga, ispu- štanje ličnih zamjenica, negaciju sa nicht i kein/keine/kein itd. (8) i. Andrea hat wenig Zeit weil sie arbeiten muss. (umj. Andrea hat wenig Zeit, weil sie arbeiten muss) (8) i. Andrea hat wenig Zeit weil sie arbeiten muss. (umj. Andrea hat wenig Zeit, weil sie arbeiten muss) (8) i. Andrea hat wenig Zeit weil sie arbeiten muss. (umj. Andrea hat wenig Zeit, weil sie arbeiten muss) ) ii. Wir waren im Kino obwohl wir zu Hause bleiben wollten. (umj. Wir waren im Kino, obwohl wir zu Hause bleiben wollten.) ii. Wir waren im Kino obwohl wir zu Hause bleiben wollten. (umj. Wir waren im Kino, obwohl wir zu Hause bleiben wollten.) 3.1. Gramatička odstupanja kod studenata prve godine učenja Lični glagolski oblik u izjavnoj rečenici – U njemačkom jeziku vrijedi pravilo da se lični glagolski oblik u izjavnoj rečenici uvijek pojavljuje na drugom mjestu, što nije slučaj u J1. Odstupanja od navedenog pravila pronalazimo u (9). j j j p j (9) i. Martina heute geht ins Möbelhaus. ins Möbelhaus.) (9) i. Martina heute geht ins Möbelhaus. (umj. Martina geht heute ins Möbelhaus.) ) ii. Heute sie kauft eine Stereoanlage. 2.1. Pravopisna odstupanja kod studenata druge godine učenja itd.) (Macan i Kolaković 2008: 39). 235 Методички Видици 4 Prndelj Šator: PRENOSNE GREŠKE KOD IZVORNIH GOVORNIKA BOSANSKOG/... iii. Und was nehmst du? iii. Und was nehmst du? Upotreba prijedloga – Prijedlozi su u J1 i u J2 nepromjenljive riječi koje zahtijevaju određene padeže. Međutim, isti prijedlozi u J1 i u J2 se ne slažu uvijek sa istim padežom (kod se slaže u J1 sa genitivom, dok se u J2 slaže sa dativom) kao u (13). (13) i. bei der Würstwaren ii. von ihrer Kinder ii. von ihrer Kinder Negacija – U njemačkom jeziku se za negiranje može koristi kein i nicht (Ich trinke keinen Saft; Ich trinke heute nicht). Postoje pravila kada se koristi jed- na negacija, a kada druga, dok to nije slučaj u b/h/s jeziku. U J1 se upotrebljava negacija ne (Ne pijem sok; Danas ne pijem). Nastaju odstupanja kao u (14). Negacija – U njemačkom jeziku se za negiranje može koristi kein i nicht (Ich trinke keinen Saft; Ich trinke heute nicht). Postoje pravila kada se koristi jed- na negacija, a kada druga, dok to nije slučaj u b/h/s jeziku. U J1 se upotrebljava negacija ne (Ne pijem sok; Danas ne pijem). Nastaju odstupanja kao u (14). (14) i. Sie trinkt nicht Orangensaft. (umj. Sie trinkt keinen Orangensaft.) ii. Martina ist keine verheiratet. (umj. Martina ist nicht verheiratet) iii. Sie hat nicht Kinder. (umj. Sie hat keine Kinder.) (14) i. Sie trinkt nicht Orangensaft. (umj. Sie trinkt keinen Orangensaft.) ii. Martina ist keine verheiratet. (umj. Martina ist nicht verheiratet) iii. Sie hat nicht Kinder. (umj. Sie hat keine Kinder.) (14) i. Sie trinkt nicht Orangensaft. (umj. Sie trinkt keinen Orangensaf (14) i. Sie trinkt nicht Orangensaft. (um (14) i. Sie trinkt nicht Orangensaft. (umj. Sie trinkt keinen Orangensaft.) ii. Martina ist keine verheiratet. (umj. Martina ist nicht verheiratet) iii i h i h i d ( j h k d ) ii. Martina ist keine verheiratet. iii. Sie hat nicht Kinder. (umj. Sie hat keine Kinder iii. Sie hat nicht Kinder. (umj. Heute kauft sie eine Stereo- ii. Heute sie kauft eine Stereoanlage. (umj. Heute kauft sie eine Stereo- anlage.) Upotreba određenog ili neodređenog člana – U J2 postoji kategorija odre- đenog i neodređenog člana (koji se slažu sa imenicom u rodu broju i padežu). U skladu s tim, postoje i striktna pravila kada se i koji član koristi, odnosno kada se član ne upotrebljava. S obzirom na to da je b/h/s jezik bez članova, za izražavanje određenosti i neodređenosti se uglavnom koriste: a) kratki i dugi oblici pridjeva; b) broj jedan i c) pokazne i nedređene zamjenice, tako da se kod studenata koji uče njemački jezik dešavaju odstupanja kao u (10). (10) i. Wien ist Hauptstadt von Österreich. (umj. Wien ist die Hauptstadt von Österreich.) ii Ich suche den Kräutertee (umj Ich suche Kräutertee ) Nastaju odstupanja kao u (11). (11) i. Wien ist der Hauptstadt von Österreich und er hat... (umj. Wien ist d d Ö h d h ) (11) i. Wien ist der Hauptstadt von Österreich und er hat... (umj. Wien ist die Hauptstadt von Österreich und sie hat ) 11) i. Wien ist der Hauptstadt von Österreich und er hat... (umj. Wien ist Ö die Hauptstadt von Österreich und sie hat...) (Imenica „grad” je u njemačkom jeziku ženskog roda, dok je u bosanskom/ p ) (Imenica „grad” je u njemačkom jeziku ženskog roda, dok je u bosanskom/ h k / k j ik šk d ) Imenica „grad” je u njemačkom jeziku ženskog roda, dok je u bosanskom/ om/srpskom jeziku muškog roda.) hrvatskom/srpskom jeziku muškog roda.) ii. Es gibt eine Sessel. (umj. Es gibt einen Sessel.) p j g ) ii. Es gibt eine Sessel. (umj. Es gibt einen Sessel.) (umj. Es gibt einen Sessel.) (umj. Es gibt einen Sessel.) 236 Методички Видици 4 МЕТОДИЧКИ ВИДИЦИ (Imenica „fotelja” je u J2 muškog roda.) iii. ein Packung Waschpulver (umj. eine Packung Waschpulver) (Imenica „pakovanje” u J1 je srednjeg roda, dok su u J2 sve imenice koje i (Imenica „fotelja” je u J2 muškog roda.) iii. ein Packung Waschpulver (umj. eine Packung Waschpulver) (Imenica „pakovanje” u J1 je srednjeg roda, dok su u J2 sve imenice koje i završavaju sufiksom -ung ženskog roda.) Prezent u 2. i 3. licu jednine – U njemačkom jeziku kod određenog broja glagola koji unutar glagolske osnove imaju samoglasnik e, u 2. i 3. (umj. Heute kauft sie eine Stereo- licu jednine dolazi do glasovne promjene e u i ili ie (u njemačkom jeziku e/i-Wechsel) (Tano- vić 2004: 139). To nije slučaj u J1, te dolazi do odstupanja kod glagola koji imaju navedenu glasovnu promjenu u prezentu jednine kao u (12). (12) i. Du sprechst auch Englisch. (umj. Du sprichst auch Englisch.) ii. Er helft Sarah bei den Hausaufgaben. (umj. Er hilft Sarah bei (12) i. Du sprechst auch Englisch. ii. Er helft Sarah bei den Hausaufgaben. den Hausaufgaben.) iii. Und was nehmst du? (umj. Und was nimmst du?) 4. TVORBENA PRENOSNA ODSTUPANJA Treću skupinu odstupanja koja su se pojavila u analiziranim testovima čine leksička odstupanja. Ona su podijeljena na leksičko-tvorbena i leksičko-semantič- ka odstupanja, iako je ponekad teško povući granicu. 3.2. Gramatička odstupanja kod studenata druge godine učenja Anja macht viele Fehler, obwohl sie schon ein Jahr Deutsch lernt.) Rod imenica – Iako se rod imenica u J1 i J2 često ne slažu, kod studenata druge godine učenja njemačkog jezika, rjeđa su ovakva odstupanja kao u (18), nego kod studenata početnika. Rod imenica – Iako se rod imenica u J1 i J2 često ne slažu, kod studenata druge godine učenja njemačkog jezika, rjeđa su ovakva odstupanja kao u (18), nego kod studenata početnika. (umj. Deutschland ist (umj. Deutschland ist (18) i. Deutschland ist eine große Land. (umj. Deutschland ist ein großes Land.) (18) i. Deutschland ist eine große Land. Zamjena veznika – Veznik kada u J1 se na njemački jezik može prevesti njemačkim wenn (za radnju koja se odvija u sadašnjosti ili se ponavljala više puta u prošlosti) i als (za radnju koja se dogodila jednom u prošlosti). U (19) studenti su pogrešno upotrebljavali veznike wenn i wann koji odgovaraju b/h/s vezniku kada. (19) i. Wenn ich aufgestanden bin, habe ich zuerst gegessen. (um 19) i. Wenn ich aufgestanden bin, habe ich zuerst gegessen. (umj. als) ii. Als ich koche, singe ich oft. (umj. wenn) ii. Als ich koche, singe ich oft. ii. Als ich koche, singe ich oft. 3.2. Gramatička odstupanja kod studenata druge godine učenja Red riječi u zavisnoj rečenici – U J2 lični glagolski oblik u zavisnoj rečeni- ci uvijek dolazi na posljednje mjesto, dok to nije slučaj u J1. Nastaju odstupanja kod studenata druge godine učenja koji već koriste složene rečenice, kao što je to slučaj u (15). (15) i. Wir sind im Kino, obwohl wir sollen zu Hause bleiben. (umj. Wir sind im Kino, obwohl wir zu Hause bleiben sollen.) (15) i. Wir sind im Kino, obwohl wir sollen zu Hause bleiben. (umj. Wir sind im Kino, obwohl wir zu Hause bleiben sollen.) ii. Du lernst schnell Deutsch, weil du lebst und arbeitest in Deutschland. (umj. Du lernst schnell Deutsch, weil du in Deutschland lebst und arbeitest.) Perfekt – U J1 perfekt se tvori od enklitičkih prezentskih oblika glagola jesam, dok se u njemačkom kao pomoćni glagol koriste glagoli biti (sein) i imati 237 Методички Видици 4 a S. Prndelj Šator: PRENOSNE GREŠKE KOD IZVORNIH GOVORNIKA BOSANSKOG/ (haben). Kod studenata druge godine učenje prisutna su odstupanja koja su rjeđa od onih u (15), kod upotrebe pomoćnog glagla haben kao u (16). ( ) p p g g g ( ) (16) i. Ich bin am Strand in der Sonne gelegen. (umj. Ich habe am Strand in der Sonne gelegen.) ii. Sie ist viele Sehenswürdigkeiten besichtigt. (umj. Sie hat viele Sehen- swürdigkeiten besichtigt.) Ispuštanje ličnih zamjenica – U J2 se samo u rijetkim slučajevima ispušta izricanje subjekta jer u tom slučaju rečenica nije gramatički ispravna. Međutim, u J1 b lič ih j i ij ž N j d j k (17) izricanje subjekta jer u tom slučaju rečenica nije gramatički ispravna. Međutim, u J1 upotreba ličnih zamjenica nije nužna. Nastaju odstupanja kao u (17). J1 upotreba ličnih zamjenica nije nužna. Nastaju odstupanja kao u (17). (17) i. Tom möchte ein neues Auto kaufen, obwohl wenig Geld hat. (umj. Tom möchte ein neues Auto kaufen, obwohl er wenig Geld hat.) (17) i. Tom möchte ein neues Auto kaufen, obwohl wenig Geld hat. (umj. Tom möchte ein neues Auto kaufen, obwohl er wenig Geld hat.) ii. Anja macht viele Fehler, obwohl schon ein Jahr Deutsch lernt. (umj. Anja macht viele Fehler, obwohl sie schon ein Jahr Deutsch lernt.) ii. Anja macht viele Fehler, obwohl schon ein Jahr Deutsch lernt. (umj. 5. LEKSIČKO-SEMANTIČKA ODSTUPANJA U prikupljenom korpusu zastupljen je veliki broj pojedinačnih primjera prenosnih leksičko-semantičkih odstupanja. Česta je pojava zamjena leksičkog elementa J2 elementom J1. Prenos leksičkih elemenata različitog značenja u J1 i J2 može se očitovati kao upotreba doslovno prevedenih riječi koje se u njemač- kom jeziku ne koriste. Uzrok je višeznačnost riječi u J1 kojim u J2 odgovaraju različite riječi, ali i homonimija. 4.1. Tvorbena odstupanja studenata prve godine učenja Za razliku od bosanskog, hrvatskog i srpskog jezika njemački jezik je sklon slaganju kao vrsti tvorbe. Nesklonost J1 za tvorbom složenica se očituje u (20), gdje je naveden primjer leksičko-tvorbenog odstupanja studenata prve godine učenja. 238 Методички Видици 4 МЕТОДИЧКИ ВИДИЦИ (20) i. Salami ist kein Milch Produkt (umj. Milchprodukt) ii. Salama nije mliječni proizvod. iii. Elektronik Abteilung (umj. Elektronikabteilung) iv. odjel za elektroniku (umj. Milchprodukt) (20) i. Salami ist kein Milch Produkt (umj. Milchprodukt) ii. Salama nije mliječni proizvod. iii. Elektronik Abteilung (umj. Elektronikabteilung) iv. odjel za elektroniku 4.2. Tvorbena odstupanja studenata druge godine učenja Studenti druge godine učenja, također, nisu skloni tvorbi složenica kao u (21). ) (21) i. Staat in Mittel Europa (umj. Mitteleuropa) ii. Srednja Evropa iii. Kulture Zentrum (umj. Kulturzentrum) iv. centar kulture (umj. Mitteleuropa) (21) i. Staat in Mittel Europa ii. Srednja Evropa iii. Kulture Zentrum iv. centar kulture (21) i. Staat in Mittel Europa ii. Srednja Evropa iii. Kulture Zentrum iv. centar kulture (umj. Kulturzentrum) 5.1. Leksičko-semantička odstupanja studenata prve godine učenja Odabir neprikladnog značenja polisemične riječi – Ukoliko se prenosi ne- prikladno značenje, najčešće se radi o polisemičnim riječima. U (22) b/h/s glagolu voljeti odgovara njemački glagol mögen, a ne lieben. (umj. mag) (umj. mag) (22) i. Ich liebe Rockmusik. (22) i. Ich liebe Rockmusik. ii.Volim rok muziku. 5.2. Leksičko-semantička odstupanja studenata druge godine učenja U radovima studenata druge godine učenja često se pojavljuje odabir riječi neprikladnog značenja kao u (26) i (27). Kod (26) lekseme Kaffee i Cafe ekvi- valentne su na ortoepskom nivou. Radi se o homonimima (Kafee znači „kafa”, a Cafe „kafić”). (umj. Cafe) i ) (26) i. Ich bin im Kaffee mit meinen Freunden. ‚ (umj. Cafi (26) i. Ich bin im Kaffee mit meinen Freunden. ‚ (umj. Cafe) ii. Ja sam u kafiću sa svojim prijateljima. ii. Ja sam u kafiću sa svojim prijateljima. (27) i.Im Norden von Deutschland hat Meer. (umj. gibt es (27) i.Im Norden von Deutschland hat Meer. (27) i.Im Norden von Deutschland hat Meer. (umj. gibt es) ii. Na sjeveru Njemačke ima more. (27) i.Im Norden von Deutschland hat Me ii. Na sjeveru Njemačke ima more. ii. Na sjeveru Njemačke ima more. a S. Prndelj Šator: PRENOSNE GREŠKE KOD IZVORNIH GOVORNIKA BOSANSKOG/ Odabir nepostojećeg značenja – Za iskazivanje starosne dobi u bosanskom/ hrvatskom/srpskom jeziku se koristi glagol imati. Međutim, ukoliko se doslovno prevede na njemački jezik, dobijamo nepostojeće značenje kao u (24). ii. Imam 24 godine. Zanimljivo je da studentice prve godine učenja, kada navode svoj spol, ko- riste imenice Frau („žena, gospođa”) i Dame („dama”) kao u (25), a ne odgova- rajući pridjev weiblich („ženski”), izveden od zastarjele imenice Weib („žensko, žena”). (25) i. Geschlecht: Frau/Dame (25) i. Geschlecht: Frau/Dame ii.Volim rok muziku. Neprikladno značenje je odabrano i kod pridjeva visok koji se trebao u (23) prevesti njemačkim pridjevom groß, te kod glagola ići koji se trebao prevesti nje- mačkim glagolom fahren. (umj. groß) (umj. groß) (23) i. Er ist hübsch und hoch. ii. On je lijep i visok. (umj.fahren) iii. Wir gehen im Sommer in die Türkei. iv. Idemo na ljeto u Tursku. 239 Методички Видици 4 (24) i. Ich habe 24 Jahre. ii. Imam 24 godine. МЕТОДИЧКИ ВИДИЦИ Rad nastavnika i lektora trebalo bi da se usredotoči na odstupanja koja se pojavljuju kod studenata, posebno se treba posvetiti onim greškama koje se pre- nose sa nižih na više razine učenja. LITERATURA Buscha, J., & Helbig, G. (1996). Deutsche Grammatik. Leipzig: Langenscheidt. Buscha, J., & Helbig, G. (1996). Deutsche Grammatik. Leipzig: Langenscheidt. Dudenredaktion. (1996). Deutsches Universalwörterbuch. Mannheim – Leipzig – Wien – Zürich: Dudenverlag. enredaktion. (1996). Deutsches Universalwörterbuch. Mannheim – Leipzig Gulešić-Machata, M., & Udier, S. (2008). Izvorna odstupanja u hrvatskome kao inojezičnome. Lahor, 1(5), 19–33. Jelaska, Z., et al. (2005). Hrvatski kao drugi i strani jezik. Zagreb: Hrvatska sveučilišna naklada. Macan, Ž., & Kolaković Z. (2008). Prijenosna odstupanja govornika njemačkoga u ovladavanju hrvatskim jezikom. Lahor, 5, 34–52. Pavličević-Franić, D. (2006). Jezičnost i međujezičnost između sustava, podsustava i komunikacije. Lahor, 1, 1–14. ović, E. (1997). Teorija nastave stranih jezika. Osijek: Pedagoški fakultet. Tanović, M. (2004). Morfologija njemačkog jezika. Mostar: Fakultet humanističkih nauka. Belma S. Prndelj-Šator 6. ZAKLJUČAK Rezultati do kojih se došlo u ovom radu ukazuju da se u procesu učenja nje- mačkog kao stranog jezika pojavljuje veći broj prenosnih grešaka koje su nastale kao posljedica prenošenja jezičkih elemenata iz dominantnog sistema J1 u J2. Potvrdila se pretpostavka da će na drugoj godini učenja neke greške nestati, ali da će se pojaviti neke nove. Među studentima prve godine česta su pravopisna odstupanja koja iščezavaju na drugoj godini (pisanje imenica malim početnim slovom). Međutim, zamjena bezvučnih i zvučnih šumnika je odstupanje koje se pojavljuje i na drugoj godini te se shodno tome treba uložiti više napora kako bi se to korigiralo. U radovima studenata druge godine bilo je manje pravopisnih grešaka, ali se pojavilo više gramatičkih i leksičkih grešaka. To se može povezati sa pove- ćanjem nastavnog gradiva i vokabulara. Na obe godine se može primijetiti kako studenti imaju poteškoće sa tvorbom složenica, čemu je sklon njemački jezik. 240 Методички Видици 4 МЕТОДИЧКИ ВИДИЦИ Summary While learning German as a second or foreign language errors are bound to occur. Some of them are result of the transfer from a dominant language system (usually the mother tongue L1). This paper analyses final papers in written language of students who are learning German as a foreign language at the University „Džemal Bijedić“ in Mo- star (Faculty of Humanities) and whose mother tongue is either Bosnian, or Croatian, or Serbian. Therefore, errors that occur as a result of an overlap between Bosnian/Croatian/ Serbian as a first and German as a second language will be compared.ii The data consisted of 70 final papers from first year students who had approximate- ly 120 hours of German, and 70 final papers from students who had altogether approxi- mately 180 hours of German (approximately 60 hours in the second year). 241 Методички Видици 4 Prndelj Šator: PRENOSNE GREŠKE KOD IZVORNIH GOVORNIKA BOSANSKOG/... Depending on which elements are transferred, a phonological and orthographic level, a grammatical level, a lexical-semantic level of transfer could be differentiated. As the data only consisted of written language, no phonetic errors could be recorded. Developmental errors were not analysed, but as it is not always easy to distinguish them from other types of errors, and since some examples included them, they were just briefly mentioned. The hypothesis tested was that some transfer errors will disappear at higher levels of German knowledge, some of them will appear less frequently, while new ones will appear with more complex language knowledge. The analysis was qualitative, not quan- titative: the investigation included types of errors that were classified with examples, and not the exact numbers of errors that were found.i The hypothesis was confirmed. While the beginners’ level of knowledge was char- acterized by various orthographic errors, second year students stopped transferring most of them. Both levels had equal variety of error types on the grammatical level. The higher level had more lexical errors then the lower level, due to a larger vocabulary and more complex semantic demands. Although comparative analysis showed that some errors caused by linguistic trans- fer that occurred while learning German as an L2 were persistant, those were, like in the case of a substitution of letters representing voiced sounds for voiceless (d/t, p/b), due to the Bosnian/Croatian/Serbian orthographic system. Key words: transfer errors, Bosnian/Croatian/Serbian as L1, German as L2, errors caused by linguistic transfer, level of transfer, dominant language system. 242 Методички Видици 4
https://openalex.org/W2744540225	https://www.nature.com/articles/s41598-017-08426-8.pdf	English	null	Circulating CD40 and sCD40L Predict Changes in Renal Function in Subjects with Chronic Kidney Disease	Scientific reports	2,017	cc-by	6,485	Circulating CD40 and sCD40L Predict Changes in Renal Function in Subjects with Chronic Kidney Disease Received: 13 March 2017 Accepted: 10 July 2017 Published: xx xx xxxx Received: 13 March 2017 Accepted: 10 July 2017 Published: xx xx xxxx Jeffrey X. Xie1, Helen Alderson2, James Ritchie2, Philip A. Kalra2, Yanmei Xie1, Kaili Ren1, Hanh Nguyen1, Tian Chen1, Pamela Brewster1, Rajesh Gupta1, Lance D. Dworkin1, Deepak Malhotra1, Christopher J. Cooper1, Jiang Tian1 & Steven T. Haller1 Soluble CD40 ligand (sCD40L) has been implicated in the development of renal injury. The CD40 receptor exists in a soluble form, sCD40R, and has been shown to function as a competitive antagonist against CD40 activation. We analyzed whether plasma levels of sCD40L and sCD40R predict changes in renal function in an all-cause chronic kidney disease (CKD) cohort. Stratification of subjects based on sCD40L and sCD40R individually, as well as in combination, demonstrated that sCD40L was directly associated with declines in estimated glomerular filtration rate (eGFR). sCD40R was negatively associated with declines in eGFR. Baseline characteristics following stratification, including systolic blood pressure, history of diabetes mellitus or peripheral vascular disease, primary renal disease classification, and angiotensin converting enzyme inhibitor or angiotensin receptor blocker usage were not significantly different. High sCD40L and low sCD40R were both found to be independent predictors of a decline in eGFR at 1-year follow-up (−7.57%, p = 0.014; −6.39%, p = 0.044). Our data suggest that circulating levels of sCD40L and sCD40R are associated with changes in renal function in patients with CKD. The CD40 decoy receptor, sCD40R, may serve as a potential therapeutic target to attenuate renal function decline. Chronic Kidney Disease (CKD) is defined as a glomerular filtration rate (GFR) below 60 mL/min per 1.73 m2 for more than three months1. A recent study estimated the prevalence of CKD in the United States at over 13%2. Several molecular pathways have been investigated for their involvement in the development of CKD, but the exact mechanisms that drive CKD progression remain unknown. CD40 was originally discovered to mediate humoral immune interactions3. However, it is now established that CD40 is expressed by a multitude of cell types including renal proximal tubule epithelial cells and is known to mediate pro-inflammatory and pro-fibrotic pathways4–7. The soluble ligand of the CD40 receptor, sCD40L, is largely derived from activated platelets8, 9. The CD40 receptor can also exist in a soluble form, sCD40R, and has been shown to function as a competitive antagonist, or decoy receptor, to CD4010–13. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports 1University of Toledo College of Medicine and Life Sciences, Toledo, OH, United States. 2Salford Royal Hospital, Salford, United Kingdom. Correspondence and requests for materials should be addressed to S.T.H. (email: Steven. haller@utoledo.edu) SCienTifiC RePorTs \| 7: 7942 \| DOI:10.1038/s41598-017-08426-8 Subjects and Methods i Continuous variables are presented as either mean ± standard deviation or medians with interquartile range (IQR), while categorical variables are presented as frequency and percent for each level. Pearson correlation coef- ficient was used to evaluate the relationship between baseline sCD40L and sCD40R. Comparisons of grouped continuous data were performed using ANOVA with post hoc pairwise comparison using Tukey-Kramer multi- ple comparisons test, and two-sample t-tests. For categorical variables, the Fisher exact test was used due to low frequency of counts for some factors. q y Subjects were dichotomized based on plasma levels of either sCD40L or sCD40R into high (greater than median) or low (less than or equal to median) groups, and were used to compare average percent change in eGFR from baseline to 1-year follow-up. Subjects were stratified by the combination of both median sCD40L and sCD40R, in order to evaluate the combined effect of sCD40L and sCD40R on CKD progression. Further stratifica- tion, such as the use of quartiles, was deemed a less favorable approach due to our sample size and the complexity of the model with a total of 16 combinations when combined sCD40L and sCD40R was evaluated. Multivariable linear regression adjusted for age, sex, baseline eGFR, systolic blood pressure (SBP), peripheral vascular disease (PVD), primary renal disease classification, and use of ACEi/ARB or aspirin medication at baseline was employed to examine the effect of sCD40L and sCD40R as dichotomous predictors of the percent change in renal function from baseline to 1-year follow-up. Backward stepwise methodology was used to select factors for the model. All multivariable models were tested for interaction between predictors and were found to be non-significant. Odds ratios and corresponding 95% confidence intervals for each predictor in the multivariable linear regression mod- els on continuous outcomes were calculated following the method proposed by Moser et al.25. g p p y Time-to-event outcomes, including the composite and secondary study endpoints, were examined using Kaplan-Meier estimates with the log-rank statistic used to compare the high and low medians for both the sCD40L and sCD40R groups. Hazard risk ratios with 95% confidence intervals were examined using the Cox proportional-hazards model for both groups adjusted for age, sex, body mass index (BMI), and baseline creati- nine and urine protein. Subjects and Methods j Patient Population. 1750 patients were recruited to the Chronic Renal Insufficiency Standards and Implementation Study (CRISIS) prior to January 31st, 2010, with a median follow up time of 4.5 years (IQR 2.9– 6.9). Baseline plasma samples were obtained from 250 randomly selected patients from this group at the time of their enrollment into CRISIS. CRISIS is a prospective observational study of outcomes in an all-cause CKD popu- lation from Salford, Greater Manchester, UK. This study was carried out in accordance with all relevant guidelines and regulations as approved by the National Health Service Research and Ethics Committee and the University of Toledo Institutional Review Board. All participants provided written informed consent. Patients referred to the nephrology clinic aged 18 years and older with a GFR <60 mL/min/1.7 m2 and without immediate need for dialysis were considered for the study. Glomerular filtration rate (eGFR) was estimated using the Modification of Diet in Renal Disease Study (MDRD) equation and the serum creatinine Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. MDRD eGFR is reported unless stated otherwise. Full methodology for CRISIS has been previously detailed22–24. Measurement of sCD40L and sCD40. Baseline plasma levels of sCD40L and sCD40R were measured by enzyme-linked immunosorbent assay (Abcam Inc, Cambridge, MA [CD40]; and R&D Systems, Minneapolis, MN [sCD40L]) at the University of Toledo according to the manufacturer recommendations. Full methodology has been described previously20. Study Aims. The primary aim of our study was to analyze whether plasma levels of sCD40L and sCD40R were associated with longitudinal changes in renal function over 4 years of follow-up. As a secondary analysis, we determined the effects of plasma levels of sCD40L and sCD40R on the composite endpoint, defined as the first occurrence of renal replacement therapy (RRT), cardiovascular or renal death, or major cardiovascular adverse events (MACE) consisting of either myocardial infarction, congestive heart failure or stroke. Full methodolo- gies for the validation of renal function measurements and definition of composite endpoint events have been described previously22–24. Statistical Analysis. Assuming a minimum detectable correlation coefficient of r = 0.17 between high lev- els of sCD40L and low levels of sCD40R with decline in percent change in eGFR from baseline, the sample size needed was 250 to achieve 80% power at one-sided 5% significance level allowing for 15% attrition. Circulating CD40 and sCD40L Predict Changes in Renal Function in Subjects with Chronic Kidney Disease g y CD40 signaling events have been linked to atherosclerosis and thrombosis, processes which are thought to develop in part due to persistent inflammation14–18. Interestingly, we have shown that subjects with atheroscle- rotic renal artery stenosis have significantly elevated levels of circulating sCD40L19. Moreover, we have shown that higher levels of baseline circulating sCD40R were associated with a higher 1-year follow-up estimated GFR (eGFR), presumably due to the ability of sCD40R to antagonize CD40-mediated signaling20. As atherosclerotic renal artery stenosis is a known cause of CKD21, the current study builds upon our previous findings by broaden- ing the scope of our study from renal artery stenosis to an all-cause CKD population.h The primary aim of the present study is to investigate whether circulating levels of sCD40L and sCD40R are associated with changes in renal function in subjects with CKD. As a secondary aim, we analyzed the effects of plasma levels of sCD40L and sCD40R on renal and cardiac mortality, rate of renal replacement therapy (RRT), myocardial infarction, stroke, and heart failure incidence. 1University of Toledo College of Medicine and Life Sciences, Toledo, OH, United States. 2Salford Royal Hospital, Salford, United Kingdom. Correspondence and requests for materials should be addressed to S.T.H. (email: Steven. haller@utoledo.edu) SCienTifiC RePorTs \| 7: 7942 \| DOI:10.1038/s41598-017-08426-8 1 www.nature.com/scientificreports/ Subjects and Methods Event rates and associated odds ratios were compared between the high and low groups for both sCD40L and sCD40R.hf The longitudinal effect of dichotomized sCD40L and sCD40R on percent change in renal function from base- line to annual follow-up out to 4 years was assessed using generalized estimating equation (GEE) analysis. The GEE model was employed to address the dependency among repeated measurements of renal function from the same subject. We opted to use GEE analysis, rather than a parametric alternative such as the generalized linear mixed-effects models (GLMM), because GEE estimates are robust for a wider class of data distributions26. The model was adjusted for the following factors: age, sex, SBP, baseline GFR, history of PVD and diabetes mellitus, primary renal disease classification, and use of angiotensin converting enzyme inhibitor (ACEi), angiotensin receptor blocker (ARB) or aspirin. Because of missing observations due to censoring by drop-outs, missed vis- its or death, weighted GEE (WGEE) was applied to adjust the analysis using the inverse probability-weighted method to account for missing observations.ii g All analyses were performed in SAS 9.4 and R 3.0. Statistical significance was defined as a p-value < 0.05. Subjects and Methods SCienTifiC RePorTs \| 7: 7942 \| DOI:10.1038/s41598-017-08426-8 2 www.nature.com/scientificreports/ Characteristics* High sCD40L (>205.0 [142.4, 324.6] pg/mL) (n = 122) Low sCD40L (≤205.0 [142.4, 324.6] pg/mL) (n = 121) P-value High sCD40R (>81.4 [50.9, 306.0] pg/mL) (n = 122) Low sCD40R (≤81.4 [50.9, 306.0] pg/mL) (n = 121) P-value Age (yr) 65.84 ± 14.99 66.05 ± 13.54 0.91 63.80 ± 15.69 68.10 ± 12.34 0.02 Male 74 (61%) 71 (59%) 0.79 76 (62%) 69 (57%) 0.48 Body mass index (kg/m2) 27.38 ± 5.68 28.41 ± 5.11 0.17 27.37 ± 4.57 28.53 ± 6.14 0.13 Smoking (current) 15 (12%) 12 (10%) 0.56 13 (11%) 14 (12%) 0.82 Systolic BP (mmHg) 136.76 ± 20.87 136.87 ± 20.18 0.97 134.8 ± 20.61 138.8 ± 20.25 0.12 Diastolic BP (mmHg) 74.79 ± 12.66 71.74 ± 11.26 0.04 73.93 ± 13.42 72.60 ± 10.53 0.39 MDRD eGFR (ml/min per 1.73 m2) 28.76 ± 14.70 29.30 ± 15.19 0.78 29.30 ± 14.62 30.02 ± 15.63 0.71 CKD-EPI eGFR (ml/min per 1.73 m2) 31.56 ± 17.67 32.02 ± 18.1 0.84 31.34 ± 17.4 32.24 ± 18.4 0.69 Urine protein (mg/dL) 60.78 ± 111.23 54.09 ± 105.96 0.63 61.96 ± 124.5 52.90 ± 89.79 0.52 Creatinine (mg/dL) 2.83 ± 1.68 2.74 ± 1.60 0.68 2.82 ± 1.61 2.75 ± 1.67 0.75 History of Myocardial infarction 18 (15%) 23 (19%) 0.40 23 (19%) 18 (15%) 0.51 History of Angina 23 (19%) 25 (21%) 0.87 25 (20%) 23 (19%) 0.92 History of CVA 12 (10%) 6 (5%) 0.22 6 (5%) 12 (10%) 0.21 History of TIA 11 (9%) 10 (8%) >0.99 7 (6%) 14 (12%) 0.16 History of Diabetes mellitus 37 (30%) 42 (35%) 0.50 33 (27%) 46 (38%) 0.09 History of Peripheral vascular disease 22 (18%) 23 (19%) 0.87 20 (16%) 25 (21%) 0.49 ACEi 43 (35%) 52 (43%) 0.24 51 (42%) 44 (36%) 0.46 ARB 31 (25%) 30 (25%) >0.99 24 (20%) 37 (31%) 0.07 ACEi or ARB 77 (64%) 71 (58%) 0.43 74 (61%) 74 (61%) >0.99 β-Blocker 36 (30%) 36 (30%) >0.99 38 (31%) 34 (28%) 0.70 Diuretic 53 (43%) 59 (49%) 0.44 61 (50%) 51 (42%) 0.27 Statin 68 (56%) 72 (60%) 0.60 63 (52%) 77 (64%) 0.08 Aspirin 55 (45%) 48 (40%) 0.44 37 (30%) 66 (55%) <0.001 Mean sCD40L (pg/mL) 479 ± 644 137 ± 37 <0.001 357 ± 652 261 ± 212 0.12 Mean sCD40R (pg/mL) 662 ± 1940 596 ± 2047 0.80 1200 ± 2695 53 ± 13 <0.001 Mean log sCD40L 5.93 ± 0.56 4.88 ± 0.30 <0.001 5.46 ± 0.76 5.35 ± 0.61 0.21 Mean log sCD40R 5.04 ± 1.41 4.82 ± 1.34 0.22 5.91 ± 1.34 3.94 ± 0.24 <0.001 Primary renal disease DM 22 (18%) 18 (15%) 0.70 16 (13%) 24 (20%) 0.59 APKD 5 (4%) 11 (9%) 0.29 10 (8%) 6 (5%) 0.39 GN/VAS 14 (11%) 18 (15%) 0.74 18 (15%) 14 (12%) 0.50 Pyelonephritis 12 (10%) 4 (3%) 0.11 10 (8%) 6 (5%) 0.39 VAS/HTN 41 (34%) 42 (35%) 39 (32%) 44 (36%) Other 28 (23%) 28 (23%) >0.99 29 (24%) 27 (22%) 0.70 Table 1 Baseline Clinical Characteristics among Participants in the CRISIS Clinical Trial Subjects are stratified Table 1. Subjects and Methods Baseline Clinical Characteristics among Participants in the CRISIS Clinical Trial. Subjects are stratified by median plasma levels of sCD40L or sCD40R. Data are expressed as the mean ± SD or number (percentage). Comparisons were evaluated using two sample t-test for continuous data and Fisher’s exact test for categorical data. Abbreviations: sCD40L, soluble CD40 ligand; sCD40R, soluble CD40 receptor; yr, year; m, meter; kg, kilogram; BP, blood pressure; MDRD, Modification of Diet in Renal Disease; CKD-EPI, Chronic Kidney Disease Epidemiology Collaboration; eGFR, estimated glomerular filtration rate; CVD, cerebral vascular accident; TIA, transient ischemic accident; ACEi, angiotensin converting enzyme inhibitor; ARB, angiotensin receptor blocker; and β-Blocker, beta adrenergic blocker DM, diabetic glomerularnephritis; APKD, adult polycystic kidney disease; GN/VAS, glomerularnephritis vasculitis; VAS/HTN, vascular hypertension. Results Study Population. Baseline levels of the study population are presented in Table 1. For analysis of this pop- ulation, eGFR, sCD40L, and sCD40R were available in 243 out of 250 total subjects. Due to extreme observations in plasma levels of sCD40L and sCD40R, natural logarithmic transformation did not achieve sufficient normality. Receiver operating characteristic (ROC) analyses did not yield a significant predictor cut point with an acceptable (>60%) area under the curve (AUC). Therefore, to reduce the effect of outliers, subjects were stratified into two groups based on their plasma levels of sCD40L: high (greater than median) and low (less than or equal to median) (>205.0 [142.4, 324.6] pg/mL and ≤ 205.0 [142.4, 324.6] pg/mL respectively). We have previously reported that the average plasma concentration of sCD40L in a cohort of 30 normal controls was 65.2 ± 7.4 pg/mL19. The high and low sCD40L groups were well matched at baseline. There were no statistically significant differences in levels SCienTifiC RePorTs \| 7: 7942 \| DOI:10.1038/s41598-017-08426-8 3 www.nature.com/scientificreports/ Figure 1. Percent Change in eGFR in the CRISIS Cohort Dichotomized by Median sCD40L or sCD40R at 1-Year Follow-up. (A) Subjects were dichotomized by median sCD40L (205.0 [142.4, 324.6] pg/mL) or (B) by median sCD40R (81.4 [50.9, 306.0] pg/mL). Figure 1. Percent Change in eGFR in the CRISIS Cohort Dichotomized by Median sCD40L or sCD40R at 1-Year Follow-up. (A) Subjects were dichotomized by median sCD40L (205.0 [142.4, 324.6] pg/mL) or (B) b median sCD40R (81.4 [50.9, 306.0] pg/mL). Figure 2. Comparison of 1-year percent change in eGFR between the combined sCD40L and sCD40R groups. Subjects were stratified into four groups based on median plasma levels of sCD40L and sCD40R. Data is presented as the mean ± 95% confidence interval. Figure 2. Comparison of 1-year percent change in eGFR between the combined sCD40L and sCD40R groups. Subjects were stratified into four groups based on median plasma levels of sCD40L and sCD40R. Data is presented as the mean ± 95% confidence interval. of sCD40R, age, sex, body mass index (BMI), or medication use. Importantly, the primary cause of CKD did not differ between the high and low sCD40L groups. However, a statistically significant difference in diastolic blood pressure (DBP) was detected between these two groups (74.8 ± 12.7 mmHg and 71.7 ± 11.3 mmHg; p = 0.04). Results Median values for sCD40L and sCD40R were not significantly different among the types of primary renal disease (S Table 1).f We also assessed the effects of plasma levels of the CD40 decoy receptor, sCD40R, on kidney function in this population. In order to conduct these analyses, subjects were dichotomized by either high or low sCD40R (>81.4 [50.9, 306.0] pg/mL and ≤ 81.4 [50.9, 306.0] pg/mL respectively). It has been reported that in a cohort of 205 control subjects, the average plasma sCD40R concentration was 41.7 ± 13.2 pg/mL27. The high and low sCD40R groups were well matched at baseline (Table 1), with the exception of age (63.8 ± 15.7 years and 68.1 ± 12.3 years; p = 0.02) and aspirin use (30% and 55%; p < 0.001). CD40 and Renal Function. Ninety-seven percent of the study cohort had moderate to end-stage-renal-disease (stages III 42%, IV 34% or V 21%). The average baseline eGFR was 32 ± 18 ml/min per 1.73 m2 and the average percent decline in eGFR at 1-year follow-up was −3.4% ± 23.5%. Only 10% had a 1-year decline of 30% or more. Subjects with high sCD40L had a greater percent decline in eGFR at 1-year follow-up compared with subjects with low sCD40L (−5.6% ± 0.21% vs 0.47% ± 0.23%, p = 0.04) (Fig. 1a). Since sCD40R is hypothesized to function as a circulating decoy receptor, we assessed the relationship between this biomarker and change in renal function at 1-year follow-up. There was a trend towards subjects with low sCD40R having a greater percent reduction in eGFR at 1-year follow-up compared with subjects with high sCD40R (−5.6% ± 0.19% vs. 0.33% ± 0.25%; p = 0.05) (Fig. 1b). We also stratified subjects based on their plasma levels of both sCD40L and sCD40R (Fig. 2). Subjects with high sCD40L/low sCD40R had significantly greater declines in eGFR at 1-year follow-up compared to subjects with low sCD40L/high sCD40R (−7.5% vs. 5.2%; p = 0.02) (Fig. 2). l bl l d f h h l h b d h Multivariable analysis was used to further assess the relationship between sCD40L, sCD40R, and changes in renal function (Table 2). Following adjustment for age, sex, SBP, baseline eGFR, PVD, diabetes, primary renal disease classification, and baseline usage of aspirin or ACEi/ARB medications, high sCD40L was independently predictive of a decline in eGFR percent change at 1-year follow-up (−7.57%, p = 0.014). Results Using High sCD40L & Low sCD40R as the reference group Abbreviations: MDRD, Modified Diet in Renal Disease study; GFR, estimated glomerular filtration rate; sCD40L, soluble CD40 ligand; sCD40R, soluble CD40 receptor; BP, blood pressure; and CI, confidence interval; and PVD, peripheral vascular disease. Predictors* Effect estimate (% change in eGFR) (95% CI) P-value Low sCD40L & High sCD40R 3.21 (0.59, 5.83) 0.016 Baseline MDRD eGFR 1.01 (0.94, 1.0) <0.001 Year (out from baseline) −1.32 (−1.80, −0.85) <0.001 PVD 1.92 (0.16, 3.67) 0.03 Table 3. Independent Predictors of Longitudinal Changes in eGFR. Only statistically significant predictors of longitudinal changes in eGFR (baseline out to 4 years) are included. The model was adjusted for, age, sex, baseline eGFR, systolic blood pressure (SBP), history of peripheral vascular disease (PVD) and diabetes mellitus, primary renal disease, and baseline ACEi/ARB or aspirin use. Using High sCD40L & Low sCD40R as the reference group Abbreviations: MDRD, Modified Diet in Renal Disease study; GFR, estimated glomerular filtration rate; sCD40L, soluble CD40 ligand; sCD40R, soluble CD40 receptor; BP, blood pressure; and CI, confidence interval; and PVD, peripheral vascular disease. predictive of a decline in eGFR percent change at 1-year follow-up (−6.39%, p = 0.044). The only other variable in our model predictive of statistically significant changes in eGFR was systolic blood pressure, which predicted a 0.20% decrease in eGFR at 1-year per unit increase in SBP (p = 0.02). As the CKD-EPI equation is also a com- monly used indicator of renal function, we tested our model using eGFR calculated with CKD-EPI-creatinine equation, which yielded similar results when compared to MDRD eGFR (S Table 2).h q y p The impact of circulating CD40 mediators on longitudinal changes (baseline and annually out to 4 years) in renal function was also assessed using a WGEE model (Table 3). Following adjustment for age, sex, base- line eGFR, SBP, PVD, diabetes, primary renal disease, and ACEi/ARB or aspirin use, our model, using the high sCD40L/low sCD40R as the comparator group, identified low sCD40L/high sCD40R as a statistically significant independent predictor of attenuation of renal function decline (3.21 ml/min per 1.73 m2 per year; p = 0.016). The other variables in our model that achieved statistical significance were baseline MDRD eGFR, PVD and years (annually out from baseline to 4 years). Results In particular, baseline eGFR is an important significant predictor in our GEE model as it addresses the dependency of longitudinal repeated measurements of renal function from the same subject. Low sCD40L/high sCD40R was predictive of attenuated renal decline in addition to the expected decline in renal function over time. CD40 and Composite Endpoint. The composite endpoint, defined as the first occurrence of renal replace- ment therapy (RRT), cardiovascular or renal death, or major adverse cardiac event (either myocardial infarction, congestive heart failure or stroke) (MACE), as well as the secondary component endpoints were examined using Kaplan-Meier estimates and Cox proportional-hazards model adjusting for age, sex, body mass index (BMI), and baseline creatinine and urine protein. No statistically significant differences in the proportion of endpoint events were observed when subjects were grouped by high or low levels of either sCD40L or sCD40R (S Fig. 1). Subjects with high sCD40L were more likely to have an endpoint event, although this trend was not statistically significant. Results Low sCD40R was also SCienTifiC RePorTs \| 7: 7942 \| DOI:10.1038/s41598-017-08426-8 4 www.nature.com/scientificreports/ Predictors Effect estimate (% change in eGFR) (95% CI) Odds Ratio (95% CI) P-value High sCD40L −7.57 (−13.60, −1.54) 1.89 (1.13, 3.10) 0.014 Low sCD40R −6.39 (−12.59, −0.18) 1.71 (1.01, 2.85) 0.044 Systolic BP −0.20 (−0.36, −0.04) 1.02 (1.00, 1.03) 0.02 Table 2. Independent Predictors of Percent Change in eGFR at 1-Year Follow-up. Model was adjusted by age, sex, baseline eGFR, systolic blood pressure (SBP), peripheral vascular disease (PVD), diabetes mellitus, primary renal disease, and baseline ACEi/ARB or aspirin use. Odds ratios were calculated for a negative percent change in eGFR at 1-year follow-up. Only significant predictors are shown. Abbreviations: eGFR, estimated glomerular filtration rate; sCD40L, soluble CD40 ligand; sCD40R, soluble CD40 receptor; BP, blood pressure; and CI, confidence interval. Table 2. Independent Predictors of Percent Change in eGFR at 1-Year Follow-up. Model was adjusted by b l l bl d ( ) h l l d ( ) d b ll Predictors Effect estimate (% change in eGFR) (95% CI) P-value Low sCD40L & High sCD40R 3.21 (0.59, 5.83) 0.016 Baseline MDRD eGFR 1.01 (0.94, 1.0) <0.001 Year (out from baseline) −1.32 (−1.80, −0.85) <0.001 PVD 1.92 (0.16, 3.67) 0.03 Table 3. Independent Predictors of Longitudinal Changes in eGFR. Only statistically significant predictors of longitudinal changes in eGFR (baseline out to 4 years) are included. The model was adjusted for, age, sex, baseline eGFR, systolic blood pressure (SBP), history of peripheral vascular disease (PVD) and diabetes mellitus, primary renal disease, and baseline ACEi/ARB or aspirin use. Using High sCD40L & Low sCD40R as the reference group Abbreviations: MDRD, Modified Diet in Renal Disease study; GFR, estimated glomerular filtration rate; sCD40L, soluble CD40 ligand; sCD40R, soluble CD40 receptor; BP, blood pressure; and CI, confidence interval; and PVD, peripheral vascular disease. Predictors Effect estimate (% change in eGFR) (95% CI) P-value Low sCD40L & High sCD40R 3.21 (0.59, 5.83) 0.016 Baseline MDRD eGFR 1.01 (0.94, 1.0) <0.001 Year (out from baseline) −1.32 (−1.80, −0.85) <0.001 PVD 1.92 (0.16, 3.67) 0.03 Table 3. Independent Predictors of Longitudinal Changes in eGFR. Only statistically significant predictors of longitudinal changes in eGFR (baseline out to 4 years) are included. The model was adjusted for, age, sex, baseline eGFR, systolic blood pressure (SBP), history of peripheral vascular disease (PVD) and diabetes mellitus, primary renal disease, and baseline ACEi/ARB or aspirin use. Discussion To the best of our knowledge, this is the largest cohort of all-cause CKD subjects in which plasma levels of both sCD40L and sCD40R were measured. Our results demonstrate that CD40 plasma mediators predict changes in renal function in subjects with CKD.f No differences in the proportion of endpoints events were found when our study population was analyzed by dichotomizing either sCD40L or sCD40R at their medians (S Fig. 1). This finding is corroborated by a recent study conducted by Rusu et al.28. However, we did observe that subjects with high sCD40L were more likely to experience an adverse event. It should be noted that our study was not sufficiently powered for composite end- point analysis. y A 2010 prospective observational study conducted by Lajer et al. in subjects with type 1 diabetic nephrop- athy demonstrated that while these subjects had statistically significant higher levels of plasma sCD40L than normo-albuminuric controls, sCD40L levels were not predictive of mortality or decreases in kidney function29. In our study, diabetes was the primary cause of CKD in 40 of the 244 subjects (Table 1). Our findings indicate that SCienTifiC RePorTs \| 7: 7942 \| DOI:10.1038/s41598-017-08426-8 5 www.nature.com/scientificreports/ levels of sCD40L were predictive of decreases in eGFR at 1-year follow-up. It is important to note, however, that our study was not sufficiently powered to specifically determine renal disease progression or mortality in subjects with diabetic nephropathy. Moreover, the baseline eGFR of the Lajer et al. study was 76 ± 33 ml/min per 1.73 m2 whereas the baseline eGFR in the present study is 29 ± 15 ml/min per 1.73 m2. This large difference in baseline eGFR may factor into the discrepancies between our respective studies. y Our results suggest that CD40 signaling is detrimental to kidney function. As discussed previously, CD40-mediated signaling events have been linked to inflammation, atherosclerosis and thrombosis6, 8, 14–16, 30, 31. Interestingly, the CD40 receptor has been demonstrated to be expressed on renal parenchymal cells, including the proximal tubules4, 5. Prior studies have linked CD40 signaling with proximal tubule inflammation and dam- age. Up-regulation of proximal tubule-associated CD40 receptor via transforming growth factor beta (TGFβ) mediated pathways led to increased destruction of proximal tubules by CD8 + cytotoxic T cells32. Others have shown that activation of proximal tubular-associated CD40 receptor is sufficient to cause a pro-fibrotic and pro-inflammatory response in vitro7, 33, 34. Discussion Importantly, inhibition of CD40 signaling has been shown to reduce the extent of renal injury in animal models of kidney disease35, 36. A recent report by our group suggests that CD40 significantly contributes to the development of renal fibrosis in experimental hypertensive nephropathy37. gi y pi p yp p p y sCD40R has been shown to directly antagonize CD40 signaling in B cells10–13. Moreover, there is evidence to suggest that elevated levels of sCD40R may partially account for end stage renal disease associated immu- nodeficiency38. Given the increasing amount of evidence implicating CD40 in renal injury, sCD40R, an endog- enous CD40 antagonist, holds promising potential as a therapeutic target. Importantly, whether sCD40R can directly inhibit CD40-mediated pro-fibrotic and pro-inflammatory signaling in the kidney has not been studied. However, we have previously demonstrated that sCD40R was inversely associated with renal function decline in an atherosclerotic renal artery stenosis cohort20. It should be noted that a 2012 study conducted by Esposito et al. demonstrated that sCD40R levels and eGFR were inversely correlated (a high level of sCD40R was associated with low eGFR and vice versa) in uremic subjects not on hemodialysis39. These opposing findings may be related to the small sample size of the Esposito study (8 subjects). In aggregate, the results from our current study support the hypothesis that sCD40R may be renoprotective. We show that subjects with high sCD40L/low sCD40R had an accelerated decline in renal function compared to subjects with low sCD40L/high sCD40R at both short-term and long-term follow-up. Furthermore, low sCD40R was found to be an independent predictor of renal function decline following adjustment for covariates such as age, sex, systolic blood pressure, baseline eGFR, history of peripheral vascular disease or diabetes, primary renal disease classification, and use of ACEi/ARB medications (Table 2). Our findings clearly necessitate the need for further mechanistic studies to better understand the func- tion of sCD40R in the kidney. y Some limitations of the design of our study need to be considered. As alluded to previously, we were unable to conduct subgroup analyses due to the size of our study. Future studies utilizing a greater sample size could enhance our understanding of the role of CD40 signaling in specific subtypes of CKD classifications. Moreover, we measured baseline plasma levels of sCD40L and sCD40R. Discussion Further studies with repeated measurements of plasma sCD40L and sCD40R are required to better elucidate the long-term effects of CD40 signaling mediators in CKD. As a retrospective observational study, we were only able to make associations between circulating CD40 mediators and changes in renal function. Future mechanistic studies are still needed to prove the causative rela- tionship between CD40 and CKD. p A growing body of both basic and clinical research implicates the CD40 pathway as a critical mediator of CKD progression. The present findings demonstrate that baseline levels of sCD40L and sCD40R are predictive of changes in renal function at both short- and long-term follow-up in a CKD cohort. Future studies are needed to determine whether interruption of the CD40 signaling, and specifically, sCD40R, can prevent progression of CKD. 1. Chapter 1: Definition and classification of CKD. Kidney international supplements 3, 19-62, doi:10.1038/kisup.2012.64 (2013). 2 C r h J t l Pr l n f hr ni kidn di in th Unit d St t J 298 2038 2047 d i 10 1001/j m 298 17 2038 (2007 2. Coresh, J. et al. Prevalence of chronic kidney disease in the United States. Jama 298, 2038 2047, doi:10.1001/jama.298.17.2038 (2007 3. van Kooten, C. & Banchereau, J. CD40-CD40 ligand. Journal of leukocyte biology 67, 2–17 (2000). j f p y gy p y gy jp 5. van Kooten, C. & Banchereau, J. Functions of CD40 on B cells, dendritic cells and other cells. Current opinion in immunology 9, 330–337 (1997). Author Contributions J.X., P.B., R.G. and S.H. wrote the main manuscript text. J.X., Y.X., K.R., H.N., T.C. and P.B. contributed equally to the preparation of figures and tables. J.X., H.A., J.R., P.K., Y.X., K.R., H.N., T.C., P.B., R.G., L.D., D.M., C.C., J.T., S.H. contributed to the conception, design and analysis of the data. J.X., H.A., J.R., P.A.K., Y.X., K.R., H.N., T.C., P.B., R.G., L.D.D., D.M., C.J.C., J.T., S.T.H. contributed to the revision and approval of the final manuscript. J.X., P.B., R.G. and S.H. wrote the main manuscript text. J.X., Y.X., K.R., H.N., T.C. and P.B. contributed equally to the preparation of figures and tables. J.X., H.A., J.R., P.K., Y.X., K.R., H.N., T.C., P.B., R.G., L.D., D.M., C.C., J.T., S.H. contributed to the conception, design and analysis of the data. J.X., H.A., J.R., P.A.K., Y.X., K.R., H.N., T.C., P.B., R.G., L.D.D., D.M., C.J.C., J.T., S.T.H. contributed to the revision and approval of the final manuscript. www.nature.com/scientificreports/ 29. Lajer, M. et al. Soluble CD40 ligand is elevated in type 1 diabetic nephropathy but not predictive of mortality, cardiovascular events or kidney function. Platelets 21, 525–532, doi:10.3109/09537104.2010.500422 (2010). y 30. Anand, S. X., Viles-Gonzalez, J. F., Badimon, J. J. & Cavusoglu, E. & Marmur, J. D. Membrane-associated CD40L and sCD40L in atherothrombotic disease. Thrombosis and haemostasis 90, 377–384, doi:10.1160/TH03-05-0268 (2003). h 31. Aukrust, P., Damas, J. K. & Solum, N. O. Soluble CD40 ligand and platelets: self-perpetuating pathogenic loop in thrombosi inflammation? Journal of the American College of Cardiology 43, 2326–2328, doi:10.1016/j.jacc.2004.03.023 (2004). l 32. Starke, A., Wuthrich, R. P. & Waeckerle-Men, Y. TGF-beta treatment modulates PD-L1 and CD40 expression in proximal renal tubular epithelial cells and enhances CD8 cytotoxic T-cell responses. Nephron. Experimental nephrology 107, e22–29, doi:10.1159/000106506 (2007).i 33. Li, H. & Nord, E. P. IL-8 amplifies CD40/CD154-mediated ICAM-1 production via the CXCR-1 receptor and p38-MAPK pathway in human renal proximal tubule cells. American journal of physiology. Renal physiology 296, F438–445, doi:10.1152/ ajprenal.90214.2008 (2009).i jp 4. van Kooten, C. et al. Possible role for CD40-CD40L in the regulation of interstitial infiltration in the kidney. Kidney international 51 711–721 (1997). 35. Kairaitis, L. et al. Blockade of CD40-CD40 ligand protects against renal injury in chronic proteinuric renal disease. Ki international 64, 1265–1272, doi:10.1046/j.1523-1755.2003.00223.x (2003). j nner, A. J. et al. CD40 Generation 2.5 Antisense Oligonucleotide T 36. Donner, A. J. et al. CD40 Generation 2.5 Antisense Oligonucleotide Treatment Attenuates Doxorubicin-induced Nephropathy Kidney Inflammation. Mol Ther Nucleic Acids 4, e265, doi:10.1038/mtna.2015.40 (2015).i ylh 7. Haller, S. T. et al. Targeted disruption of Cd40 in a genetically hypertensive rat model attenuates renal fibrosis and proteinuria independent of blood pressure. Kidney international 91, 365–374 (2017).i 38. Contin-Bordes, C., Lacraz, A. & de Precigout, V. Potential role of the soluble form of CD40 in deficient immunological function of dialysis patients: new findings of its amelioration using polymethylmethacrylate (PMMA) membrane. NDT Plus 3, i20–i27, doi:10.1093/ndtplus/sfq033 (2010). p q 39. Esposito, P. et al. Mechanisms underlying sCD40 production in hemodialysis patients. Cellular immunology 278, 10–15, doi:10.1016/j.cellimm.2012.06.007 (2012). www.nature.com/scientificreports/ www.nature.com/scientificreports/ 5. Schonbeck, U. & Libby, P. CD40 signaling and plaque instability. Circulation research 89, 1092–1103 (2001).l & Libby, P. CD40 signaling and plaque instability. Circulation resea 6. Phipps, R. P. Atherosclerosis: the emerging role of inflammation and the CD40-CD40 ligand system. Proceedings of the Nationa Academy of Sciences of the United States of America 97, 6930–6932 (2000).h y f f f 7. Heeschen, C. et al. Soluble CD40 ligand in acute coronary syndromes. The New England journal of medicine 348, 1104–1111 doi:10.1056/NEJMoa022600 (2003).l J ( ) 18. Libby, P. & Simon, D. I. Inflammation and thrombosis: the clot thickens. Circulation 103, 1718–1720 (2001). yl 19. Haller, S. et al. Platelet activation in patients with atherosclerotic renal artery stenosis undergoing stent revascularization journal of the American Society of Nephrology: CJASN 6, 2185–2191, doi:10.2215/CJN.03140411 (2011).f j f y f p gy 20. Haller, S. T. et al. Effect of CD40 and sCD40L on renal function and survival in patients with renal artery stenosis. Hypertension 61, 894–900, doi:10.1161/HYPERTENSIONAHA.111.00685 (2013). 1. Preston, R. A. & Epstein, M. Ischemic renal disease: an emerging cause of chronic renal failure and end-stage renal disease. Hypertens 15, 1365–1377 (1997). yp , ( ) 2. Ritchie, J. et al. Extreme Elevations in Blood Pressure and All-Cause Mortality in a Referred CKD Population: Results from the CRISIS Study. International journal of hypertension 2013, 597906, doi:10.1155/2013/597906 (2013).ih 23. Hoefield, R. A. et al. The use of eGFR and ACR to predict decline in renal function in people with diabetes. Nephrology, dialysis, transplantation: official publication of the European Dialysis and Transplant Association - European Renal Association 26, 887–892, doi:10.1093/ndt/gfq526 (2011). g q ( ) 24. Hoefield, R. A. et al. Factors associated with kidney disease progression and mortality in a referred CKD population. American journal of kidney diseases: the official journal of the National Kidney Foundation 56, 1072–1081, doi:10.1053/j.ajkd.2010.06.010 (2010). fi 25. Moser, B. K. & Coombs, L. P. Odds ratios for a continuous outcome variable without dichotomizing. Stat Med 23, 1843– doi:10.1002/sim.1776 (2004). 26. Tang, W., He, H. and Tu, X.M. in Applied Categorical and Count Data Analysis (Chapman & Hall/CRC, 2012).h 27. Chen, J. M. et al. The association of CD40 polymorphisms with CD40 serum levels and risk of systemic lupus erythematosus. BMC Genet 16, 121, doi:10.1186/s12863-015-0279-8 (2015). 8. Rusu, C. et al. Soluble CD40 ligand in haemodialysis patients: survival impact and cardiovascular prognostic role. Biomarkers, 1–7 doi:10.1080/1354750X.2016.1201531 (2016). Referencesi g f y gy 4. Li, H. & Nord, E. P. CD40 ligation stimulates MCP-1 and IL-8 production, TRAF6 recruitment, and MAPK activation in proxima tubule cells. American journal of physiology. Renal physiology 282, F1020–1033, doi:10.1152/ajprenal.00291.2001 (2002). j f p y gy p y gy j 5. van Kooten, C. & Banchereau, J. Functions of CD40 on B cells, dendritic cells and other cells. Current opinion in immunology 9 330–337 (1997).l 6. Rizvi, M., Pathak, D., Freedman, J. E. & Chakrabarti, S. CD40-CD40 ligand interactions in oxidative stress, inflammation and vascular disease. Trends in molecular medicine 14, 530–538, doi:10.1016/j.molmed.2008.09.006 (2008).lif j 7. Pontrelli, P. et al. CD40L proinflammatory and profibrotic effects on proximal tubular epithelial cells: role of NF-kappaB and lyn Journal of the American Society of Nephrology: JASN 17, 627–636, doi:10.1681/ASN.2005020202 (2006). 8. Andre, P., Nannizzi-Alaimo, L., Prasad, S. K. & Phillips, D. R. Platelet-derived CD40L: the switch-hitting player of cardiovascula disease. Circulation 106, 896–899 (2002). 9. Mazzei, G. J. et al. Recombinant soluble trimeric CD40 ligand is biologically active. The Journal of biological chemistry 270 7025–7028 (1995). 10. Contin, C. et al. Potential role of soluble CD40 in the humoral immune response impairment of uraemic patients. Immunology 110, 131–140 (2003). 11. van Kooten, C. et al. B cells regulate expression of CD40 ligand on activated T cells by lowering the mRNA level and throug release of soluble CD40. European journal of immunology 24, 787–792, doi:10.1002/eji.1830240402 (1994). 12. Schwabe, R. F., Engelmann, H., Hess, S. & Fricke, H. Soluble CD40 in the serum of healthy donors, patients with chronic renal failure, haemodialysis and chronic ambulatory peritoneal dialysis (CAPD) patients. Clinical and experimental immunology 117, 153–158 (1999). 3. Eshel, D. et al. Characterization of natural human antagonistic soluble CD40 isoforms produced through alternative splicing Molecular immunology 46, 250–257, doi:10.1016/j.molimm.2008.08.280 (2008). gy j ( ) 14. Lutgens, E., Lievens, D., Beckers, L., Donners, M. & Daemen, M. CD40 and its ligand in atherosclerosis. Trends in cardiovascular medicine 17, 118–123, doi:10.1016/j.tcm.2007.02.004 (2007). SCienTifiC RePorTs \| 7: 7942 \| DOI:10.1038/s41598-017-08426-8 6 Additional Information Supplementary information accompanies this paper at doi:10.1038/s41598-017-08426-8 Supplementary information accompanies this paper at doi:10.1038/s41598-017-08426-8h Competing Interests: The authors declare that they have no competing interests. Competing Interests: The authors declare that they have no competing interests. ublisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and nstitutional affiliations. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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 Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted 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 license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2017 7 SCienTifiC RePorTs \| 7: 7942 \| DOI:10.1038/s41598-017-08426-8
https://openalex.org/W2556409681	https://bsd.biomedcentral.com/track/pdf/10.1186/s13293-016-0109-3	English	null	Sex differences in outcomes after stroke among patients with low total cholesterol levels: a large hospital-based prospective study	Biology of sex differences	2,016	cc-by	6,932	© 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. Abstract Background: Previous studies have shown that total cholesterol (TC) levels are associated with stroke outcomes, but sex differences in the association between TC levels, especially a low TC level, and ischemic stroke outcomes are unknown. We aimed to assess the sex differences in stroke outcomes among patients with atherothrombotic infarctions and low TC levels in China. Methods: This study recruited patients with atherothrombotic infarctions from Tianjin, China, between May 2005 and September 2014. Patients with low TC levels (defined as TC <4.22 mmol/L) were analyzed in this study. Sex differences in stroke subtypes, severity, risk factors, and outcomes at 3 and 12 months after stroke were compared. Results: Overall, 1587 patients with low TC levels were recruited to this study from among 6407 patients with atherothrombotic infarctions listed in a stroke registry. Women were more likely than men to have posterior circulation infarcts, severe stroke, hypertension, and obesity but less likely to be current smokers or to consume alcohol. There were no sex differences in stroke outcomes. Older age and severe stroke were common risk factors for poor outcomes after stroke in this study. The presence of diabetes mellitus was an independent predictor of low mortality at 12 months after stroke, possibly because a drug commonly used to treat diabetes, metformin, enhances angiogenesis. Obesity was the determinant of the recurrence and dependency rates at 12 months after stroke. Conclusions: These findings suggest that patients (both men and women) with atherothrombotic infarction who have low TC levels would not benefit from receiving statin treatment. Therefore, it is crucial to explore the impact of statin treatment on outcomes in Asian patients, especially Chinese patients with atherothrombotic and low TC levels, in order to improve outcomes after stroke and reduce the disease burden. Keywords: Total cholesterol, Ischemic stroke, Outcomes, Sex differences third most common cause of death overall, the third in urban areas, and the second in rural areas [3]. Moreover, stroke is also a leading cause of functional impairments, with 20% of survivors requiring institutional care after 3 months and 15–30% being permanently disabled [4]. Sex differences in outcomes after stroke among patients with low total cholesterol levels: a large hospital-based prospective study Guanen Zhou1,2, Zhongping An1,2, Wenjuan Zhao1,2, Yan Hong1,2, Haolin Xin1,2, Xianjia Ning3,4 and Jinghua Wang3,4 Correspondence: tjhhazp@sina.com 1Department of Neurology, Tianjin Huanhu Hospital, 6 Jizhao Road, Jinnan District, Tianjin 300350, China 2Tianjin Key Laboratory of Cerebral Vascular and Neurodegenerative Disease, Tianjin 300350, China Full list of author information is available at the end of the article Zhou et al. Biology of Sex Differences (2016) 7:62 DOI 10.1186/s13293-016-0109-3 Zhou et al. Biology of Sex Differences (2016) 7:62 DOI 10.1186/s13293-016-0109-3 Open Access Stroke subtypes Stroke subtypes were defined as total anterior circulation infarct (TACI), partial anterior circulation infarct (PACI), posterior circulation infarct (POCI), and lacunar infarct (LACI) according to the Oxfordshire Community Stroke Project (OCSP) classification criteria [17]. Ethics, consent, and permissions The study was approved by the Ethics Committee for Medical Research at Tianjin Huanhu Hospital, and writ- ten informed consent was obtained from each partici- pant during recruitment. Neurological function deficits and stroke severity y Neurological function deficits were defined using the National Institutes of Health stroke scale (NIHSS), Barthel index (BI) [18], and modified Rankin scale (mRS) on admission [19]. Stroke severity was catego- rized into three groups on the basis of the NIHSS score: mild (NIHSS ≤7), moderate (NIHSS between 8 and 16), and severe (NIHSS ≥17) [20]. Background Although age-standardized rates of stroke mortality have decreased worldwide in the past few decades, the global burden of stroke disability-adjusted life-years is a crucial health issue due to the increasing absolute number of stroke survivors [1, 2]. In 2014, in China, stroke was the High total cholesterol (TC) level is a well-documented risk factor for coronary disease [5, 6]. Moreover, hyperchol- esterolemia has been well-documented as a modifiable risk factor for ischemic stroke [7], although a lower TC level was shown to be an independent predictor of hemorrhagic stroke in previous studies [8, 9]. The association between * Correspondence: tjhhazp@sina.com 1Department of Neurology, Tianjin Huanhu Hospital, 6 Jizhao Road, Jinnan District, Tianjin 300350, China 2Tianjin Key Laboratory of Cerebral Vascular and Neurodegenerative Disease, Tianjin 300350, China Full list of author information is available at the end of the article Page 2 of 8 Zhou et al. Biology of Sex Differences (2016) 7:62 Zhou et al. Biology of Sex Differences (2016) 7:62 TC levels and stroke outcomes is controversial. A large number of studies have indicated that high TC levels were associated with better stroke outcomes [7, 10, 11], but the reverse trend was observed in other studies [12, 13]. To ensure data quality, three groups of senior trained neurologists (the assessment group, the follow-up group, and the quality control group) were responsible for deter- mining the nervous system score at admission, for the reex- amination (including of neurological score, risk factor management, and directing the treatment and rehabilita- tion) during follow-up, and for a sampled confirmation of 20% of all patients each month, respectively. TC levels and stroke outcomes is controversial. A large number of studies have indicated that high TC levels were associated with better stroke outcomes [7, 10, 11], but the reverse trend was observed in other studies [12, 13]. With recent economic development, the incidence of stroke in China has increased dramatically [14]; however, large-scale studies of the association between TC level and stroke outcomes in China are rare, especially in pa- tients with atherothrombotic infarction. Therefore, we aimed to assess the sex differences in the associations between low TC levels on admission and long- term stroke outcomes after acute ischemic stroke (AIS) in patients with atherothrombotic infarction in China. Definitions of outcomes Stroke outcomes were described on the basis of mortal- ity, recurrence, and dependency rates at 3 and 12 months after stroke. Outcomes were assessed using face-to-face or telephone follow-up interviews. Death was defined as all-cause cumulative death at the corresponding follow- up time points after stroke, and this information was collected from medical records or patients’ family mem- bers by telephone follow-up. Recurrence was defined as new-onset vascular events (stroke, myocardial infarction, and venous thrombosis) 30 days after the initial stroke in all survivors who completed follow-up using face-to- face interviews or telephone calls. Dependency was de- fined as an mRS score >2 among all survivors who underwent follow-up using face-to-face interview or telephone calls [21]. Risk factors Stroke risk factors included a medical history of hyperten- sion (defined as a self-reported history of hypertension or the use of antihypertension drugs), diabetes mellitus (DM, defined as a history of DM or the use of hypoglycemic medications at discharge), atrial fibrillation (AF, defined as a history of AF, confirmed by at least one electrocardiogram, or the presence of arrhythmia during hospitalization), and obesity (body mass index ≥30 kg/m2) and modifiable life- style factors, including current smoking status and alcohol consumption. Methods This was a hospital-based follow-up study using the Stroke Registry System that we developed in 2005 in Tianjin Huanhu Hospital, a specialized neurological hospital in Tianjin, China. All consecutive patients with first-ever AIS who were admitted to the Stroke Unit at Tianjin Huanhu Hospital within 72 h of stroke onset between May 2005 and September 2014 were recruited to this study. A clinical diagnosis of stroke was made according to the World Health Organization criteria and confirmed by neuroimag- ing (computed tomography/magnetic resonance imaging) [15]. Cases of transient ischemic attack were excluded from this study. The study originally included all patients with atherothrombotic infarction classified according to the Trial of Org 10172 in Acute Stroke Treatment (TOAST) for large-artery atherothrombosis and small-artery occlusion (SAO) [16], who were treated using statins and for whom data on TC level on admission were available. The patients were further categorized into two groups according to TC level on admission: the low-TC group, defined as patients with a TC level <4.22 mmol/L, and the non-low-TC group, defined as patients with a TC level ≥4.22 mmol/L. For this study, the final study population included only patients with low TC. Statistical analysis Age is presented as mean (standard deviation), and NIHSS, BI, and mRS scores are presented as medians (interquartile ranges). These continuous variables were compared be- tween men and women using the Student t test or the Mann-Whitney U test, as appropriate. At the different follow-up time periods after stroke, categorical variables, including stroke subtype, stroke severity, risk factors, and outcomes, are presented as number (percentage), and the trends were compared using chi-squared tests. Associations between the relevant risk factors and outcomes in men and women were assessed individually using univariate and multivariate logistic regression models and are presented as unadjusted and adjusted (by age, stroke severity, stroke sub- types, and risk factors) odds ratios (ORs), respectively, with 95% confidence intervals (CIs). All statistical analyses were performed using SPSS version 15.0 (SPSS Inc., Chicago, IL), Patient selection Of the 7565 AIS patients recruited between May 2005 and September 2014, 392 patients with cardioembolic stroke, 284 patients with other stroke and stroke of un- determined causes, and 482 patients without a TC level recorded on admission were excluded, resulting in 6407 patients with atherothrombotic infarction that were in- cluded. Of these, there were 1587 patients with low TC levels. After excluding those patients who did not complete follow-up, the response rate was 97.4% at 3 months and 94.9% at 12 months (Fig. 1). Follow-up period and two-tailed P values <0.05 were considered statistically significant. Follow-up was conducted according to a predetermined procedure. Trained neurologists reexamined patients in the outpatient department at 3 and 12 months after stroke. All patients completed follow-up by face-to-face interview, except for patients who were reexamined in their neighboring hospitals; these patients completed follow-up by telephone. Data collection and group assignments Detailed information on ischemic stroke subtype, stroke severity, previous history of diseases, stroke risk factors, laboratory examination results, and outcomes at 3 and 12 months after stroke were obtained from a standard- ized questionnaire and recorded in the Stroke Registry System. Page 3 of 8 Zhou et al. Biology of Sex Differences (2016) 7:62 Page 3 of 8 Sex differences in outcomes among patients with atherothrombotic infarction and low TC levels Table 2 shows that women had significantly higher recur- rence and dependency rates at 12 months than men did; the corresponding rates were 33.2 vs. 25.1% (P = 0.010) for recurrence rate at 12 months and 31.3 vs. 24.5% (P = 0.031) for dependency rate at 12 months. However, there were no significant differences in mortality at 3 and 12 months after stroke or in recurrence and dependency rates at 3 months after stroke. at 3 and 12 months after stroke. Moreover, OCSP classifica- tion, diabetes mellitus (DM), arterial stenosis, and alcohol drinking were associated with mortality, and sex, obesity, and current smoking were associated with recurrence and dependency rates (Table 3). Results of the univariate analysis indicated that age and stroke severity were significantly associated with outcomes p y The sex differences in recurrence and dependency rates became non-significant after adjustment for age, severity, subtype, and risk factors. Older age and stroke severity were independent risk factors for stroke outcomes. A low risk of mortality was observed in patients with DM at 12 months after stroke, but a positive association was found between obesity and the recurrence and dependency rates at 12 months after stroke. The risk of mortality decreased by 49% at 12 months in patients with DM (P = 0.013). How- ever, the risk increased by 77% for recurrence (P = 0.008) and by 87% for dependency (P = 0.004) at 12 months in patients with obesity (Table 4). Sex differences in clinical features among patients with atherothrombotic infarction Of the 1578 patients with low TC levels, 1272 (80.2%) were men and 315 (19.8%) were women. Women were more likely than men to have PACI (33.0 vs. 31.5%, P = 0.006), hypertension (80.6 vs. 71.4%, P < 0.001), and obesity (18.1 vs. 7.9%, P < 0.001), but men were more likely than women to have mild stroke (69.9 vs. 64.8%, P < 0.001), to be current smokers (49.4 vs. 13.0%, P < 0.001), or to drink alcohol Fig. 1 Flow diagram of participants Page 4 of 8 Page 4 of 8 Zhou et al. Biology of Sex Differences (2016) 7:62 (26.3 vs. 1.3%, P < 0.001). Moreover, women showed poorer neurological function (Table 1). Table 2 Sex differences in outcomes among atherosclerotic stroke patients with low TC level Table 2 Sex differences in outcomes among atherosclerotic stroke patients with low TC level Outcomes Men Women P At 3 months after stroke Mortality, n (%) 63 (6.3) 13 (5.7) 0.736 Recurrence, n (%) 94 (10.0) 24 (11.1) 0.630 Dependency, n (%) 99 (10.5) 25 (11.6) 0.659 At 12 months after stroke Mortality, n (%) 95 (8.8) 23 (8.6) 0.945 Recurrence, n (%) 253 (25.1) 83 (33.2) 0.010 Dependency, n (%) 242 (24.5) 76 (31.3) 0.031 Sex differences in outcomes among patients with atherothrombotic infarction and low TC levels Table 1 Sex differences in clinical characteristics and risk factors among patients with low TC level and atherosclerotic stroke Characteristics Total Men Women P Cases, n (%) 1587 1272 (29.5) 315 (15.1) – Age, year, mean (SD) 64.41 (11.40) 64.17 (11.42) 65.40 (11.28) 0.087 OCSP, n (%) PACI 895 (56.4) 728 (57.2) 167 (53.0) 0.177 TACI 83 (5.2) 62 (4.9) 21 (6.7) 0.201 LACI 104 (6.6) 81 (6.4) 23 (7.3) 0.549 POCI 505 (31.8) 401 (31.5) 104 (33.0) 0.006 Stroke severity Mild 1093 (68.9) 889 (69.9) 204 (64.8) <0.001 Moderate 367 (23.1) 289 (22.7) 78 (24.8) 0.442 Severe 127 (8.0) 94 (7.4) 33 (10.5) 0.071 Neurological functiona NIHSS 5 (7) 5 (7) 6 (7) 0.006 BI 60 (50) 65 (50) 55 (50) 0.004 mRS 3 (2) 3 (2) 3 (2) 0.007 Risk factors, n (%) Hypertension 1162 (73.2) 908 (71.4) 254 (80.6) 0.001 Diabetes 472 (29.7) 366 (28.8) 106 (33.7) 0.090 Atrial fibrillation 71 (4.5) 52 (4.1) 19 (6.0) 0.135 Artery stenosis 416 (26.2) 341 (26.8) 75 (23.8) 0.279 Obesity 157 (9.9) 100 (7.9) 57 (18.1) <0.001 Current smoking 669 (42.2) 628 (49.4) 41 (13.0) <0.001 Alcohol drinking 339 (21.4) 335 (26.3) 4 (1.3) <0.001 OCSP Oxfordshire Community Stroke Project, TACI total anterior circulation infarct, PACI partial anterior circulation infarct, POCI posterior circulation infarct, LACI lacunar infarct, NIHSS National Institute of Health stroke scale, BI Barthel index, mRS modified Rankin scale aData were presented as median with interquartile range Table 1 Sex differences in clinical characteristics and risk factors among patients with low TC level and atherosclerotic stroke Discussion This is the first report to demonstrate sex differences in clinical features, risk factors, and outcomes among patients with atherothrombotic infarction and low TC levels. Women were more likely than men to have PACI, poor neurological function, hypertension, and obesity, but men were more likely than women to be current smokers or to drink alcohol. There were significantly higher recurrence and dependency rates at 12 months in women than in men. However, the sex differences in stroke outcomes disap- peared after adjustment for age, stroke severity, subtype, and risk factors. Previous studies have indicated that women tend to have strokes at an older age than men do [22–24]. It has been reported that women were more likely than men to have severe stroke [23, 25], but this result was not found in other studies [26, 27]. Moreover, a greater prevalence of hypertension, DM, atrial fibrillation, dyslipidemia, and obesity has been reported for women in previous studies [20, 28, 29]. Consistent with these studies, in the present study, we found that women were more likely than men to have POCI, severe stroke, hypertension, and obesity but less Zhou et al. Discussion Biology of Sex Differences (2016) 7:62 Page 5 of 8 Page 5 of 8 Table 3 Unadjusted OR with 95% CI of outcome determinants at 3 and 12 months after stroke among patients with low TC level in univariate analysis Factors Reference Mortality Recurrence Dependency 3 months 12 months 3 months 12 months 3 months 12 months Men Women 0.94 (0.56, 1.60) 0.98 (0.61, 1.58) 1.02 (0.66, 1.57) 1.48 (1.10, 2.00)* 1.10 (0.73, 1.66) 1.40 (1.03, 1.91)* Age – 1.07 (1.05, 1.10)* 1.07 (1.05, 1.10)* 1.03 (1.01, 1.05)* 1.02 (1.01, 1.04)* 1.03 (1.02, 1.05)* 1.02 (1.01, 1.04)* OCSP POCI PACI 0.73 (0.47, 1.15) 0.94 (0.62, 1.44) 0.97 (0.66, 1.42) 1.03 (0.78, 1.36) 1.00 (0.69, 1.46) 1.05 (0.79, 1.39) TACI 1.91 (0.90, 4.02) 2.46 (1.21, 5.02)* 1.72 (0.84, 3.51) 1.70 (0.93, 3.09) 1.86 (0.93, 3.72) 1.65 (0.89, 3.08) LACI 0.25 (0.06, 1.06) 0.44 (0.15, 1.25) 0.56 (0.23, 1.35) 0.74 (0.43, 1.27) 0.65 (0.28, 1.47) 0.70 (0.40, 1.22) Stroke severity Mild Moderate 5.02 (2.82, 8.93)* 3.80 (2.31, 6.26)* 1.88 (1.27, 2.77)* 2.32 (1.74, 3.08)* 1.85 (1.26, 2.72)* 2.31 (1.73, 3.09)* Severe 32.18 (18.02, 57.47)* 32.44 (18.99, 55.43)* 3.74 (2.11, 6.66)* 2.44 (1.39, 4.30)* 4.43 (2.55, 7.67)* 2.15 (1.16, 4.00)* Hypertension No 0.71 (0.46, 1.11) 0.73 (0.49, 1.08) 1.08 (0.73, 1.61) 1.24 (0.93, 1.64) 1.16 (0.78, 1.72) 1.26 (0.94, 1.69) Diabetes No 0.57 (0.34, 0.95)* 0.56 (0.35, 0.90)* 0.95 (0.65, 1.39) 1.12 (0.85, 1.47) 0.89 (0.61, 1.29) 1.20 (0.91, 1.57) AF No 1.24 (0.49, 3.17) 1.66 (0.73, 3.77) 0.63 (0.22, 1.75) 1.05 (0.55, 2.02) 0.59 (0.21, 1.65) 0.93 (0.46, 1.85) Artery stenosis No 0.50 (0.29, 0.88)* 0.59 (0.36, 0.97)* 1.14 (0.78, 1.66) 1.08 (0.81, 1.42) 1.10 (0.75, 1.59) 1.09 (0.82, 1.45) Obesity No 0.50 (0.20, 1.25) 0.45 (0.18, 1.12) 0.90 (0.49, 1.63) 1.84 (1.23, 2.76)* 0.85 (0.47, 1.54) 1.94 (1.29, 2.91)* Current smoking No 0.79 (0.52, 1.22) 0.85 (0.58, 1.26) 0.71 (0.50, 1.02) 0.74 (0.57, 0.95)* 0.68 (0.47, 0.96)* 0.72 (0.56, 0.94)* Alcohol drinking No 0.57 (0.32, 1.04) 0.55 (0.32, 0.94)* 0.67 (0.42, 1.07) 0.84 (0.62, 1.14) 0.60 (0.37, 0.96)* 0.84 (0.62, 1.15) OR odds ratios, CI confidence intervals; Presented P<0.05 in univariate analysis Table 3 Unadjusted OR with 95% CI of outcome determinants at 3 and 12 months after stroke among patients with low TC level in univariate analysis an increased risk of ischemic stroke have been reported in several studies [31, 32], but a clear association was not found in others [33–37]. OR odds ratios, CI confidence intervals; Presented P<0.05 after adjusted by covariates Discussion dependency and recurrence rates occurred in female pa- tients with low TC levels on admission, and all patients received statin treatment after stroke. Thus, the benefit of statins for improving outcomes after stroke in Asian populations, especially in Chinese people, needs to be explored further. TC levels were associated with increased risk of severe stroke, TACI, and poor functional outcomes in patients with ischemic stroke who had received pre-stroke statin treatment, and the short-term and long-term mortality rates were significantly higher in patients with low chol- esterol levels [41]. p There are several limitations in this study. First, all patients were from a local neurological hospital in Tianjin, China, and may not represent all stroke pa- tients in China. Second, data on statin use before stroke onset were lacking, which may have affected the evaluation of TC level on stroke outcomes. How- ever, the aim of this study was to evaluate the differ- ences in stroke outcomes between men and women with low TC levels. Thus, it is not likely that the lack of information regarding previous statin use had a major impact on the results. Third, there were a few patients who completed follow-up by telephone (2.8% at 3 months and 12.2% at 12 months), which may have introduced an assessment bias due to a measurement disparity. Moreover, the differences in baseline characteristics (higher prevalence of DM at 3 months; older age, higher frequency of severe stroke, and higher prevalence of DM at 12 months) may have partially affected the assessment of the as- sociation between outcomes and risk factors. Finally, the TC compositions were not measured in this study, which may have affected the evaluation of stroke outcomes among patients with a low TC level. Poor outcomes after AIS have been reported in pa- tients with low cholesterol levels [9, 40, 41]. In particu- lar, a negative or non-significant association between TC level and mortality was observed in patients with ische- mic stroke aged 70 years or older [35]. Higher TC levels were also associated with lower short-term mortality after stroke; the neuroprotective role of cholesterol may have contributed to this finding [11, 42, 43]. Another study indicated that the lower mortality after stroke among patients with higher cholesterol levels attributed to hypercholesterolemia could be linked to minor strokes (mainly small-vessel stroke) with good outcomes [44]. Discussion A positive association between TC levels and atherothrombotic infarction has been reported in previous studies [38, 39]. Other studies have indicated that a higher TC level increased the risk of cerebral infarction likely to be current smokers and to consume alcohol. The delayed time to hospital admission in women could explain the greater frequency of severe stroke [30]. High cholesterol level is an identified risk factor for cor- onary heart disease, but its role in stroke remains contro- versial. The associations between high serum TC levels and Table 4 Adjusted OR with 95% CI of outcome determinants at 3 and 12 months after stroke among patients with low TC level in multivariate analysis Factors Reference Mortality Recurrence Dependency 3 months 12 months 3 months 12 months 3 months 12 months Men Women – – – 1.27 (0.92, 1.75) – 1.18 (0.84, 1.64) Age – 1.06 (1.03, 1.08)* 1.06 (1.04, 1.08)* 1.03 (1.01, 1.04)* 1.02 (1.01, 1.03)* 1.03 (1.01, 1.04)* 1.02 (1.01, 1.03)* OCSP POCI PACI – 0.77 (0.47, 1.25) – – – – TACI – 0.80 (0.33, 1.99) – – – – LACI – 0.59 (0.19, 1.86) – – – – Stroke severity Mild Moderate 4.86 (2.72, 8.70)* 3.77 (2.25, 6.30)* 1.86 (1.26, 2.75)* 2.27 (1.70, 3.03)* 1.84 (1.24, 2.71)* 2.28 (1.70, 3.05)* Severe 26.23 (14.47, 47.55)* 27.46 (15.39, 49.02)* 3.39 (1.89, 6.06)* 2.27 (1.28, 4.02)* 3.92 (2.24, 6.85)* 1.96 (1.05, 3.68)* Diabetes No – 0.51 (0.30, 0.87)* – – – – Artery stenosis No 0.68 (0.37, 1.24) 0.73 (0.42, 1.28) – – – – Obesity No – – – 1.77 (1.17, 2.70)* – 1.87 (1.23, 2.84)* Current smoking No – – – 0.92 (0.70, 1.22) 0.90 (0.60, 1.35) 0.89 (0.67, 1.19) Alcohol drinking No – 0.47 (0.42, 1.49) – – 0.76 (0.45, 1.30) – OR odds ratios, CI confidence intervals; *Presented P<0.05 after adjusted by covariates th 95% CI of outcome determinants at 3 and 12 months after stroke among patients with low TC level in Table 4 Adjusted OR with 95% CI of outcome determinants at 3 and 12 months after stroke among patient multivariate analysis Page 6 of 8 Page 6 of 8 Zhou et al. Biology of Sex Differences (2016) 7:62 [9, 40], but sex differences in the association between TC level and stroke outcomes are not well-known. [9, 40], but sex differences in the association between TC level and stroke outcomes are not well-known. Discussion Several large-scale studies demonstrated that lower TC levels were associated with higher all-cause mortality and that higher TC levels were associated with lower all- cause mortality [45, 46]. Furthermore, a U-shaped asso- ciation between TC level and dependency after AIS was shown in a group of people of very old age; patients with moderate TC levels had the most favorable outcomes after AIS in patients aged >80 years [47]. Moreover, our previous study indicated that low cholesterol levels among patients with atherothrombotic infarction receiv- ing statin treatment increased long-term dependency and recurrence rates, but not mortality rates [48]. Author details 1 f 21. Banks JL, Marotta CA. Outcomes validity and reliability of the modified Rankin scale: implications for stroke clinical trials: a literature review and synthesis. Stroke. 2007;38(3):1091–6. 1Department of Neurology, Tianjin Huanhu Hospital, 6 Jizhao Road, Jinnan District, Tianjin 300350, China. 2Tianjin Key Laboratory of Cerebral Vascular and Neurodegenerative Disease, Tianjin 300350, China. 3Department of Epidemiology, Tianjin Neurological Institute, Tianjin 300052, China. 4 22. Petrea RE, Beiser AS, Seshadri S, Kelly-Hayes M, Kase CS, Wolf PA. Gender differences in stroke incidence and poststroke disability in the Framingham heart study. Stroke. 2009;40:1032–7. 4Department of Neurology, Tianjin Medical University General Hospital, Tianjin 300052, China. 23. Gall SL, Donnan G, Dewey HM, Macdonell R, Sturm J, Gilligan A, et al. Sex differences in presentation, severity, and management of stroke in a population-based study. Neurology. 2010;74:975–81. Tianjin 300052, China. Consent for publication Not applicable. 18. Fi M, Dw B. Functional evaluation: the Barthel index. Md State Med J. 1965; 14:61–5. Received: 18 June 2016 Accepted: 17 October 2016 24. Niewada M, Kobayashi A, Sandercock PA, Kamiński B, Członkowska A. International Stroke Trial Collaborative Group. Influence of gender on baseline features and clinical outcomes among 17,370 patients with confirmed ischaemic stroke in the international stroke trial. Neuroepidemiology. 2005;24:123–8. Ethics approval and consent to participate 19. de Haan R, Limburg M, Bossuyt P, van der Meulen J, Aaronson N. The clinical meaning of Rankin ‘handicap’ grades after stroke. Stroke. 1995;26:2027–30. The study was approved by the Ethics Committee for Medical Research at Tianjin Huanhu Hospital, and a written informed consent was obtained from each participant during recruitment. 20. Kim J-S, Lee K-B, Roh H, Ahn M-Y, Hwang H-W. Gender differences in the functional recovery after acute stroke. J Clin Neurol. 2010;6:183–8. Funding This study was funded by Tianjin Health Bureau of Science and Technology Fund Key Projects (contract: 13KG120 and 2015KG109). 12. von Budingen HC, Baumgartner RW, Baumann CR, Rousson V, Siegel AM, Georgiadis D. Serum cholesterol levels do not influence outcome or recovery in acute ischemic stroke. Neurol Res. 2008;30:82–4. Acknowledgements Acknowledgements We thank all the participants in this study. 10. Pan SL, Lien IN, Chen TH. Is higher serum total cholesterol level associated with better long-term functional outcomes after noncardioembolic ischemic stroke? Arch Phys Med Rehabil. 2010;91:913–8. We thank all the participants in this study. 11. Vauthey C, de Freitas GR, van Melle G, Devuyst G, Bogousslavsky J. Better outcome after stroke with higher serum cholesterol levels. Neurology. 2000; 54:1944–9. Availability of data and materials The datasets during and/or analyzed during the current study are available from the corresponding author on reasonable request. 13. Cui R, Iso H, Yamagishi K, Saito I, Kokubo Y, Inoue M, et al. High serum total cholesterol levels is a risk factor of ischemic stroke for general Japanese population: the JPHC study. Atherosclerosis. 2012;221:565–9. References Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet. 2012;380:2095–128. 2. Murray CJ, Vos T, Lozano R, Naghavi M, Flaxman AD, Michaud C, et al. Disability-adjusted life-years (DALYs) for 291 diseases and injuries in 21 regions, 1990–2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet. 2012;380:2197–223. 3. National Health and Family Planning Commission of the People’s Republic of China. China health and family planning statistics yearbook. Beijing: China Union Medical University Press; 2015. 4. Goldstein LB, Bushnell CD, Adams RJ, Appel LJ, Braun LT, Chaturvedi S, et al. Heart disease and stroke statistics—2010 update: a report from the American Heart Association. Circulation. 2010;121:e46–215. 5. Castelli WP, Anderson K, Wilson PW, Levy D. Lipids and coronary heart disease: the Framingham study. Ann Epidemiol. 1992;2:23–8. 6. Nam B-H, Kannel W, D’Agostino RB. Search for an optimal atherogenic lipid risk profile: from the Framingham study. Am J Cardiol. 2006;97:372–5. 7. Markaki I, Nilsson U, Kostulas K, SjÖstrand C. High cholesterol levels are associated with improved long-term survival after acute ischemic stroke. J Stroke Cerebrovasc. 2014;23:e47–53. 8. Tirschwell DL, Smith NL, Heckbert SR, Lemaitre RN, Longstreth Jr WT, Psaty BM. Association of cholesterol with stroke risk varies in stroke subtypes and patient subgroups. Neurology. 2004;63:1868–75. 9. Suzuki K, Izumi M, Sakamoto T, Hayashi M. Blood pressure and total cholesterol level are critical risks especially for hemorrhagic stroke in Akita, Japan. Cerebrovasc Dis. 2011;31:100–6. 26. Lai SM, Duncan PW, Dew P, Keighley J. Sex differences in stroke recovery. Prev Chronic Dis. 2005;2:A13. 2. Murray CJ, Vos T, Lozano R, Naghavi M, Flaxman AD, Michaud C, et al. Disability-adjusted life-years (DALYs) for 291 diseases and injuries in 21 regions, 1990–2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet. 2012;380:2197–223. 27. Barrett KM, Brott TG, Brown Jr RD, Frankel MR, Worrall BB, Silliman SL, et al. Sex differences in stroke severity, symptoms, and deficits after first-ever ischemic stroke. J Stroke Cerebrovasc Dis. 2007;16:34–9. 28. Di Carlo A, Lamassa M, Baldereschi M, Pracucci G, Basile AM, Wolfe CD, et al Sex differences in the clinical presentation, resource use, and 3-month outcome of acute stroke in Europe: data from a multicenter multinational hospital based registry. Stroke. 2003;34:1114–9. 3. National Health and Family Planning Commission of the People’s Republic of China. Authors’ contributions 14. Wang J, An Z, Li B, Yang L, Tu J, Gu H, et al. Increasing stroke incidence and prevalence of risk factors in a low-income Chinese population. Neurology. 2015;84(4):374–81. GZ was involved in the data collection, data interpretation, and manuscript drafting. ZA obtained the funding for this study and was involved in the conception and design, data interpretation, and critical review for this article. WZ, YH, and HX were involved in the data collection, case diagnosis, and confirmation for this article. XN and JW were involved in the data analysis, conception and design, data interpretation, and critical review for this article. All authors read and approved the final manuscript. 15. World Health Organization Task Force on Stroke and Other Cerebrovascular Disorders: Stroke–1989. Recommendations on stroke prevention, diagnosis, and therapy. Report of the WHO task force on stroke and other cerebrovascular disorders. Stroke. 1989;20(10):1407–31. 16. Adams HP, Bendixen BH, Kappelle LJ, Biller J, Love BB, Gordon DL, et al. Classification of subtype of acute ischemic stroke: definitions for use in a multicenter clinical trial: TOAST: Trial of Org 10172 in Acute Stroke Treatment. Stroke. 1993;24:35–41. Competing interests Competing interests The authors declare that they have no competing interests. 17. Bamford J, Sandercock P, Dennis M, Burn J, Warlow C. Classification and natural history of clinically identifiable subtypes of cerebral infarction. Lancet. 1991;337:1521–6. Conclusions This large, hospital-based, prospective study was the first to report sex differences in outcomes at 3 and 12 months after stroke among patients with athero- thrombotic infarction and low TC levels. Women were more likely than men to have POCI, severe stroke, hypertension, and obesity and were less likely to smoke or consume alcohol. There were no sex dif- ferences in stroke outcomes. Older age and severe stroke were common risk factors for poor outcomes after stroke in this study. DM was an independent predictor of low mortality at 12 months after stroke, which could be explained by metformin’s mediation of enhancing angiogenesis. Obesity was the determin- ant of recurrence and dependency rates at 12 months after stroke. These findings suggest that patients (both men and women) with atherothrombotic infarction and low TC levels would not benefit from receiving statin treatment. Therefore, it is crucial to explore the impact of statin treatment on outcomes in Asian pa- tients, especially Chinese atherothrombotic infarction patients, with low TC levels in order to improve out- comes after stroke and reduce the disease burden. Consistent with the results of previous studies, in the present study, higher recurrence and dependency rates at 12 months after stroke were observed for women than for men. The sex differences in recurrence and depend- ency rates became statistically non-significant after ad- justment for covariates. Moreover, older age and stroke severity were independent risk factors for stroke out- comes in this study. A low risk of mortality was ob- served in patients with DM at 12 months after stroke, but a positive association was found between obesity and recurrence and dependency rates at 12 months after stroke. The negative association between DM and mor- tality at 12 months after stroke could be explained by treating patients with metformin, which mediates en- hanced angiogenesis [49]. Statin therapy has become the most important ad- vancement in stroke prevention since aspirin and blood pressure-lowering therapies were introduced. Clinical trials have shown that lowering cholesterol levels can re- duce the incidence of stroke in high-risk populations and in patients with a stroke or transient ischemic attack [37, 50]. However, in this study, worse long-term Page 7 of 8 Page 7 of 8 Page 7 of 8 Zhou et al. Biology of Sex Differences (2016) 7:62 References 1. Rafael L, Mohsen N, Kyle F, Stephen L, Shibuya KenjiAboyans V, Abraham J, et al. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet. 2012;380:2095–128. 1. Rafael L, Mohsen N, Kyle F, Stephen L, Shibuya KenjiAboyans V, Abraham J, et al. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet. 2012;380:2095–128. 1. Rafael L, Mohsen N, Kyle F, Stephen L, Shibuya KenjiAboyans V, Abraham J, et al. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet. 2012;380:2095–128. 2. Murray CJ, Vos T, Lozano R, Naghavi M, Flaxman AD, Michaud C, et al. Disability-adjusted life-years (DALYs) for 291 diseases and injuries in 21 regions, 1990–2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet. 2012;380:2197–223. 3. National Health and Family Planning Commission of the People’s Republic of China. China health and family planning statistics yearbook. Beijing: China Union Medical University Press; 2015. 4. Goldstein LB, Bushnell CD, Adams RJ, Appel LJ, Braun LT, Chaturvedi S, et al. Heart disease and stroke statistics—2010 update: a report from the American Heart Association. Circulation. 2010;121:e46–215. 5. Castelli WP, Anderson K, Wilson PW, Levy D. Lipids and coronary heart disease: the Framingham study. Ann Epidemiol. 1992;2:23–8. 6. Nam B-H, Kannel W, D’Agostino RB. Search for an optimal atherogenic lipid risk profile: from the Framingham study. Am J Cardiol. 2006;97:372–5. 7. Markaki I, Nilsson U, Kostulas K, SjÖstrand C. High cholesterol levels are associated with improved long-term survival after acute ischemic stroke. J Stroke Cerebrovasc. 2014;23:e47–53. 8. Tirschwell DL, Smith NL, Heckbert SR, Lemaitre RN, Longstreth Jr WT, Psaty BM. Association of cholesterol with stroke risk varies in stroke subtypes and patient subgroups. Neurology. 2004;63:1868–75. 9. Suzuki K, Izumi M, Sakamoto T, Hayashi M. Blood pressure and total cholesterol level are critical risks especially for hemorrhagic stroke in Akita, Japan. Cerebrovasc Dis. 2011;31:100–6. 25. Roquer J, Campello AR, Gomis M. Sex differences in first-ever acute stroke. Stroke. 2003;34:1581–5. y p y j y et al. 9. Suzuki K, Izumi M, Sakamoto T, Hayashi M. Blood pressure and total cholesterol level are critical risks especially for hemorrhagic stroke in Akita, Japan. Cerebrovasc Dis. 2011;31:100–6. References Harmsen P, Lappas G, Rosengren A, Wilhelmsen L. Long-term risk factors for stroke. Twenty-eight years of follow-up of 7457 middle-aged men in Goteborg, Sweden. Stroke. 2006;37:1663–7. 37. Wattanakit K, Folsom AR, Chambless LE, Nieto FJ. Risk factors for cardiovascular event recurrence in the Atherosclerosis Risk in Communities (ARIC) study. Am Heart J. 2005;149:606–12. 37. Wattanakit K, Folsom AR, Chambless LE, Nieto FJ. Risk factors for cardiovascular event recurrence in the Atherosclerosis Risk in Communities (ARIC) study. Am Heart J. 2005;149:606–12. 38. Ohira T, Shahar E, Chambless LE, Rosamond WD, Mosley TH, Folsom AR. Risk factors for ischemic stroke subtypes: the atherosclerosis risk in communities study. Stroke. 2006;37:2493–8. 38. Ohira T, Shahar E, Chambless LE, Rosamond WD, Mosley TH, Folsom AR. Risk factors for ischemic stroke subtypes: the atherosclerosis risk in communities study. Stroke. 2006;37:2493–8. 39. Tanizaki Y, Kiyohara Y, Kato I, Iwamoto H, Nakayama K, Shinohara N, et al. Incidence and risk factors for subtypes of cerebral infarction in a general population: the Hisayama study. Stroke. 2000;31:2616–22. 40. Iso H, Jacobs Jr DR, Wentworth D, Neaton JD, Cohen JD. Serum cholesterol levels and six-year mortality from stroke in 350,977 men screened for the Multiple Risk Factor Intervention Trial. N Engl J Med. 1989;320:904–10. 41. Koton S, Molshatzki N, Bornstein NM, Tanne D. Low cholesterol, statins and outcomes in patients with first-ever acute ischemic stroke. Cerebrovasc Dis. 2012;34(3):213–20. 42. Dyker AG, Weir CJ, Lees KR. Influence of cholesterol on survival after stroke: retrospective study. BMJ. 1997;314:1584–8. 43. Zuliani G, Cherubini A, Atti AR, Ble´ A, Vavalle C, Todaro FD, et al. Low cholesterol levels are associated with short-term mortality in older patients with ischemic stroke. J Gerontol M Sci. 2004;59A:293–7. 44. Olsen TS, Christensen RH, Kammersgaard LP, Andersen KK. Higher total serum cholesterol levels are associated with less severe strokes and lower all-cause mortality: ten-year follow-up of ischemic strokes in the Copenhagen Stroke study. Stroke. 2007;38:2646–51. 45. Schatz IJ, Masaki K, Katsuhiko Y, Chen R, Rodrigues BL, Curb JD. Cholesterol and all-cause mortality in elderly people from the Honolulu Heart Program: a cohort study. Lancet. 2001;358:351–5. 46. Brescianini S, Maggi S, Farchi G, Mariotti S, Di Carlo A, Baldereschi M, et al. Low total cholesterol and increased risk of dying: are low levels clinical warning signs in the elderly? Results from the Italian Longitudinal Study on Aging. J Am Geriatr Soc. 2003;51:1991–6. 47. Zhou et al. Biology of Sex Differences (2016) 7:62 References China health and family planning statistics yearbook. Beijing: China Union Medical University Press; 2015. 4. Goldstein LB, Bushnell CD, Adams RJ, Appel LJ, Braun LT, Chaturvedi S, et al. Heart disease and stroke statistics—2010 update: a report from the American Heart Association. Circulation. 2010;121:e46–215. 29. Galassi A, Reynolds K, He J. Metabolic syndrome and risk of cardiovascular disease: a meta-analysis. Am J Med. 2006;119:812–9. 30. Mehndiratta P, Wasay M, Mehndiratta MM. Implications of female sex on stroke risk factors, care, outcome and rehabilitation: an Asian perspective. Cerebrovasc Dis. 2015;39:302–8. 31. Lindenstrom E, Boysen G, Nyboe J. Influence of total cholesterol, high density lipoprotein cholesterol, and triglycerides on risk of cerebrovascular disease: the Copenhagen City Heart study. Br Med J. 1994;309:11–5. 7. Markaki I, Nilsson U, Kostulas K, SjÖstrand C. High cholesterol levels are associated with improved long-term survival after acute ischemic stroke. J Stroke Cerebrovasc. 2014;23:e47–53. 32. Benfante R, Yano K, Hwang LJ, Curb JD, Kagan A, Ross W. Elevated serum cholesterol is a risk factor for both coronary heart disease and thromboembolic stroke in Hawaiian Japanese men. Stroke. 1994;25:814–20. 33. Horenstein RB, Smith DE, Mosca L. Cholesterol predicts stroke mortality in the Women’s Pooling Project. Stroke. 2002;33:1863–8. 32. Benfante R, Yano K, Hwang LJ, Curb JD, Kagan A, Ross W. Elevated serum cholesterol is a risk factor for both coronary heart disease and thromboembolic stroke in Hawaiian Japanese men. Stroke. 1994;25:814–20. 8. Tirschwell DL, Smith NL, Heckbert SR, Lemaitre RN, Longstreth Jr WT, Psaty BM. Association of cholesterol with stroke risk varies in stroke subtypes and patient subgroups. Neurology. 2004;63:1868–75. 33. Horenstein RB, Smith DE, Mosca L. Cholesterol predicts stroke mortality in the Women’s Pooling Project. Stroke. 2002;33:1863–8. 9. Suzuki K, Izumi M, Sakamoto T, Hayashi M. Blood pressure and total cholesterol level are critical risks especially for hemorrhagic stroke in Akita, Japan. Cerebrovasc Dis. 2011;31:100–6. 34. Kurth T, Everett BM, Buring JE, Kase CS, Ridker PM, Gaziano JM. Lipid levels and the risk of ischemic stroke in women. Neurology. 2007;68:556–62. Page 8 of 8 Page 8 of 8 Zhou et al. Biology of Sex Differences (2016) 7:62 35. Prospective Studies Collaboration, Lewington S, Whitlock G, Clarke R, Sherliker P, Emberson J. Blood cholesterol and vascular mortality by age, sex, and blood pressure: a meta-analysis of individual data from 61 prospective studies with 55000 vascular deaths. Lancet. 2007;370:1829–39. prospective studies with 55000 vascular deaths. Lancet. 2007;370:182 36. References Cha JK, Lim JH, Kim DH, Nah HW, Park HS, Choi JH, et al. Prognostic factors for long-term poor outcomes after acute ischemic stroke in very old age (>80 years) patients: total cholesterol level might differently influence long- term outcomes after acute ischemic stroke at ages above 80 years. Geriatr Gerontol Int. 2015;15(11):1227–33. 48. Zhao W, An Z, Hong Y, Zhou G, Guo J, Zhang Y, Yang Y, Ning X, Wang J. Low total cholesterol level is the independent predictor of poor outcomes in patients with acute ischemic stroke: a hospital-based prospective study. BMC Neurol. 2016;16:36. 49. Venna VR, Li J, Hammond MD, Mancini NS, McCullough LD. Chronic metformin treatment improves post-stroke angiogenesis and recovery after experimental stroke. Eur J Neurosci. 2014;39(12):2129–38. 50. Prospective studies collaboration. Cholesterol, diastolic blood pressure, and stroke: 13,000 strokes in 450,000 people in 45 prospective cohorts. Lancet. 1995;346:1647–53. 50. Prospective studies collaboration. Cholesterol, diastolic blood pressure, and stroke: 13,000 strokes in 450,000 people in 45 prospective cohorts. Lancet. 1995;346:1647–53. 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https://openalex.org/W343720152	http://masujournal.org/store_file/archive/99-1-3-26-33.pdf	English	null	Screening of Sunflower Genotypes for Drought Tolerance Based on Certain Morpho-Physiological Parameters	Madras Agricultural Journal	2,012	cc-by	6,431	Screening of Sunflower Genotypes for Drought Tolerance Based on Certain Morpho-Physiological Parameters A. Geetha, P. Saidaiah, A. Sivasankar, J. Suresh, Lakshmi Prayaga and G. Anuradha Department of Plant Physiology, College of Agriculture, Acharya N.G. Ranga Agricultural University, Rajendranagar, Hyderabad (A.P.) – 500 030, India. A. Geetha, P. Saidaiah, A. Sivasankar, J. Suresh, Lakshmi Prayaga and G. Anuradha Department of Plant Physiology, College of Agriculture, Acharya N.G. Ranga Agricultural University, Rajendranagar, Hyderabad (A.P.) – 500 030, India. A study was conducted to screen twenty nine sunflower genotypes for tolerance to drought under field conditions based on morpho - physiological characteristics. Moisture stress treatment was imposed at flower bud initiation stage (irrigation withheld for 20 days from 40 DAS to 60 DAS) where as, control plots were irrigated at 10 days intervals throughout the crop growth period. Results revealed that water stress showed repressing effect on plant height, total leaf area, SPAD chlorphyll meter reading, chlorophyll fluorescence (Fv/Fm), total dry matter weight at harvest, capitulum diameter per plant, capitulum weight per plant, seed yield per plant, drought susceptibility index and harvest index. However, genotypic variation was significant for characters studied. Based on drought susceptibility index and various morpho- physiological traits, eight genotypes viz., TSF-103, RSF-107, TSF-106, ASF-104, DSF-104, SH- 491, RSF-106 and DSF-111 were selected as promising lines under water limited situation. These lines may further be used in stress physiology studies and drought resistance breeding. Key words: Drought, Genotypes, Morphological, Physiological traits, Screening, Sunflower. treatment irrigation withheld from 40 DAS to 60 DAS. This period of drought was imposed, when crop was at flower bud initiation stage. The treatments comprised of 29 lines. Each genotype was sown in two rows at 5 m length with spacing of 60 x 30 cm. Two to three seeds were sown per hill to achieve uniform stand. Thinning was done at two weeks after sowing to retain one seedling per hill. Recommended package of practices (seed rate, weeding,fertilizer dosage-30 kg N, 60 kg P2O5, 30 kg K2O per hectare. Fifty percent nitrogen and entire doses of phosphorous and potassium was applied at the time of planting as band placement at the side of seed rows. The remaining 50% N was applied as top dressing in two equal splits, first at 35 days after planting and second dose at fortnight later of first dose and need based plant protection measures) of crop were followed to raise a healthy crop. Screening of Sunflower Genotypes for Drought Tolerance Based on Certain Morpho-Physiological Parameters The data were recorded on plant height, total leaf area, SPAD chlorophyll meter reading, chlorophyll fluorescence (Fv/Fm), total dry matter weight at harvest, capitulum diameter per plant, capitulum weight per plant, seed yield per plant, drought susceptibility index and harvest index at five days after imposition of stress and fifteen days after release of stress whereas, yield and yield related parameters were recorded. Plant height (cm) was measured from base of the plant to the terminal bud of the plant. Total leaf area was estimated by Sunflower has the maximum potential for bridging the edible oil gap in India as its seed contain high oil contents ranging from 35 to 40 per cent. Physiological changes in plants, which occur in response to water stress conditions decrease photosynthesis and respiration (Human et al., 1990) and as a result, overall production of crop is decreased. Although, sunflower has good potential for drought tolerance because of its well developed system, decrease in plant height, 100-seed weight, head diameter and seed yield per plant under water stress conditions has been observed (Ravishankar et al., 1991).The objective of the present study was to investigate the effect of water stress on morpho-physiological traits in sunflower genotypes. The information collected will be useful in planning the future breeding strategies for the improvement of sunflower cultivars for drought resistance. Madras Agric. J., 99 (1-3): 26-33, March 2012 https://doi.org/10.29321/MAJ.10.100008 Madras Agric. J., 99 (1-3): 26-33, March 2012 https://doi.org/10.29321/MAJ.10.100008 Corresponding author email: geethagri_100@yahoo.co.in Materials and Methods The experiment was laid out in factorial Randomized Block Design with two factors and 29 treatments which were replicated thrice during rabi, 2009-10 at College Farm, College of Agriculture, ANGRAU, Rajendranagar, Hyderabad. Control (irrigated) and water stress were used as factors. Control plots were irrigated at 10 days intervals throughout the crop growth period whereas, in stress Corresponding author email: geethagri_100@yahoo.co.in 27 measuring length and width of top, middle and bottom leaves using the formula. Table 1. Mean of plant height (cm) of sunflower genotypes during stress and after stress as influenced by moisture stress genotypes during stress and after stress as influenced by moisture stress Genotype Five days after Fifteen days after imposition of release of stress stress Control Stress Mean Control Stress Mean RSF-101 54.33 52.67 53.50 102.00 76.00 89.00 TSF-103 71.00 55.67 63.33 167.00 121.67 144.33 ASF-107 78.00 76.67 77.33 132.33 112.67 122.50 DSF-114 74.67 65.67 70.17 150.00 113.33 131.67 SH-177 73.00 70.33 66.67 130.00 107.67 118.83 DSF-104 84.83 82.43 83.63 162.00 132.00 147.00 RSF-106 49.33 45.00 47.17 138.00 119.33 128.67 DSF-111 82.00 70.10 76.05 187.33 140.00 163.67 RSF-107 71.00 69.00 67.50 177.67 140.00 158.83 ASF-104 60.33 53.20 56.77 167.67 130.33 149.00 TSF-106 64.17 49.70 56.93 162.67 113.67 138.17 SH-491 50.50 37.33 43.92 162.67 112.00 137.33 M-1029 77.00 46.67 61.83 114.67 79.67 97.17 GP-812-5 45.67 35.67 40.67 131.67 97.67 114.67 GP-247-4 100.33 83.00 91.67 135.33 108.67 122.00 GP4-2605 86.67 59.67 73.17 99.67 65.33 82.50 GP-69 60.33 41.83 51.08 95.67 48.00 71.83 GP4-2935 102.67 67.13 84.90 180.33 126.33 153.33 GP-978 97.00 69.40 83.20 113.33 94.00 103.67 DK-3849 72.67 56.77 64.72 126.33 98.33 112.33 GP9-515-7-3 115.33 85.00 100.17 137.33 98.67 118.00 GP4-2885 55.00 45.33 50.17 82.00 63.00 72.50 RHA-274 82.33 64.00 73.17 114.67 96.00 105.33 GP4-187 87.33 64.10 75.72 131.67 107.00 119.33 GP-2793 73.33 60.83 67.08 134.00 114.67 124.33 GP4-2704 59.33 54.73 57.03 118.67 95.33 107.00 EC-512690 53.00 20.00 36.50 117.00 80.33 98.67 GP9-846-4-4 76.67 60.00 68.33 137.33 99.00 118.17 GP9-38-C-2-1 87.33 60.40 73.87 149.00 107.33 128.17 Mean 73.49 58.67 66.08 136.48 103.38 119.93 CD at 5% 1.06 0.85 for treatments CD at 5% 9.03 7.25 for genotypes CD at 5% for 10.70 8.60 T x G Leaf area (cm2) = Length (cm) x Width(cm) x 0.90 . Leaf area (cm2) = Length (cm) x Width(cm) x 0.90 . Chlorophyll concentration was assessed using a chlorophyll meter (SPAD-502, Minolta, Japan). Materials and Methods Measurements were taken at three points of each leaf (upper, middle and lower part). Average of these three readings was considered as SPAD reading of the leaf. The optimal and effective quantum yields of PSII were measured using the fluorometer OS-500 (Opti- Science, USA). Total dry matter accumulation (g m-2) of harvested plants were separated into stem, leaf, petiole and capitulum and kept in brown paper bags and dried to a constant weight in hot air oven at 80æ%C for 48 hours. Each component of the plant was weighed in gram. Capitulum diameter (cm) of the mature head at its maximum width was measured and its dry weight was taken to get single capitulum weight (g). Seed yield per plant (g) was determined after threshing the seeds and allowing it to dry up to 9-10% moisture content. Weight of total seeds of the ten heads is measured in each treatment, averaged and expressed in gram (g). Drought susceptibility index (S) was calculated according to Fischer and Maurer (1978). S= (1-Y/YP) / (1-X d/Xp) S= (1-Y/YP) / (1-X d/Xp) Where, Y is the achenes yield per head of a given genotype under drought, YP is the achenes yield per head of he same genotype under irrigation, Xd is the mean achenes yield of all the genotypes within group (inbred or parent) under drought, Xp is the achenes yield per head of all genotypes within group under irrigation. Harvest index was estimated as the proportion of total dry matter production Partitioned to economic parts expressed in (%) These results are in accordance with observations of several researchers who reported reduction in plant height under stress condition (Nezami et al., 2008 and Shao et al., 2008). Drought stress has led to reduction in stem cell’s water potential to a lower level needed for cell elongation and consequently shorter internodes and stem height (Nezami et al., 2008). The reduction in plant height was associated with a decline in the cell enlargement and more leaf senescence in A. esculentus under water stress (Bhatt and Srinivasa Rao, 2005). Harvest Economical yield per plant index = x 100 (%) Biological yield per plant Harvest Economical yield per plant index = x 100 (%) Biological yield per plant Results and Discussion Plant height was reduced when drought was imposed at flower bud initiation stage. The percent reduction in plant height was more during fifteen days after stress recovery when compared to five days after imposition of stress (Table1). Differences among genotypes were significant at 15 days after stress recovery. Genotypes DSF-111 and GP4-2935 under control condition and DSF-111 and RSF-107 under stressful condition, were at par and significantly superior over other genotypes. However, the interaction data revealed that genotype DSF-111 recorded maximum height followed by RSF -107. Total leaf area was significantly affected by stress treatment imposed at flower bud initiation stage. Higher percent reduction was resulted at fifteen days after release of stress (32.4 %) when compared to 5 days after imposition of stress (31.7%) (Table 2). At 15 days after release of stress GP9-515-7-3 under control and GP9-515-7-3 and GP4-2704 in stress treatment exhibited higher total leaf area over other genotypes. Maximum and minimum values of total 28 Table 2. Results and Discussion Mean of total leaf area (cm2 plant-1) of sunflower genotypes as influenced by moisture stress Genotype Five days after imposition of stress Fifteen days after release of stress Control Stress Mean % decrease Control Stress Mean % decrease RSF-101 4188 3060 3624 27 5314 4670 4992 14 TSF-103 4344 3279 3812 25 8036 4294 6165 87 ASF-107 5401 4324 4862 20 7178 6392 6785 12 DSF-114 4271 3393 3832 21 7471 5488 6480 36 SH-177 4410 2669 3540 39 1095 1001 1048 9 DSF-104 4439 2925 3682 34 7012 6240 6626 12 RSF-106 3184 2557 2870 20 6251 5659 5955 10 DSF-111 5755 3944 4850 31 7310 4780 6045 53 RSF-107 6615 4321 5468 35 11973 9047 10510 32 ASF-104 8493 5203 6848 39 9775 8453 9114 16 TSF-106 8650 3179 5915 63 9151 4768 6959 92 SH-491 4003 2185 3094 45 9172 5771 7472 59 M-1029 9248 8019 8634 13 10991 10031 10511 10 GP-812-5 2011 1756 1884 13 7102 6303 6703 13 GP-247-4 6252 3110 4681 50 7772 4985 6379 56 GP4-2605 5751 4536 5143 21 7191 6756 6974 6 GP-69 3827 2076 2952 46 7898 4208 6053 88 GP4-2935 9267 3167 6217 66 10887 8525 9706 28 GP-978 5687 3935 4811 31 6684 6123 6403 9 DK-3849 8133 4719 6426 42 12549 5525 9037 127 GP9-515-7-3 9862 9354 9608 5 18905 12822 15864 47 GP4-2885 5834 2877 4356 51 8519 4831 6675 76 RHA-274 4798 4451 4625 7 6950 5941 6446 17 GP4-187 4736 4302 4519 9 8584 7569 8076 13 GP-2793 4595 4232 4414 8 11196 10155 10676 10 GP4-2704 7776 6499 7138 16 12892 12217 12555 6 EC-512690 5930 3905 4918 34 11106 7334 9220 51 GP9-846-4-4 7494 4727 6111 37 9251 7800 8525 19 GP9-38-C-2-1 4975 3399 4187 32 5633 4222 4927 33 Mean 5860 4004 4932 32 8753 6618 7685 32 CD at 5% for treatments 387 249 CD at 5% for genotypes 1475 950 CD at 5% for T x G 2085 1343 leaf area were recorded in GP9-515-7-3 and SH- 177 respectively in interactions. Wullschleger et al.( 2005), Farooq et al.( 2009) and Manivannan et al. (2007 and 2008) concluded that water stress reduces the leaf area by limiting size of individual leaf, prevents the leaf growth and leaf cell expansion due to reduction in turgour pressure and accelerates leaf senescence process in sunflower. Results and Discussion SPAD chlorophyll meter readings declined in stress treatment when stress was imposed at flower bud initiation stage. Water stress at stress imposition period decreased SPAD value from 4.7% to 0.3% to stress recovery period compared with respective controls (Table 3). At stress recovery period, GP4- 2885 under control and RHA-274 under both stress and interactions recorded significantly more SPAD meter reading and GP-247-4 recorded less SPAD meter values. Sawhney and Singh (2002) found that chlorophyll content of flag leaf in several wheat genotypes was reduced towards the end of growing season. SPAD chlorophyll meter reading, a reflection of leaf chlorophyll/leaf nitrogen declined in stress treatment of present investigation due to degradation of leaf chlorophyll content. Maximum quantum efficiency of PS-II (Fv/Fm) was found reduced under drought condition. Reduction in Fv/Fm by stress at 45 DAS was 6.7 per cent in comparison with control (Table 4). In general, fluorescence value declined at recovery period (7.5%) compared to stress imposition period. At stress release period (75 DAS), ASF-107, DSF-114 and SH- 177 followed by TSF-103 and GP4-2885 under control recorded higher fluorescence over most of the other genotypes, whereas under stress Table 2. Results and Discussion Mean of total leaf area (cm2 plant-1) of sunflower genotypes as influenced by moisture stress Genotype Five days after imposition of stress Fifteen days after release of stress Control Stress Mean % decrease Control Stress Mean % decrease RSF-101 4188 3060 3624 27 5314 4670 4992 14 TSF-103 4344 3279 3812 25 8036 4294 6165 87 ASF-107 5401 4324 4862 20 7178 6392 6785 12 DSF-114 4271 3393 3832 21 7471 5488 6480 36 SH-177 4410 2669 3540 39 1095 1001 1048 9 DSF-104 4439 2925 3682 34 7012 6240 6626 12 RSF-106 3184 2557 2870 20 6251 5659 5955 10 DSF-111 5755 3944 4850 31 7310 4780 6045 53 RSF-107 6615 4321 5468 35 11973 9047 10510 32 ASF-104 8493 5203 6848 39 9775 8453 9114 16 TSF-106 8650 3179 5915 63 9151 4768 6959 92 SH-491 4003 2185 3094 45 9172 5771 7472 59 M-1029 9248 8019 8634 13 10991 10031 10511 10 GP-812-5 2011 1756 1884 13 7102 6303 6703 13 GP-247-4 6252 3110 4681 50 7772 4985 6379 56 GP4-2605 5751 4536 5143 21 7191 6756 6974 6 GP-69 3827 2076 2952 46 7898 4208 6053 88 GP4-2935 9267 3167 6217 66 10887 8525 9706 28 GP-978 5687 3935 4811 31 6684 6123 6403 9 DK-3849 8133 4719 6426 42 12549 5525 9037 127 GP9-515-7-3 9862 9354 9608 5 18905 12822 15864 47 GP4-2885 5834 2877 4356 51 8519 4831 6675 76 RHA-274 4798 4451 4625 7 6950 5941 6446 17 GP4-187 4736 4302 4519 9 8584 7569 8076 13 GP-2793 4595 4232 4414 8 11196 10155 10676 10 GP4-2704 7776 6499 7138 16 12892 12217 12555 6 EC-512690 5930 3905 4918 34 11106 7334 9220 51 GP9-846-4-4 7494 4727 6111 37 9251 7800 8525 19 GP9-38-C-2-1 4975 3399 4187 32 5633 4222 4927 33 Mean 5860 4004 4932 32 8753 6618 7685 32 CD at 5% for treatments 387 249 CD at 5% for genotypes 1475 950 CD at 5% for T x G 2085 1343 l SPAD t l S h d Si h (2002) Mean of total leaf area (cm2 plant-1) of sunflower genotypes as influenced by moisture stress less SPAD meter values. Sawhney and Singh (2002) found that chlorophyll content of flag leaf in several wheat genotypes was reduced towards the end of growing season. Results and Discussion SPAD chlorophyll meter reading, a reflection of leaf chlorophyll/leaf nitrogen declined in stress treatment of present investigation due to degradation of leaf chlorophyll content. leaf area were recorded in GP9-515-7-3 and SH- 177 respectively in interactions. Wullschleger et al.( 2005), Farooq et al.( 2009) and Manivannan et al. (2007 and 2008) concluded that water stress reduces the leaf area by limiting size of individual leaf, prevents the leaf growth and leaf cell expansion due to reduction in turgour pressure and accelerates leaf senescence process in sunflower. leaf area were recorded in GP9-515-7-3 and SH- 177 respectively in interactions. Wullschleger et al.( 2005), Farooq et al.( 2009) and Manivannan et al. (2007 and 2008) concluded that water stress reduces the leaf area by limiting size of individual leaf, prevents the leaf growth and leaf cell expansion due to reduction in turgour pressure and accelerates leaf senescence process in sunflower. Maximum quantum efficiency of PS-II (Fv/Fm) was found reduced under drought condition. Reduction in Fv/Fm by stress at 45 DAS was 6.7 per cent in comparison with control (Table 4). In general, fluorescence value declined at recovery period (7.5%) compared to stress imposition period. At stress release period (75 DAS), ASF-107, DSF-114 and SH- 177 followed by TSF-103 and GP4-2885 under control recorded higher fluorescence over most of the other genotypes, whereas under stress SPAD chlorophyll meter readings declined in stress treatment when stress was imposed at flower bud initiation stage. Water stress at stress imposition period decreased SPAD value from 4.7% to 0.3% to stress recovery period compared with respective controls (Table 3). At stress recovery period, GP4- 2885 under control and RHA-274 under both stress and interactions recorded significantly more SPAD meter reading and GP-247-4 recorded 29 Table 3. Results and Discussion Mean of SPAD chlorophyll meter reading of sunflower genotypes as influenced by moisture stress Genotype Five days after imposition of stress Fifteen days after release of stress Control Stress Mean % decrease Control Stress Mean % decrease RSF-101 40.07 32.23 36.15 19.55 30.83 27.17 29.00 11.89 TSF-103 39.07 40.43 39.75 -3.50 32.40 35.13 33.77 -8.44 ASF-107 41.93 40.03 40.98 4.53 32.50 30.60 31.55 5.85 DSF-114 40.97 39.27 40.12 4.15 37.57 37.44 37.51 0.33 SH-177 40.30 36.00 38.15 10.67 39.60 36.90 38.25 6.82 DSF-104 44.60 41.33 42.97 7.32 35.60 33.00 34.30 7.30 RSF-106 40.33 36.13 38.23 10.41 34.77 34.00 34.38 2.21 DSF-111 43.00 38.73 40.87 9.92 31.67 31.67 31.67 0.00 RSF-107 38.80 39.13 38.97 -0.86 34.53 34.13 34.33 1.16 ASF-104 40.00 36.40 38.20 9.00 32.90 32.00 32.45 2.74 TSF-106 39.77 36.30 38.03 8.72 32.00 32.00 32.00 0.00 SH-491 44.57 38.93 41.75 12.64 33.03 32.94 32.99 0.28 M-1029 34.63 30.07 32.35 13.19 29.37 29.33 29.35 0.11 GP-812-5 42.33 39.57 40.95 6.54 37.43 35.10 36.27 6.23 GP-247-4 42.40 38.30 40.35 9.67 27.00 26.73 26.87 0.99 GP4-2605 39.57 39.43 39.50 0.34 36.53 34.83 35.68 4.65 GP-69 44.57 39.00 41.78 12.49 37.60 37.33 37.47 0.71 GP4-2935 41.97 39.83 40.90 5.08 39.27 37.27 38.27 5.09 GP-978 40.53 40.97 40.75 -1.07 29.00 29.67 29.33 -2.30 DK-3849 40.10 40.47 40.28 -0.91 38.33 40.00 39.17 -4.35 GP9-515-7-3 38.13 40.17 39.15 -5.33 33.27 34.53 33.90 -3.81 GP4-2885 40.97 37.70 39.33 7.97 45.43 37.67 41.55 17.09 RHA-274 40.27 42.87 41.57 -6.46 39.63 45.33 42.48 -14.38 GP4-187 40.33 40.80 40.57 -1.16 40.33 43.33 41.83 -7.44 GP-2793 37.00 36.37 36.68 1.71 39.40 39.00 39.20 1.02 GP4-2704 37.37 32.30 34.83 13.56 37.10 34.13 35.62 8.00 EC-512690 38.43 41.00 39.72 -6.68 23.33 34.87 29.10 -49.43 GP9-846-4-4 43.40 43.00 43.20 0.92 30.80 27.60 29.20 10.39 GP9-38-C-2-1 43.07 46.23 44.65 -7.35 32.57 36.93 34.75 -13.41 Mean 40.64 38.72 39.68 4.71 34.61 34.51 34.56 0.31 CD at 5% for treatments 0.23 NS CD at 5% for genotypes 0.89 1.03 CD at 5% for T x G 1.26 1.45 Table 3. Mean of SPAD chlorophyll meter reading of sunflower genotypes as influenced by moistu stress Mean of SPAD chlorophyll meter reading of sunflower genotypes as influenced by moisture Greater plant fresh and dry weights under water limited conditions are desirable characters.A common adverse effect of water stress on crop plants is the reduction in fresh and dry biomass production (Farooq et al., 2009). Results and Discussion Diminished biomass due to water stress was observed in almost all genotypes of sunflower (Tahir and Mehid, 2001). However, some genotypes showed better stress tolerance than the others. Drought induced at flower bud initiation stage cause significant reduction in dry weights. The percent reduction in dry weight in stress treatment was 21.9 per cent compared to its control (Table 5). M-1029 exhibited highest total dry weights in control, stress and interaction of genotype with treatments at harvest and lowest dry weight was reported by DSF-114 in treatments as well as in interaction. condition, DSF-114 and GP4-187 exhibited significant and superior fluorescence value. While in mean effect, DSF-114 recorded maximum Fv/Fm value followed by condition, DSF-114 and GP4-187 exhibited significant and superior fluorescence value. While in mean effect, DSF-114 recorded maximum Fv/Fm value followed by GP4-187 and SH-177. The genotypes with high values of Fv/Fm are associated with the resistance of the photosynthetic processes to water deficit (Pankoviæ et al., 1999), whereas genotypes with low value of Fv/Fm under drought stress decreases the flux of electron flow out of photo system–II , which consequently lowers the rates of ATP and NADPH2 formation and in turn leads to slower enzymatic conversion of CO2 into organic carbon, thereby yield (Reddy et al., 2004). Lower fluorescence is either due to a smaller antenna cross-section or to a process increasing the non-radioactive energy dissipation (Konstantina et al., 2004). 30 Table 4. Results and Discussion Mean of chlorophyll fluorescence (Fv/Fm) of sunflower genotypes as influenced by moisture stress Genotype Five days after imposition of stress Fifteen days after release of stress Control Stress Mean % decrease Control Stress Mean % decrease RSF-101 0.76 0.71 0.73 6.17 0.63 0.51 0.57 18.52 TSF-103 0.72 0.67 0.70 6.05 0.64 0.56 0.60 12.04 ASF-107 0.72 0.66 0.69 8.37 0.65 0.59 0.62 10.16 DSF-114 0.75 0.70 0.73 6.67 0.65 0.63 0.64 3.08 SH-177 0.74 0.72 0.73 1.81 0.65 0.61 0.63 6.19 DSF-104 0.75 0.63 0.69 15.18 0.58 0.51 0.55 12.00 RSF-106 0.74 0.68 0.71 7.24 0.61 0.57 0.59 6.52 DSF-111 0.72 0.67 0.70 6.05 0.53 0.51 0.52 4.38 RSF-107 0.68 0.65 0.67 3.45 0.50 0.47 0.49 6.62 ASF-104 0.70 0.67 0.69 3.35 0.61 0.57 0.59 7.61 TSF-106 0.66 0.66 0.66 -1.02 0.57 0.58 0.58 -0.58 SH-491 0.60 0.56 0.58 6.70 0.56 0.54 0.55 3.59 M-1029 0.67 0.67 0.67 0.50 0.57 0.54 0.55 5.29 GP-812-5 0.65 0.64 0.65 2.04 0.58 0.53 0.56 9.14 GP-247-4 0.71 0.65 0.68 7.98 0.63 0.52 0.57 17.99 GP4-2605 0.61 0.58 0.59 4.40 0.59 0.54 0.56 9.04 GP-69 0.72 0.69 0.71 3.26 0.53 0.50 0.52 5.63 GP4-2935 0.69 0.70 0.70 -1.44 0.62 0.63 0.62 -1.08 GP-978 0.72 0.68 0.70 4.65 0.56 0.53 0.55 5.92 DK-3849 0.76 0.67 0.71 11.89 0.60 0.51 0.55 15.56 GP9-515-7-3 0.76 0.66 0.71 13.22 0.60 0.54 0.57 11.05 GP4-2885 0.65 0.62 0.63 4.64 0.64 0.50 0.57 22.28 RHA-274 0.66 0.63 0.65 4.04 0.59 0.55 0.57 6.25 GP4-187 0.68 0.66 0.67 2.94 0.65 0.62 0.64 4.62 GP-2793 0.66 0.58 0.62 12.12 0.55 0.52 0.54 5.45 GP4-2704 0.73 0.61 0.67 17.27 0.58 0.53 0.55 8.09 EC-512690 0.72 0.70 0.71 3.69 0.61 0.58 0.59 5.46 GP9-846-4-4 0.70 0.65 0.68 6.22 0.56 0.54 0.55 4.73 GP9-38-C-2-1 0.68 0.60 0.64 11.82 0.58 0.53 0.56 7.51 Mean 0.70 0.65 0.68 6.28 0.59 0.55 0.57 8.16 CD at 5% for treatments 0.003 0.004 CD at 5% for genotypes 0.011 0.013 CD at 5% for T x G 0.016 0.019 diameter may be due to reduction in LAI and insufficient photo assimilates required for development of head. Similar results are found by researchers in several crops including soybean (Specht et al., 2001), Poncirus trifoliatae seedlings (Wu et al., 2008), common bean and green gram (Webber et al., 2006) and Petroselinum crispum (Petropoulos et al., 2008). Results and Discussion Capitulum diameter was highly reduced when drought was imposed at flower bud initiation stage when compared to non-stress. Stress recorded 32.2 per cent reduction in capitulum diameter. At harvest stage, SH-491 followed by DSF-111 and RSF-107 under control condition and RSF- Similar results are found by researchers in several crops including soybean (Specht et al., 2001), Poncirus trifoliatae seedlings (Wu et al., 2008), common bean and green gram (Webber et al., 2006) and Petroselinum crispum (Petropoulos et al., 2008). Capitulum diameter was highly reduced when drought was imposed at flower bud initiation stage when compared to non-stress. Stress recorded 32.2 per cent reduction in capitulum diameter. At harvest stage, SH-491 followed by DSF-111 and RSF-107 under control condition and RSF- 107 and TSF-103 under stress condition showed higher capitulum diameter. In combined effect, RSF- 107 recorded highest capitulum diameter followed by DSF -111, SH-491, while ASF-104 recorded lowest capitulum diameter. The reduction of capitulum Capitulum weight was highly reduced when drought was imposed at flower bud initiation stage. Maximum capitulum weight was recorded in control (74.78g) and was significantly superior to stress treatment (52.72 g) (Table 6). Genotype DK-3849 and SH-491 exhibited more capitulum weight in non stress, whereas SH-491 recorded highest capitulm weight both in stress and interaction and were superior over other genotypes. While DSF- 114 recorded lowest capitulum weight in both the treatments and mean effect. Poor photosynthetic performance and reduction in assimilatory structure 31 Table 5. Mean of total dry matter weight (g plant-1) per plant at harvest and capitulum diameter (cm) per plant of sunflower genotypes as influenced by moisture stress Table 5. Results and Discussion Mean of total dry matter weight (g plant-1) per plant at harvest and capitulum diameter (cm) per plant of sunflower genotypes as influenced by moisture stress per plant of sunflower genotypes as influenced by moisture stress Genotype Total dry matter weight (g plant-1) per plant at harvest Capitulum diameter (cm) per plant Control Stress Mean % decrease Control Stress Mean % decrease RSF-101 203.73 170.00 186.86 16.55 15.33 11.00 13.17 28.26 TSF-103 308.32 234.67 271.49 23.89 28.00 19.63 23.82 29.88 ASF-107 242.76 198.30 220.53 18.31 26.33 19.33 22.83 26.58 DSF-114 144.05 114.78 129.42 20.32 10.67 6.17 8.42 42.19 SH-177 348.19 292.00 320.10 16.14 26.67 11.73 19.20 56.00 DSF-104 207.89 187.07 197.48 10.02 19.33 14.00 16.67 27.59 RSF-106 159.00 123.53 141.26 22.31 13.67 8.67 11.17 36.59 DSF-111 359.23 299.17 329.20 16.72 30.67 19.33 25.00 36.96 RSF-107 319.40 252.67 286.03 20.89 30.00 21.27 25.63 29.11 ASF-104 251.67 215.25 233.46 14.47 9.67 5.93 7.80 38.62 TSF-106 291.95 257.33 274.64 11.86 21.67 19.33 20.50 10.77 SH-491 484.50 346.87 415.69 28.41 31.67 18.00 24.83 43.16 M-1029 544.77 414.00 479.38 24.00 30.67 15.17 22.92 50.54 GP-812-5 233.67 187.67 210.67 19.69 14.17 10.67 12.42 24.71 GP-247-4 184.63 156.83 170.73 15.06 20.97 15.60 18.28 25.60 GP4-2605 214.92 187.33 201.13 12.83 11.33 9.10 10.22 19.71 GP-69 293.56 255.00 274.28 13.13 14.70 11.90 13.30 19.05 GP4-2935 368.72 324.17 346.44 12.08 25.67 16.33 21.00 36.36 GP-978 224.88 171.67 198.27 23.66 14.93 9.63 12.28 35.49 DK-3849 504.62 319.50 412.06 36.68 28.83 13.33 21.08 53.76 GP9-515-7-3 306.17 240.00 273.08 21.61 23.67 18.10 20.88 23.52 GP4-2885 177.10 147.17 162.14 16.90 13.57 11.03 12.30 18.67 RHA-274 183.35 146.96 165.16 19.85 10.93 7.97 9.45 27.13 GP4-187 224.06 183.17 203.61 18.25 13.50 11.17 12.33 17.28 GP-2793 293.23 230.67 261.95 21.33 16.20 13.13 14.67 18.93 GP4-2704 221.48 196.00 208.74 11.51 15.33 11.13 13.23 27.39 EC-512690 279.09 249.64 264.36 10.55 18.67 12.89 15.78 30.93 GP9-846-4-4 249.00 189.67 219.33 23.83 16.43 13.37 14.90 18.66 GP9-38-C-2-1 241.33 188.33 214.83 21.96 13.90 9.67 11.78 30.46 Mean 279.28 220.11 249.70 21.19 19.56 13.26 16.41 32.19 CD at 5% for treatments 2.96 0.47 CD at 5% for genotypes 11.26 1.80 CD at 5% for T x G 15.92 2.55 sink size (mainly number of seeds) and seed weight. It may be related with decreased photosynthetic efficiency by degradation of chlorophyll, lower production and translocation of organic material from source to sink (Amrutha et al., 2007). Results and Discussion leads to carbohydrates and mineral deficiency which cause abortions of ovaries, pollen sterility leading to production of less achenes results in reduction in capitulum weight (Rauf and Sadaqat, 2007). Seed yield per plant was reduced when stress was imposed at flower bud initiation stage. The percent reduction in seed yield during stress was 27.8 compared to control (Table 6). Significant variation was noticed among the genotypes studied with respect to seed yield. SH-491 followed by DK-3849 under control and SH-491 under stress recorded significantly superior seed yield in comparison to rest of the genotypes. However, genotype x treatments data revealed that SH-491 recorded highest seed yield and significantly superior over the rest of the genotypes. The decrease in yield under stress might be due to decreased There were significant differences among the genotypes in DSI values. Genotype GP9-38-C-2-1 recorded lowest (0.20) drought susceptible index (Table 7). A higher value of susceptibility index indicates higher susceptibility of a genotype to the stress. Results and Discussion Higher drought susceptibility index of some genotype under water stress situations is due to degradation of membrane system, poor photosynthetic performance, failure to produce anti oxidants defense mechanism, inability to maintain water potential or lack of production of osmolytes, poor translocation of assimilates to developing 32 Table 6.Mean of capitulum weight (g) per plant and seed yield (g) per plant of sunflower genotypes as influenced by moisture stress Table 6.Mean of capitulum weight (g) per plant and seed yield (g) per plant of sunflower genotypes as influenced by moisture stress Table 6.Mean of capitulum weight (g) per plant and seed yield (g) per plant of sunflower genotypes as influenced by moisture stress Genotype Capitulum weight (g) per plant Seed yield (g) per plant Control Stress Mean % decrease Control Stress Mean % decrease RSF-101 48.67 39.00 43.83 19.86 25.17 14.98 20.08 40.46 TSF-103 83.00 59.17 71.09 28.71 44.92 40.06 42.49 10.82 ASF-107 81.89 40.67 61.28 50.34 40.00 23.00 31.50 42.50 DSF-114 38.67 19.00 28.83 50.86 11.43 8.37 9.90 26.78 SH-177 101.70 83.33 92.52 18.06 66.53 32.73 49.63 50.80 DSF-104 60.55 50.07 55.31 17.31 24.60 20.36 22.48 17.24 RSF-106 44.53 32.63 38.58 26.74 19.27 15.30 17.28 20.59 DSF-111 105.00 84.67 94.83 19.37 87.17 66.97 77.07 23.17 RSF-107 105.41 82.00 93.71 22.21 67.67 59.67 63.67 11.82 ASF-104 41.74 31.41 36.57 24.75 19.27 15.60 17.43 19.03 TSF-106 73.00 62.33 67.67 14.61 38.00 32.00 35.00 15.79 SH-491 131.33 85.67 108.50 34.77 102.00 81.03 91.52 20.56 M-1029 120.38 71.00 95.69 41.02 95.17 51.00 73.08 46.41 GP-812-5 56.67 40.33 48.50 28.82 35.42 15.33 25.38 56.71 GP-247-4 60.64 41.67 51.16 31.29 32.95 23.58 28.26 28.45 GP4-2605 61.58 52.33 56.96 15.02 25.66 23.45 24.55 8.60 GP-69 51.33 41.33 46.33 19.48 54.67 48.49 51.58 11.29 GP4-2935 91.26 68.00 79.63 25.48 66.20 40.20 53.20 39.27 GP-978 69.68 52.00 60.84 25.38 54.33 49.33 51.83 9.20 DK-3849 133.63 74.00 103.82 44.62 100.25 48.78 74.51 51.34 GP9-515-7-3 91.33 69.67 80.50 23.72 47.33 21.33 34.33 54.93 GP4-2885 53.51 38.67 46.09 27.74 23.65 14.33 18.99 39.39 RHA-274 41.33 31.67 36.50 23.39 9.93 8.47 9.20 14.77 GP4-187 58.48 42.33 50.41 27.61 35.72 33.33 34.53 6.68 GP-2793 78.33 56.00 67.17 28.51 45.08 36.25 40.67 19.59 GP4-2704 75.43 48.00 61.72 36.37 41.77 38.26 40.01 8.40 EC-512690 76.33 45.00 60.67 41.05 37.77 33.26 35.52 11.94 GP9-846-4-4 63.67 46.33 55.00 27.23 31.00 24.00 27.50 22.58 GP9-38-C-2-1 69.67 40.67 55.17 41.63 30.33 28.67 29.50 5.49 Mean 74.78 52.72 58.89 42.50 45.28 32.69 38.99 27.80 CD at 5% for treatments 0.99 0.80 CD at 5% for genotypes 3.77 3.07 CD at 5% for T x G 5.33 4.34 sinks ultimately leading to reduction in yield under stress conditions compared to irrigated conditions. Results and Discussion Mean of drought susceptibility index (DSI) and harvest index % of sunflower genotypes as influenced by moisture stress Konstantina, K., Petar Lambrev, Georgy Georgiev, Vasilii Goltsevc and Miroslav Karabaliev. 2004. Evaluation of chlorophyll fluorescence and membrane injury in the leaves of barley cultivars under osmotic stress. Bioelectrochemistry. 63: 121-124. Geno Drought Harvest index (%) Susceptibility type Index Control Stress Mean % decrease RSF-101 1.47 23.96 22.95 23.45 4.22 TSF-103 0.38 26.92 25.22 26.07 6.33 ASF-107 1.53 33.73 20.50 27.12 39.22 DSF-114 1.01 26.88 16.64 21.76 38.12 SH-177 1.85 29.28 28.57 28.92 2.44 DSF-104 0.63 29.22 26.80 28.01 8.26 RSF-106 0.75 28.12 26.49 27.30 5.80 DSF-111 0.85 29.23 28.30 28.76 3.17 RSF-107 0.43 33.06 32.49 32.77 1.72 ASF-104 0.69 16.59 14.61 15.60 11.93 TSF-106 0.55 25.02 24.27 24.64 2.98 SH-491 0.75 27.11 24.71 25.91 8.86 M-1029 1.68 22.13 17.16 19.65 22.49 GP-812-5 2.06 24.29 21.50 22.90 11.49 GP-247-4 1.03 32.95 26.59 29.77 19.32 GP4-2605 0.24 28.69 27.95 28.32 2.57 GP-69 0.33 17.51 16.19 16.85 7.51 GP4-2935 1.40 24.77 21.02 22.89 15.12 GP-978 0.33 31.00 30.38 30.69 2.01 DK-3849 1.84 26.49 23.19 24.84 12.47 GP9-515-7-3 1.98 29.81 29.07 29.44 2.49 GP4-2885 1.38 30.25 26.30 28.27 13.08 RHA-274 0.54 22.55 21.61 22.08 4.17 GP4-187 0.21 26.13 23.11 24.62 11.56 GP-2793 0.72 26.77 24.26 25.52 9.37 GP4-2704 0.29 34.07 24.48 29.27 28.15 EC-512690 0.45 27.36 18.03 22.70 34.09 GP9-846-4-4 0.83 25.56 24.39 24.98 4.59 GP9-38-C-2-1 0.20 29.03 21.58 25.31 25.66 Mean 0.91 26.80 19.54 23.17 27.07 CD at 5% - 0.48 for treatments CD at 5% 0.52 1.83 for genotypes CD at 5% - 2.60 for T x G susceptible lines. These lines may be studied further using molecular markers to identify stress tolerant markers and used in development of drought tolerant cultivars using appropriate breeding methods. Nezami, A., Khazaeia, H.R., Boroumand Rezazadehb, Z. and Hosseinic, A. 2008. Effects of drought stress and defoliation on sunflower (Helianthus annuus) in controlled conditions. Desert, 12:99-104. Pankoviæ, D., Sakaè, Z., Kevrešan, S., Plesnièar, M. 1999. Acclimation to long-term water deficit in the leaves of two sunflower hybrids: photosynthesis, electron transport and carbon metabolism. J. Expl. Botany. 330: 127-138. Petropoulos, S.A., Dimitra Daferera., M.G., Polissiou. and Passam, H.C. 2008. The effect of water deficit stress on the growth, yield and composition of essential oils of parsley. Scientia Horti.,115:393-397. Prabhudeva, T.V., Chalapathi, M.V., Thimmegowda, S., Devakumar, N., Rao, G.G. and Mallikarjuna, K. 1998. Results and Discussion Soil moisture stress and drought susceptibility index in sunflower. Indian Agriculturist. 42: 287-289. Rauf, S. and Sadaqat, H.A. 2007. Effect of varied water regimes on root length, dry matter partitioning and endogenous plant growth regulators in (Helianthus annuus L.) Plant interactions 2:41-51. Rauf, S. and Sadaqat, H.A. 2008. Effect of varied water regimes on root length, dry matter partitioning and endogenous plant growth regulators in sunflower (Helianthus anuus L.).J.Plant Interactions.2: 41-51. Ravishankar, K.V., Shanker, R.U., Ravishankar, H.M., Kumar, M.U. and Prasad, T.G. 1991. Development of drought tolerant sunflower for semiarid tracts of India : duration of genotypes influence their performance under imposed moisture stress. Helia. 14 : 77-85. Reddy, A.R., Chaitanya, K.V., Vivekanandan, M. 2004. Drought-induced responses of photosynthesis and antioxidant metabolism in higher plants. J. Plant Physiol., 161: 1189-1202. Shao, H.B., Chu, L.Y., Shao, M.A., Abdul Jaleel, C. and Hong-Mei, M. 2008. Higher plant antioxidants and redox signaling under environmental stresses. Comp Rend Biol., 331: 433–441. susceptible lines. These lines may be studied further using molecular markers to identify stress tolerant markers and used in development of drought tolerant cultivars using appropriate breeding methods. susceptible lines. These lines may be studied further using molecular markers to identify stress tolerant markers and used in development of drought tolerant cultivars using appropriate breeding methods. Specht, J.E., Chase, K., Macrander, M., Graef, G.L., Chung, J., Markwell, J.P., Germann, M., Orf, J.H. and Lark, K. J. 2001. Soybean response to water. A QTL analysis of drought tolerance. Crop Sci., 41: 493–509. Results and Discussion Moisture stress treatment imposed at flower bud initiation stage recorded decrease in harvest index (27.1%) compared to irrigated treatment (Table 7). Control, stress and interactions showed significant differences among genotypes for HI values. GP4-2704 followed by ASF-107 and RSF-107 in control and GP- 247-4 and RSF-107 in stress showed higher harvest index over rest of other genotypes. However, in genotype x treatments interaction, RSF-107 recorded maximum harvest index, which was significantly superior over other genotypes, whereas lowest harvest index was recorded in ASF-104. Exposure of sunflower plants to drought stress at bud initiation stage was more detrimental to seed and biological yield than at seed filling stage (Prabhudeva et al., 1998). Higher harvest index was obtained due to better translocation of photosynthates to the reproductive part under drought stress (Rauf and Sadaqat, 2008). obtained due to better translocation of photosynthates to the reproductive part under drought stress (Rauf and Sadaqat, 2008). sinks ultimately leading to reduction in yield under stress conditions compared to irrigated conditions. Moisture stress treatment imposed at flower bud initiation stage recorded decrease in harvest index (27.1%) compared to irrigated treatment (Table 7). Control, stress and interactions showed significant differences among genotypes for HI values. GP4-2704 followed by ASF-107 and RSF-107 in control and GP- 247-4 and RSF-107 in stress showed higher harvest index over rest of other genotypes. However, in genotype x treatments interaction, RSF-107 recorded maximum harvest index, which was significantly superior over other genotypes, whereas lowest harvest index was recorded in ASF-104. Exposure of sunflower plants to drought stress at bud initiation stage was more detrimental to seed and biological yield than at seed filling stage (Prabhudeva et al., 1998). Higher harvest index was The results indicated that water stress at flower bud initiation stage negatively affected plant height, total leaf area, total dry weight at harvesting, SPAD reading, chlorophyll fluorescence (Fv/Fm) , yield and yield related parameters. However, some genotypes performed better under drought stress than others. No genotype was tolerant to all the characters studied. Candidate genes tolerant to particular trait should be identified by breeders and those genes have to be incorporated in high yielding varieties. Based on DSI, genotypes TSF-103, RSF-107, TSF-106, ASF-104, DSF-104, SH-491, RSF-106, DSF- 111 were selected as tolerant and SH-177, ASF- 107, RSF-101,DSF-114 were selected as 33 Table 7. References Tahir, M.H.N. and Mehid, S.S. 2001. Evaluation of open pollinatedsunflower (Helianthus annuus L.) populations under water stress and normal conditions. Intnl J..Agric. Biol., 3: 236–238. Amrutha, R., Muthulaksmi, S., Baby Rani, W., Indira, W. K. and Mareeswari, P. 2007. Alleviation of High Temperature Stress in Sunflower (Helianthus Annus L.) by Plant Growth Regulators and Chemicals. Res. J. Agric.. Biol. Sci., 3: 1658-1662. Webber, M., Barnett, J., Finlayson, B. and Wang, M. 2006. Pricing China’s Irrigation Water. Working Paper, School of Anthropology, Geography and Environmental Studies, the University of Melbourne, Victoria, Australia. Bhatt, R.M. and Srinivasa Rao, N.K. 2005. Influence of pod load response of okra to water stress. Indian J.Plant Physiol., 10: 54–59. Wu, Q.S., Xia, R.X. and Zou, Y.N. 2008. Improved soil structure and citrus growth after inoculation with three arbuscular mycorrhizal fungi under drought stress. European J. Soil Biol., 44: 122-128. Farooq, M., Wahid, A., Kobayashi, N., Fujita, D. and Basra, S.M.A. 2009. Plant drought stress: effects, mechanisms and management. Agron. Sustainable Development. 29: 185-212. Wullschleger, S.D.,Yin, T.M., Difazio, S.P., Tschaplinski, T. J., Gunter, L.E., Davis, M.F, and Tuskan, G. A. 2005. Phenotypic variation in growth and biomass distribution for two advanced-generation pedigrees of hybrid poplar. Canadian J. Forest Res., 35: 1779–1789. Human, J.J., Toit, D., Bezuidenhout, H.D. and Bruyn, L.P.D. 1990. The influence of plant water stress on net photosynthesis and yield of sunflower (Helianthus annuus L.). J. Agron. Crop Sci., 164: 231-241. Received: November 11, 2011; Accepted: February 3, 2012
https://openalex.org/W4223457642	https://link.springer.com/content/pdf/10.1007/s10577-022-09687-4.pdf	English	null	The non-Mendelian behavior of plant B chromosomes	Chromosome research	2,022	cc-by	7,316	Chromosome Res (2022) 30:229–239 https://doi.org/10.1007/s10577-022-09687-4 Chromosome Res (2022) 30:229–239 https://doi.org/10.1007/s10577-022-09687-4 Chromosome Res (2022) 30:229–239 https://doi.org/10.1007/s10577-022-09687-4 REVIEW Keywords Chromosome drive · Nondisjunction · Supernumerary B chromosome · Asymmetric cell division Abstract B chromosomes, also known as supernu- merary chromosomes, are dispensable elements in the genome of many plants, animals, and fungi. Many B chromosomes have evolved one or more drive mecha- nisms to transmit themselves at a higher frequency than predicted by Mendelian genetics, and these mechanisms counteract the tendency of non-essential genetic elements to be lost over time. The frequency of Bs in a population results from a balance between their effect on host fitness and their transmission rate. Here, we will summarize the findings of the drive process of plant B chromosomes, focusing on maize and rye. Abbreviations A A chromosome Ab10 abnormal A chromosome 10 B B chromosome CENH3 centromere-specific histone H3 Gen generative NCR nondisjunction control region PMC pollen mother cell Veg vegetative The non‑Mendelian behavior of plant B chromosomes Jianyong Chen · James A. Birchler · Andreas Houben Received: 10 January 2022 / Revised: 14 March 2022 / Accepted: 16 March 2022 / Published online: 12 April 2022 © The Author(s) 2022 Keywords Chromosome drive · Nondisjunction · Supernumerary B chromosome · Asymmetric cell division Keywords Chromosome drive · Nondisjunction · Supernumerary B chromosome · Asymmetric cell division Introduction Responsible editors: Stacey Hanlon and Amanda Larracuente Supplementary Information The online version contains supplementary material available at https://doi. org/10.1007/s10577-022-09687-4. J. Chen · A. Houben () Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Corrensstrasse 3, 06466 Seeland, Germany e-mail: houben@ipk-gatersleben.de J. A. Birchler () Division of Biological Sciences, University of Missouri, Columbia, MO 65211, USA e-mail: birchlerj@missouri.edu The B chromosome (B) is a masterwork of evolution. This accessory chromosome was named after their distinctions from A chromosomes (As), the stand- ard chromosomes in eukaryotes. Bs are not required for the normal growth and development of organ- isms, yet they are found in all eukaryotic phyla (Burt and Trivers 2006; Jones 1991; Kimura and Kayano 1961). Depending on the species, Bs may vary in behavior and DNA/chromatin composition prop- erties (reviewed in Camacho et al. 2000; Douglas and Birchler 2017; Houben et al. 2013; Jones 1995; Birchler and Yang, 2021). Generally, it is assumed that Bs are derived from As, either from the same or from a related species (for related studies, see Responsible editors: Stacey Hanlon and Amanda Larracuente Supplementary Information The online version contains supplementary material available at https://doi. org/10.1007/s10577-022-09687-4. J. Chen · A. Houben () Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Corrensstrasse 3, 06466 Seeland, Germany e-mail: houben@ipk-gatersleben.de J. A. Birchler () Division of Biological Sciences, University of Missouri, Columbia, MO 65211, USA e-mail: birchlerj@missouri.edu ol.: (011 123456789) 3 230 J. Chen et al. Fig. 1 The non-Mendelian behavior of B chromosomes of dif- ferent species during the live cycle of plants. The chromosome drive mechanism acts (a) pre-meiotically, (b) meiotically, and/ or (c) post-meiotically. Controlled elimination of B chromo- somes occurs during early embryogenesis Fig. 1 The non-Mendelian behavior of B chromosomes of dif- ferent species during the live cycle of plants. The chromosome drive mechanism acts (a) pre meiotically (b) meiotically and/ or (c) post-meiotically. Controlled elimination of B chromo- somes occurs during early embryogenesis or (c) post-meiotically. Controlled elimination of B chromo- somes occurs during early embryogenesis Fig. 1 The non-Mendelian behavior of B chromosomes of dif- ferent species during the live cycle of plants. Pre‑meiotic and meiotic drive of Bs the transmission of P. nodosum Bs increases through the female side (Bosemark 1957). ( ) The maximum number of B chromosomes that can accumulate in an individual varies among spe- cies (e.g., maize and rye could possess up to 34 and 6 Bs, respectively) and likely depends on the bal- ance between drive efficiency and adverse effects like reduced fertility and vigor caused by the B. How- ever, Bs without drive also exist. According to the transmission data of Bs from about 70 species, only about 60% of Bs can drive (Jones 1995). Bs without drive likely counteract their dispensable nature by providing beneficial features to the host species. For instance, in Allium schoenoprasum, the germination rate and survival of B-containing individuals was higher than that of individuals without Bs in drought conditions (Plowman and Bougourd 1994). Super- numerary chromosomes of the blast fungus Magna- porthe oryzae contribute to adaptive evolution by carrying a virulence-related gene and participating in genome rearrangement (Langner et al. 2021). B chromosome polymorphism could be interpreted as a dynamic system in which the frequency of Bs in a population continually shifts due to an “arms race” between the standard A and supernumerary B chro- mosomes (Camacho et al. 2000). Accumulation of Bs before meiosis has been reported in only a few plant species, and it is more common in animals (Austin et al. 2009). In Crepis pannonica, Crepis conyzaefolia, and Crepis capillaris, a higher number of Bs in pollen mother cells (PMCs) were found compared with their number in root meristems (Fröst 1964; Fröst and Östergren 1959; Rutishauser and Rothlisberger 1966). Nondisjunction of B sister chromatids occurs during inflorescence development in a genotype-dependent manner (Fig. 1(a)). As a result, the inflorescences of many C. capillaris plants carrying Bs are mosaics with PMCs having varying numbers of Bs (Parker et al. 1989; Rutishauser and Rothlisberger 1966). The asymmetry of female meiosis and cell division to produce the egg cell in some plants and vertebrates provides an opportunity for genetic drive. Only one product of meiosis becomes an egg nucleus, and the other three products do not contribute genetically to the next generation. This sets the stage for mecha- nisms that cause chromosomes to preferentially end up in the egg nucleus as opposed to the other prod- ucts of meiosis (Fig. 1(b)). Introduction The chromosome drive mechanism acts (a) pre-meiotically, (b) meiotically, and/ In contrast to Gregor Mendel’s first and second laws (the law of segregation and the law of inde- pendent assortment), the transmission of Bs in many species is greater than 0.5, a phenomenon known as genetic drive. Although the drive is one of the most important features of many B chromosomes, insights about the drive mechanism exist at the cellular level only for a few cereals. Dependent on the species, the drive mechanisms act pre-meiotically, meiotically, and/or post-meiotically (Fig. 1). Examples of species possessing a B chromosome drive are listed in Suppl. Table 1. Bs from the same genus often share a simi- lar drive mechanism, but there are exceptions. For instance, the Bs of Phleum phleoides undergo non- disjunction during the first pollen division (Bosemark 1956), while the Bs of Phleum nodosum undergo a normal division at the first and second pollen mito- sis and no accumulation of Bs was found on the male side (Bosemark 1957; Fröst 1969). On the contrary, rye (Martis et al. 2012), Aegilops speltoides (Ruban et al. 2020), and maize (Blavet et al. 2021)), but fol- low their own evolutionary pathway (Beukeboom 1994; Camacho et al. 2000). So far, B chromosomes have been found in 2087 plant species, accounting for 2.68% of 77,958 species with a known chromosome number (D’Ambrosio et al. 2017). Two criteria are used to distinguish Bs from the As and other special chromosomes: Bs are dispensa- ble and do not pair with any member of the standard A chromosome complement (Jones and Rees 1982). Most Bs do not confer obvious advantages under standard growth conditions on the host plant and have no or slight effects on the host when their numbers are low. But, exceptions exist, e.g., studies of the plant pathogen Zymoseptoria tritici demonstrate that some Bs influence the fitness of the fungus during host infection in a cultivar-dependent manner (Habig et al. 2017). In rye, the B chromosome may contribute to heat tolerance during meiosis (Pereira et al. 2017). 1 Vol:. 3 (1234567890) The non‑Mendelian behavior of plant B chromosomes 231 Pre‑meiotic and meiotic drive of Bs In Plantago serraria, Tril- lium grandiflorum, and Lilium callosum, Bs segregate preferentially to a cell later on, giving rise to the egg cell during meiosis. During meiosis I (MI), the major- ity of the Bs were seen lying outside the MI plate and on the micropylar side, the side which would give rise to the egg cell (Kayano 1957). No mechanism of numerical increase of Bs to the next generation was found on the male side in the same species. Nondisjunction in favor of a chromosome Nondisjunction of sister chromatids is likely a key component of the B chromosome drive in many spe- cies. Nondisjunction occurs when both sister chroma- tids migrate to the same daughter cell during division. This can happen if the sister chromosomes are held together post replication by DNA-DNA topological entanglement and the cohesion complex. Lagging chromosomes are also known to arise from error- prone kinetochore-microtubule interactions (reviewed by Kamenz and Hauf 2017). While nondisjunction of A chromosomes causes aneuploid and often results in cell death or genetic instability, controlled nondis- junction of Bs at a defined developmental stage, e.g., the first pollen grain mitosis, allows Bs to accumulate in the generative nuclei. Notably, despite this lagging of Bs, the cell cycle progresses, and no cell death occurs. Whether the intrinsic mitotic spindle assem- bly checkpoint is impaired or Bs escape the check- point control remains unknown. Although not involving a B chromosome, a well- studied mechanism of meiotic drive represents the abnormal A chromosome 10 (Ab10) of maize (Rhoades 1942; Longley 1945). With the aid of a kinesin-14 motor located on Ab10, large blocks of heterochromatin called knobs that are variably pre- sent on many corn chromosomes moves ahead of the rest of the genome to the micropylar direction at the anaphase of MI and MII. This causes chromosomes with large knobs, including Ab10, to be positioned in the lower cell, which will go on to form an egg (Dawe et al. 2018). Different from female meiosis, male mei- osis produces four spores (tetrads), which all lead to gametes that compete to fertilize an egg cell and drive is not observed for Ab10 through the male. The drive of maize Bs and Ab10 occurs at different life cycle 1 ol.: (01 3 123456789) 232 J. Chen et al. stages, one in meiosis (Ab10) and the other at the sec- ond pollen mitosis, so the drive mechanisms are dif- ferent. But, Ab10 is another example of a dispensable genetic element that persists in maize populations via genetic drive. Different from female meiosis, male meiosis pro- duces four spores (tetrads), which all lead to gametes stages, one in meiosis (Ab10) and the other at the sec- ond pollen mitosis, so the drive mechanisms are dif- ferent. Nondisjunction in favor of a chromosome Because only a single B chromosome is frequently present in individuals within populations that carry B chromosomes, B chromosomes may ben- efit from mechanisms or features that enable them to transit meiosis as a univalent chromosome. In order to reduce its meiotic loss, the rye B increases its ability to form B bivalents at the metaphase of MI (Jiménez et al. 1997). However, different from the drive, rye Bs moderate their polymorphic transmission rates to reach the gain/loss balance since too many Bs would affect the fitness of the host plants (Puertas et al. 1998). The maize B has the property to help its mei- otic transmission as a univalent (Carlson and Rose- man, 1992). This property is dependent on regions of the B chromosome (Carlson and Roseman, 1992) itself as well as the genetic background (Gonzalez- Sanchez et al. 2007). The loss of maize B univalent in a low transmission line is due to the misorienta- tion of the Bs during metaphase-anaphase I; on the other hand, the B univalent in the high transmission line is always correctly oriented (González-Sánchez et al. 2007). that have equal opportunity to fertilize an egg cell. Unpaired chromosomes such as those present in ane- uploids are often lost due to their irregular behavior at meiosis I. Because only a single B chromosome is frequently present in individuals within populations that carry B chromosomes, B chromosomes may ben- efit from mechanisms or features that enable them to transit meiosis as a univalent chromosome. In order to reduce its meiotic loss, the rye B increases its ability to form B bivalents at the metaphase of MI (Jiménez et al. 1997). However, different from the drive, rye Bs moderate their polymorphic transmission rates to reach the gain/loss balance since too many Bs would affect the fitness of the host plants (Puertas et al. 1998). The maize B has the property to help its mei- otic transmission as a univalent (Carlson and Rose- man, 1992). This property is dependent on regions of the B chromosome (Carlson and Roseman, 1992) itself as well as the genetic background (Gonzalez- Sanchez et al. 2007). The loss of maize B univalent in a low transmission line is due to the misorienta- tion of the Bs during metaphase-anaphase I; on the other hand, the B univalent in the high transmission line is always correctly oriented (González-Sánchez et al. 2007). Nondisjunction in favor of a chromosome Nondisjunction is not common on the female side in those plants whose Bs exhibit nondisjunction in pollen grains. For example, crossing results indicated no drive of Bs of Anthoxanthum aristatum (Östergren 1947), Festuca pratensis (Bosemark 1954), P. phle- oides (Bosemark 1956), and Ae. speltoides (Mendel- son and Zohary 1972) occurring on the female side. On the other hand, the B chromosome of rye could drive during the development of female gameto- phytes. The nondisjunction of rye Bs was observed at the first mitosis in the embryo sac (Håkansson 1948). i The drive of the rye B works equally well when the B was introduced as an additional chromosome into Secale vavilovii (Puertas et al. 1985), hexaploid wheat (Endo et al. 2008; Lindström 1965; Müntzing 1970; Niwa et al. 1997), or hypo-pentaploid Triticale (Kishikawa and Suzuki 1982). Thus, the rye B con- trols the nondisjunction process by itself (Matthews and Jones 1983; Romera et al. 1991). The heterochromatic end of the rye long B arm is involved in the control of nondisjunction. B chro- mosomes lacking the so-called nondisjunction con- trol region (NCR) undergo normal disjunction at the first pollen mitosis (Fig. 2b). This region acts in trans because nondisjunction occurs for the NCR-deficient B (defB) if a standard B (Lima-de-Faria 1962) or the NCR of the long arm of the B (Endo et al. 2008) is present in the same cell containing a NCR-deficient B (Fig. 2c). Thus, defB carries the cis-acting element(s) responsible for nondisjunction. Although the Giemsa Nondisjunction in favor of a chromosome But, Ab10 is another example of a dispensable genetic element that persists in maize populations via genetic drive.f Different from female meiosis, male meiosis pro- duces four spores (tetrads), which all lead to gametes 1 Vol:. 3 (1234567890) 3 ( 3 567890) 233 The non‑Mendelian behavior of plant B chromosomes A. speltoides during the first pollen mitosis, Bs undergo nondisjunction and then will be included in the generative nucleus or remain lagging so that most vegetative nuclei receive A chromosomes only (Fig. 2a). A quantitative flow cytometric approach revealed that independent of the number of Bs pre- sent in the mother plant, Bs accumulate in the gen- erative nuclei to > 93% in Ae. speltoides (Wu et al. 2019). Similarly, in rye, the nondisjunction of Bs is a highly efficient process in all kinds of populations, from Turkey (93%), Iran (92%), South Korea (93%), Japan (96%) (Niwa and Sakamoto 1995), and China (88%) (Niwa and Sakamoto 1996). In weedy rye (Secale segetale) from Pakistan, a 95% B nondis- junction frequency was found (Niwa and Sakamoto 1996). At the second pollen mitosis, B sister chro- matids normally divide like standard chromosomes, and each sperm nucleus contains the same number of Bs (Fig. 2a). Fig. 2 The segregation behavior of rye and Ae. speltoides B chromosomes. a–c Drive of the rye B occurs during the first pollen grain mitosis. Veg, vegetative nucleus; Gen, gen- erative nucleus. a Standard B behavior during pollen develop- ment. Mature pollen of rye with Bs after FISH with B-repeat (in green) and A chromosome–specific 5S rDNA (in red). Bs accumulate in sperm nuclei due to nondisjunction and asym- metric spindle formation. b No drive occurs if the nondisjunc- tion control region (NCR) of the B is missing. c Standard B with a complete NCR and deficient B variants. Drive of both B variants occurs (Endo et al. 2008). NCR acts in trans. d Non- disjunction of the Ae. speltoides B in proto-root cells during early embryogenesis results in micronucleation and complete elimination. In contrast to B chromosome drive, a symmetri- cal cell division occurs as part of the chromosome elimination process. e Different types of chromosomes. The rye B’s NCR is shown in green ◂ that have equal opportunity to fertilize an egg cell. Unpaired chromosomes such as those present in ane- uploids are often lost due to their irregular behavior at meiosis I. Post‑meiotic drive of Bs The post-meiotic accumulation of Bs is the best- analyzed drive process and is frequent in plants, where the formation of mature pollen involves two post-meiotic divisions that result in genera- tive and vegetative nuclei (Fig. 1(c)). In rye and 1 ol.: (01 3 123456789) 234 J. Chen et al. unresolved sister chromatid cohesion of Bs leads to nondisjunction, micronucleation, and subsequent elimination during early embryogenesis of proto-root cells (Ruban et al. 2020) (Fig. 2d). Not only in plants but also in animals, nondisjunction of specific chro- mosomes leads to programmed DNA elimination. The extended cohesion between sister chromatids at the distal end at anaphase results in a partial loss of the sea lamprey (Petromyzon marinus) genome dur- ing early embryogenesis (Smith et al. 2021; Timo- shevskiy et al. 2016). A programmed chromosome elimination process also occurs in Sciara species; paternal X chromosomes undergo elimination during embryonic development since nondisjunction of them at the distal ends gives rise to their retardation after anaphase segregation (Escribá and Goday 2013). banding–positive NCR is a hot spot of B-specific sat- ellite DNAs, containing at least 8 different satellite repeats (Blunden et al. 1993; Carchilan et al. 2007; Klemme et al. 2013; Sandery et al. 1990; Wu et al. 2019), this region also encodes transcriptionally active protein-coding genes (Chen, Boudichevskaia et al. unpublished). banding–positive NCR is a hot spot of B-specific sat- ellite DNAs, containing at least 8 different satellite repeats (Blunden et al. 1993; Carchilan et al. 2007; Klemme et al. 2013; Sandery et al. 1990; Wu et al. 2019), this region also encodes transcriptionally active protein-coding genes (Chen, Boudichevskaia et al. unpublished). The NCR replicates later than the rest of the entire genome (Klemme et al. 2013; Lima-de-Faria and Jaworska 1972). The peculiarity of the NCR lies in the fact that, contrary to the Giemsa-positive subtelomeric heterochromatic regions of As, this domain is simultaneously marked by trimethylated histone H3K4 and trimethylated H3K27 (Carchilan et al. 2007), an unusual combination of apparently conflicting post-translational histone modifications. Unknown is whether nonrepressive (H3K4me) and repressive (H3K27me) histone modifications coexist within the same nucleosome or whether they occupy alternate nucleosomes. It is possible that in Ae. speltoides and rye, the cohesion between B sister chromatids during first pollen mitosis is stronger than the microtubule trac- tion force required to divide chromatids. But why does the cohesion differ between A and B chroma- tids? Post‑meiotic drive of Bs A B chromosome–specific composition of (peri) centromere sequences was observed for the Bs of rye (Banaei-Moghaddam et al. 2012), Ae. speltoides (Wu et al. 2019), maize (Jin et al. 2005; Lamb et al. 2005), F. pratensis (Ebrahimzadegan et al. 2019), the daisy Brachycome dichromosomatica (Leach et al. 1995), and the grasshopper Xyleus discoideus angu- latus (Bernardino et al. 2017). It is likely that the B-specific (peri)centromere sequence composition is functionally involved in the drive of Bs. In addition, heterochromatin, checkpoint control, release of cohe- sion, or related yet unidentified proteins may differ between A and B chromosomes and result in different segregation dynamics of Bs. It is tempting to specu- late that the extended cohesion of B sister chromatids is also part of their behavior during meiosis if occur- ring as a univalent. Unlike As, rye B univalents split sister chromatids less frequently at anaphase I (Man- zanero et al. 2000). Does a dysfunctional centromere cause or con- tribute to the mechanisms of nondisjunction of Bs? In rye and Ae. speltoides, no major differences in the CENH3 (CENPA) signal size were found between A and B centromeres and the interaction between B centromeres and tubulin fibers was observed (Banaei- Moghaddam et al. 2012; Wu et al. 2019). Thus, a dif- ferent centromere activity of Bs might be excluded. An important hint regarding the mechanism of chro- mosome drive comes from the finding that the micro- tubule spindle of both species is asymmetrical during the first pollen mitosis, in accordance with previous studies in other species (Banaei-Moghaddam et al. 2012; Borg et al. 2009; Wu et al. 2019). It is likely that the inclusion of lagging Bs into the generative nucleus is caused by the fact that the equatorial plate is nearer to the generative pole. Spindle asymmetry as a component of the drive process has also been suggested for the Bs of the lily L. callosum (Kimura and Kayano 1961), the Asteraceae C. capillaris (Rut- ishauser and Rothlisberger 1966), and the grasshop- per Myrmeleotettix maculatus (Robinson and Hewitt 1976). But, also standard meiotic mouse A chromo- somes with a stronger centromere harness the spindle asymmetry to drive (Akera et al. 2017). Notably, con- trary to drive, the targeted loss of Bs is also caused by nondisjunction. However, in contrast to drive, a symmetrical cell division occurs as part of the chro- mosome elimination process. In Ae. speltoides, the 1 3 :. (1234567890) Nondisjunction of Bs at the second pollen mitosis and preferential fertilization Maize, which belongs to the subfamily Panicoideae, evolved a different B drive mechanism. Bs of maize will undergo nondisjunction during the sec- ond pollen division (Roman 1947). In addition, the 1 3 . (1234567890) 1 Vol:. The non‑Mendelian behavior of plant B chromosomes 235 Fig. 3 Drive of the maize B chromosome. a At the first pol- len mitosis, the maize B behaves like As. Consequently, both vegetative (Veg) and generative nuclei (Gen) contain Bs. Non- disjunction of the B occurs at second pollen mitosis, and one sperm nucleus accumulates the Bs. b Mature pollen of a +2B maize plant after FISH using a B-specific repeat (in red). c At the fertilization process, the sperm with Bs preferentially fertilizes the egg, which results in an embryo with Bs and endosperm without Bs. d Different types of chromosomes maize plant after FISH using a B-specific repeat (in red). c At the fertilization process, the sperm with Bs preferentially fertilizes the egg, which results in an embryo with Bs and endosperm without Bs. d Different types of chromosomes Fig. 3 Drive of the maize B chromosome. a At the first pol- len mitosis, the maize B behaves like As. Consequently, both vegetative (Veg) and generative nuclei (Gen) contain Bs. Non- disjunction of the B occurs at second pollen mitosis, and one sperm nucleus accumulates the Bs. b Mature pollen of a +2B joining with the polar nuclei more often than with the egg (Carlson 1999). nondisjunction of maize Bs occurs during the first pollen division, but its frequency is very low (Rusche et al. 1997). Nondisjunction results in one sperm nucleus containing duplicate Bs and a sperm nucleus without Bs (Fig. 3a, b). Thus, at the subsequent dou- ble fertilization, the sperm nuclei with Bs will ferti- lize with the egg and the other one without Bs will fuse with two polar nuclei with the preference for the egg being about 67% in most lines (Roman 1948) (Fig. 3c). In short, the drive process of maize Bs takes two steps: nondisjunction and preferential fertiliza- tion. Interestingly, some lines of maize have random fertilization with regard to the presence/absence of a B chromosome (Carlson 1969) or even a reversal of preferential fertilization with the B-containing sperm The maize B chromosome has a B chromo- some–specific repeat that is in and around the cen- tromere (Alfenito and Birchler 1993; Jin et al. Nondisjunction of Bs at the second pollen mitosis and preferential fertilization 2005; Blavet et al. 2021). Sequence analysis indicates that it is the only unique sequence in minichromosomes derived from the maize B that are still capable of nondisjunction, thus implicating it as the target for mediating the process. The repeat spans several megabases and is heterogeneous but generally has a segment with homology to heterochromatic knobs (Alfenito and Birchler 1993; Hsu et al. 2003) as well as degenerate telomere repeat arrays. 1 ol.: (01 3 123456789) 236 J. Chen et al. mitosis—the same mitosis at which the B centromere nondisjoins. The consequence is that the knobbed chromosomes break at this mitosis at a high fre- quency. Because the maize B chromosome and knobs are both late replicating in S phase, the hypothesis put forward to explain this interaction was that the high- loss line interacts with the B chromosome to cause an even more delayed replication of both, resulting in the chromosomal breakage. The nondisjunction of the maize B centromere requires trans-acting factors elsewhere on the chro- mosome (Roman 1947; Ward 1973a; Lin 1978; Auger and Birchler 2002). One of these is located near the very distal tip of the long arm. The sequence information locates the responsible region to one with 34 predicted protein-encoding genes (Blavet et al. 2021), although other sources of a trans-factor are possible from this segment. The control of nondisjunction, increased recom- bination of the A complement, univalent stability, and interaction with knobs all appear to be affected by genes encoded on the maize B chromosome, and indeed, various regions have been attributed to these functions (Birchler and Yang 2021). The sequence of the maize B chromosome revealed 758 predicted protein encoding genes, many of which are expressed (Huang et al. 2016), but did not reveal any synteny with a potential progenitor region in the A chromo- somes. Instead, the sequence revealed that the para- logues shared with the A chromosomes were dis- persed and had widely different divergence times (Blavet et al. 2021). Thus, it appears that the maize B chromosome has been in the evolutionary lineage for millions of years during which the initial genes have deteriorated beyond recognition. The current gene repertoire is likely to have resulted from a continuous transposition of genes from the A complement over millions of years. Many predicted genes on the maize B show evidence of relaxed purifying selection. Nondisjunction of Bs at the second pollen mitosis and preferential fertilization How- ever, some of the genes likely have evolved to per- form functions for the perpetuation of the B despite its nonvital nature. In addition to the nondisjunction and preferential fertilization, the maize B has other properties that help perpetuate it. It has long been known that the B chromosome will foster increased recombination in the A chromosomes, particularly on the male side and apparently in pericentromeric heterochromatin (Ayonoadu and Rees 1968; Rhoades 1968; Han- son 1969; Nel 1973; Ward 1973b; Robertson 1984; Carlson et al. 1993). The presumed reason for this stimulation is that it conditions recombination in its own highly heterochromatic regions to facilitate faithful separation in meiosis I (Carlson 1994). It is notable that the increase is greater on the male side, which immediately precedes the aspects of the drive mechanism involving nondisjunction at the second pollen mitosis. Another property of the maize B that aids its perpetuation is its stabilization as a univalent in meiosis (Carlson and Roseman 1992; Gonzalez- Sanchez et al. 2007). The maize B chromosome can find itself alone in meiosis, which with most chromosomes will lead to a high frequency of loss. This process also appears to involve the B-specific repeat in the centromeric region (Gonzalez-Sanchez et al. 2007), which shows a precocious attachment to the meiotic spindle before the normal centromere analogous to abnormal chromosome 10, described above. There is, nevertheless, a clear distinction because the maize B chromosome does not induce the heterochromatic knobs on the A chromosomes to show preferential segregation. 1 3 l:. (1234567890) Outlook is a holder of a China Scholarship Council (CSC) fellowship (No. 202006850005). Funding Open Access funding enabled and organized by Projekt DEAL. J.B. was supported by NSF grant IOS-1545780. A.H. received financial support from the DFG (HO 1779/30- 1). Publication costs were supported by DFG (HE9114/1-1). Blunden R, Wilkes TJ, Forster JW, Jimenez MM, Sandery MJ, Karp A, Jones RN (1993) Identification of the E3900 family, a second family of rye B chromosome specific repeated sequences. Genome 36:706–711i Open Access This article is licensed under a Creative Com- mons 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 Crea- tive 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/. Borg M, Brownfield L, Twell D (2009) Male gameto- phyte development: a molecular perspective. J Exp Bot 60:1465–1478 Bosemark NO (1954) On accessory chromosomes. In Fes- tuca pratensis: I. Cytological investigations. Hereditas 40:346–376 Bosemark NO (1956) Cytogenetics of accessory chromosomes in Phleum phleoides. Hereditas 42:443–466 Bosemark NO (1957) Further studies on accessory chromo- somes in grasses. Hereditas 43:236–297l Burt A, Trivers R (2006) Genes in conflict: the biology of self- ish genetic elements. The Belknap Press of Harvard Uni- versity Press, Cambridge, Massachusetts, London, pp 602 Camacho JPM, Sharbel TF, Beukeboom LW (2000) B-chromo- some evolution. Philos T Roy Soc B 355:163–178 Carchilan M, Delgado M, Ribeiro T, Costa-Nunes P, Caperta A, Morais-Cecílio L, Jones RN, Viegas W, Houben A (2007) Transcriptionally active heterochromatin in rye B chromosomes. The Plant Cell 19:1738–1749f Outlook B chromosome sequences are notoriously difficult to assembly due to their complex mosaic composition of A chromosome–derived and organelle-derived DNA fragments and their high repeat composition (Blavet et al. 2021). However, the recent develop- ment of accurate long-read sequencing by PacBio circular consensus sequencing (CCS), HiC, optical mapping, and nanopore sequencing greatly improved the sequence assembly of large genome species at the chromosome scale, and the first nearly complete plant B chromosome sequence became available (Blavet et al. 2021). The accessibility of genomic B The maize B chromosome and the heterochro- matic knobs have an apparent antagonism, given that there is a negative correlation between the presence of knobs and B chromosomes in Native American collections of maize (Longley 1938; Bianchi et al. 1963). Indeed, in a line described by Rhoades et al. (1967) and Rhoades and Dempsey (1972), the pres- ence of B chromosomes will cause the knobs in the A chromosome to remain adhered at the second pollen 3 (1234567890) 1 Vol:. The non‑Mendelian behavior of plant B chromosomes 237 Bernardino ACS, Cabral-de-Mello DC, Machado CB, Pala- cios-Gimenez OM, Santos N, Loreto V (2017) B chromo- some variants of the grasshopper Xyleus discoideus angu- latus are potentially derived from pericentromeric DNA. Cytogenet Genome Res 152:213–221 sequences in combination with tissue-specific tran- scriptome data of B chromosome mutants with and without drive will greatly speed up the quest for genes controlling the drive of B chromosomes. A detailed understanding of the molecular mechanism underlying the tissue and chromosome type–specific drive of Bs will provide clues about the process of chromosome nondisjunction, which is a major cause of genetic diseases across species. Beukeboom LW (1994) Bewildering Bs - an impression of the 1st B-Chromosome Conference. Heredity 73:328–336 Bianchi A, Ghatnekar MV, Ghidoni A (1963) Knobs in Italian maize. Chromosoma 14:601–617 Birchler JA, Yang H (2021) The supernumerary B chromosome of maize: drive and genomic conflict. Open Biology 11:210197i l Blavet N, Yang H, Su H, Solansky P, Douglas RN, Karafiatova M, Simkova L, Zhang J, Liu Y, Hou J, Shi X, Chen C, El-Walid M, McCaw ME, Albert PS, Gao Z, Zhao C, Ben- Zvi G, Glick L et al (2021) Sequence of the supernumer- ary B chromosome of maize provides insight into its drive mechanism and evolution. Proc. Natl. Acad. Sci., USA 118:e2104254118 Acknowledgements J.C. is a holder of a China Scholarship Council (CSC) fellowship (No. 202006850005). Acknowledgements J.C. References Genetics 46:1699–1712 Endo TR, Nasuda S, Jones N, Dou Q, Akahori A, Wakimoto M, Tanaka H, Niwa K, Tsujimoto H (2008) Dissection of rye B chromosomes, and nondisjunction properties of the dissected segments in a common wheat background. Genes Genet Syst 83:23–30 Kishikawa H, Suzuki A (1982) Cytological study on hypo- pentaploid triticale with four B chromosomes of rye. The Japanese Journal of Genetics 57:17–24 Klemme S, Banaei-Moghaddam AM, Macas J, Wicker T, Novák P, Houben A (2013) High-copy sequences reveal distinct evolution of the rye B chromosome. New Phytol 199:550–558 Escribá MC, Goday C (2013) Histone H3 phosphorylation and elimination of paternal X chromosomes at early cleavages in sciarid flies. J Cell Sci 126:3214–3222 l Fröst S (1964) Further studies of accessory chromosomes in Crepis conyzaefolia. Hereditas 52:237–239 Lamb JC, Kato A, Birchler JA (2005) Sequences associated with A chromosome centromeres are present throughout the maize B chromosome. Chromosoma 113:337–349 Fröst S (1969) The inheritance of accessory chromosomes in plants, especially in Ranunculus acris and Phleum nodo- sum. Hereditas 61:317–326 Langner T, Harant A, Gomez-Luciano LB, Shrestha RK, Malmgren A, Latorre SM, Burbano HA, Win J, Kamoun S (2021) Genomic rearrangements generate hypervariable mini-chromosomes in host-specific isolates of the blast fungus. PLoSGenet 17:e1009386 Fröst S, Östergren G (1959) Crepis pannonica and Crepis conyzaefolia - two more species having accessory chro- mosomes. Hereditas 45:211–214 González-Sánchez M, González-García M, Vega J, Rosato M, Cuacos M, Puertas M (2007) Meiotic loss of the B chromosomes of maize is influenced by the B univalent co-orientation and the TR-1 knob constitution of the A chromosomes. Cytogenet Genome Res 119:282–290 Leach CR, Donald TM, Franks TK, Spiniello SS, Hanrahan CF, Timmis JN (1995) Organization and origin of a B chromosome centromeric sequence from Brachycome dichromosomatica. Chromosoma 103:708–714 Lima-de-Faria A (1962) Genetic interaction in rye expressed at the chromosome phenotype. Genetics 47:1455 Habig M, Quade J, Stukenbrock EH (2017) Forward genetics approach reveals host genotype-dependent importance of accessory chromosomes in the fungal wheat pathogen Zymoseptoria tritici. Mbio 8:e01919–e01917 Lima-de-Faria A, Jaworska H (1972) The relation between the chromosome size gradient and the sequence of DNA rep- lication in rye. Hereditas 70:39–57 Håkansson A (1948) Behaviour of accessory rye chromo- somes in the embryo-sac. Hereditas 34:35–59 Lin BY (1978) Regional control of nondisjunction of the B chromosome in maize. Genetics 90:613–627 Lindström J (1965) Transfer to wheat of accessory chromo- somes from rye. References Akera T, Chmatal L, Trimm E, Yang K, Aonbangkhen C, Che- noweth DM, Janke C, Schultz RM, Lampson MA (2017) Spindle asymmetry drives non-Mendelian chromosome segregation. Science 358:668–672 Carlson WR (1969) Factors affecting preferential fertilization in maize. Genetics 62:543–554f Carlson WR (1994) Crossover effects of B chromosomes may be “selfish”. Heredity 72:636–638 i Carlson WR (1999) Reversal of preferential fertilization. Maize Newsletter 73:39 Alfenito MR, Birchler JA (1993) Molecular characterization of a maize B chromosome centric sequence. Genetics 135:589–597 Carlson WR, Roseman RR (1992) A new property of the maize B chromosome. Genetics 131:211–223 Auger DL, Birchler JA (2002) Maize tertiary trisomic stocks derived from B-A translocations. Heredity 93:42–47l Carlson WR, Roseman RR, Zheng YZ (1993) Localizing a region on the B-chromosome that influences crossing over. Maydica 38:107–113 Austin B, Trivers R, Burt A (2009) Genes in conflict: the biol- ogy of selfish genetic elements. Harvard University Pressl D’Ambrosio U, Alonso-Lifante MP, Barros K, Kovařík A, de Xaxars GM, Garcia S (2017) B-chrom: a database on B-chromosomes of plants, animals and fungi. New Phytol 216:635–642 i Ayonoadu A, Rees H (1968) The influence of B chromosomes on chiasma frequencies in Black Mexican Sweet corn. Genetica 39:75–81 Banaei-Moghaddam AM, Schubert V, Kumke K, Weibeta O, Klemme S, Nagaki K, Macas J, Gonzalez-Sanchez M, Heredia V, Gomez-Revilla D, Gonzalez-Garcia M, Vega JM, Puertas MJ, Houben A (2012) Nondisjunction in favor of a chromosome: the mechanism of rye B chromosome drive during pollen mitosis. Plant Cell 24:4124–4134 Dawe RK, Lowry EG, Gent JI, Stitzer MC, Swentowsky KW, Higgins DM, Ross-Ibarra J, Wallace JG, Kanizay LB, Alabady M (2018) A kinesin-14 motor activates neo- centromeres to promote meiotic drive in maize. Cell 173:839–850 1 ol.: (01 3 123456789) 238 J. Chen et al. Douglas RN, Birchler J (2017) B chromosomes. In: Chromosome structure and aberrations. Springer, New Delhi, pp 13–39 Kayano H (1957) Cytogenetic studies in Lilium callosum III. Preferential segregation of a supernumerary chromosome in EMCs. Proceedings of the Japan Academy 33:553–558 Ebrahimzadegan R, Houben A, Mirzaghaderi G (2019) Repeti- tive DNA landscape in essential A and supernumerary B chromosomes of Festuca pratensis Huds. Sci Rep 9:19989 Kimura M, Kayano H (1961) The maintenance of supernumer- ary chromosomes in wild populations of Lillium callosum by preferential segregation. References ( 3 (1234567890) The non‑Mendelian behavior of plant B chromosomes 239 Niwa K, Horiuchi G, Hirai Y (1997) Production and characteri- zation of common wheat with B chromosomes of rye from Korea. Hereditas 126:139–146 Roman H (1947) Mitotic nondisjunction in the case of inter- changes involving the B-type chromosome in maize. Genetics 32:391–409 Niwa K, Sakamoto S (1995) Origin of B chromosomes in culti- vated rye. Genome 38:307–312 Roman H (1948) Directed fertilization in maize. Proc. Natl. Acad. Sci. USA 34:36–42 Niwa K, Sakamoto S (1996) Detection of B chromosomes in rye collected from Pakistan and China. Hereditas 124:211–216 Romera F, Jimenez M, Puertas M (1991) Genetic control of the rate of transmission of rye B chromosomes. I. Effects in 2B× 0B crosses. Heredity 66:61–65 Östergren G (1947) Heterochromatic B-chromosomes in Anth- oxanthum. Hereditas 33:261–296 Ruban A, Schmutzer T, Wu DD, Fuchs J, Boudichevskaia A, Rubtsova M, Pistrick K, Melzer M, Himmelbach A, Schubert V, Scholz U, Houben A (2020) Supernumerary B chromosomes of Aegilops speltoides undergo precise elimination in roots early in embryo development. Nat Commun 11:2764 Parker J, Jones G, Edgar L, Whitehouse C (1989) The popula- tion cytogenetics of Crepis capillaris. II. The stability and inheritance of B-chromosomes. Heredity 63:19–27 Pereira HS, Delgado M, Viegas W, Rato JM, Barão A, Caperta AD (2017) Rye (Secale cereale) supernumer- ary (B) chromosomes associated with heat tolerance during early stages of male sporogenesis. Ann Bot 119:325–337 Rusche ML, Mogensen HL, Shi L, Keim P, Rougier M et al (1997) B chromosome behavior in maize pollen as deter- mined by a molecular probe. Genetics 147:1915–1921 Rutishauser A, Rothlisberger E (1966) Boosting mechanism of B chromosomes in Crepis capillaris. Chromosomes Today 1:28–30 Plowman AB, Bougourd SM (1994) Selectively advantageous effects of B-chromosomes on germination behavior in Allium schoenoprasum L. Heredity 72:587–593 Sandery MJ, Forster JW, Blunden R, Jones RN (1990) Iden- tification of a family of repeated sequences on the rye B chromosome. Genome 33:908–913 Puertas M, Romera F, De La Peña A (1985) Comparison of B chromosome effects on Secale cereale and Secale vavilovii. Heredity 55:229–234 Smith JJ, Timoshevskiy VA, Saraceno C (2021) Programmed DNA elimination in vertebrates. Annual Review of Ani- mal Biosciences 9:173–201 Puertas MJ, González-Sánchez M, Manzanero S, Romera F, Jiménez MM (1998) Genetic control of the rate of transmission of rye B chromosomes. IV. Localization of the genes controlling B transmission rate. References Hereditas 54:149–155 Houben A, Banaei Moghaddam AM, Klemme S (2013) Biol- ogy and evolution of B chromosomes. In: Plant genome diversity, physical structure, behaviour and evolution of plant genomes. Edited: I K Leich, Springer Press, pp 149–166 Longley AE (1945) Abnormal segregation during megasporo- genesis in maize. Genetics 30:100–113 Longley AE (1938) Chromosomes of maize from North Amer- ican Indians. J Agric Res 56:177–195 Hsu FC, Wang CJ, Chen CM, Hu HY, Chen CC (2003) Molec- ular characterization of a family of tandemly repeated DNA sequences, TR-1, in heterochromatic knobs of maize and its relatives. Genetics 164:1087–1097 Manzanero S, Arana P, Puertas MJ, Houben A (2000) The chromosomal distribution of phosphorylated histone H3 differs between plants and animals at meiosis. Chromo- soma 109:308–317 Huang W, Du Y, Zhao X, Jin W (2016) B chromosome con- tains active genes and impacts the transcription of A chro- mosomes in maize (Zea mays L.). BMC Plant Biol 16:88 Martis MM, Klemme S, Banaei-Moghaddam AM, Blattner FR, Macas J, Schmutzer T, Scholz U, Gundlach H, Wicker T, Simkova H, Novak P, Neumann P, Kubalakova M, Bauer E, Haseneyer G, Fuchs J, Dolezel J, Stein N, Mayer KF, Houben A (2012) Selfish supernumerary chromosome reveals its origin as a mosaic of host genome and organel- lar sequences. Proc Natl Acad Sci USA 109:13343–13346 Jiménez MM, Romera F, González-Sánchez M, Puertas MJ (1997) Genetic control of the rate of transmission of rye B chromosomes. III. Male meiosis and gametogenesis. Heredity 78:636–644 Jin W, Lamb JC, Vega JM, Dawe RK, Birchler JA (2005) Molecular and functional dissection of the maize B chro- mosome centromere. Plant Cell 17:1412–1423 Matthews R, Jones R (1983) Dynamics of the B chromosome polymorphism in rye IIEstimates of parameters. Heredity 50:119–137 Jones RN (1991) B-chromosome drive. American Naturalist 137:430–442 Mendelson D, Zohary D (1972) Behaviour and transmission of supernumerary chromosomes in Aegilops speltoides. Heredity 29:329 Jones RN (1995) Tansley Review No 85, B chromosomes in plants. New Phytol 131:411–434 Jones RN, Rees H (1982) B chromosomes, 1st edn. Academic Press, London New York Müntzing A (1970) Chromosomal variation in the Lindström strain of wheat carrying accessory chromosomes of rye. Hereditas 66:279–285i Kamenz J, Hauf S (2017) Time to split up: dynamics of chro- mosome separation. Trends Cell Biol 27:42–54 Nel PM (1973) The modification of crossing over in maize by extraneous chromosomal elements. Theor Appl Genet 43:196–202 1 Vol:. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References Heredity 80:209–213 Timoshevskiy VA, Herdy JR, Keinath MC, Smith JJ (2016) Cellular and molecular features of developmentally pro- grammed genome rearrangement in a vertebrate (sea lam- prey: Petromyzon marinus). PLoS Genet 12:e1006103 Rhoades MM (1942) Preferential segregation in maize. Genet- ics 27:395–407 Ward EJ (1973a) Nondisjunction: localization of the controlling site in the maize B chromosome. Genetics 73:387–391 Rhoades MM (1968) In: Peacock WJ, Brock RD (eds) Studies on the cytological basis of crossing over. In: Replication and recombination in genetic material. Australian Acad- emy of Science, pp 229–241 Ward EJ (1973b) The heterochromatic B chromosome of maize: the segments affecting recombination. Chromo- soma 43:177–186 Wu D, Ruban A, Fuchs J, Macas J, Novak P, Vaio M, Zhou Y, Houben A (2019) Nondisjunction and unequal spindle organization accompany the drive of Aegilops speltoides B chromosomes. New Phytol 223:1340–1352 Rhoades MM, Dempsey E (1972) On the mechanism of chro- matin loss induced by the B chromosome of maize. Genet- ics 71:73–96 Rhoades MM, Dempsey E, Ghidoni A (1967) Chromosome elimination in maize induced by supernumerary B chro- mosomes. Proc. Natl. Acad. Sci. USA 57:1626–1632f Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Robertson DS (1984) Different frequency in the recovery of crossover products from male and female gametes of plants hypoploid for a B-A translocation. Genetics 107:117–130 Robinson PM, Hewitt GM (1976) Annual cycles in the inci- dence of B chromosomes in the grasshopper Myrme- leotettix maculatus (Acrididae: Orthoptera). Heredity 36:399–412 1 3 l.: (0123456789)
https://openalex.org/W2130887055	https://cp.copernicus.org/articles/8/149/2012/cp-8-149-2012.pdf	English	null	Impact of oceanic processes on the carbon cycle during the last termination	Climate of the past	2,012	cc-by	19,571	Climate of the Past Climate of the Past Correspondence to: N. Bouttes (n.bouttes@reading.ac.uk) Correspondence to: N. Bouttes (n.bouttes@reading.ac.uk) Received: 31 May 2011 – Published in Clim. Past Discuss.: 14 June 2011 Revised: 10 December 2011 – Accepted: 12 December 2011 – Published: 20 January 2012 Received: 31 May 2011 – Published in Clim. Past Discuss.: 14 June 2011 Revised: 10 December 2011 – Accepted: 12 December 2011 – Published: 20 January 2012 Abstract. During the last termination (from ∼18 000 years ago to ∼9000 years ago), the climate signiﬁcantly warmed and the ice sheets melted. Simultaneously, atmospheric CO2 increased from ∼190 ppm to ∼260 ppm. Although this CO2 rise plays an important role in the deglacial warming, the reasons for its evolution are difﬁcult to explain. Only box models have been used to run transient simulations of this carbon cycle transition, but by forcing the model with data constrained scenarios of the evolution of temperature, sea level, sea ice, NADW formation, Southern Ocean vertical mixing and biological carbon pump. More complex models (including GCMs) have investigated some of these mecha- nisms but they have only been used to try and explain LGM versus present day steady-state climates. sinking of salty water and therefore breaks down the deep stratiﬁcation and releases carbon from the abyss. Based on this scenario, it is possible to simulate both the amplitude and timing of the long-term CO2 increase during the last ter- mination in agreement with ice core data. The atmospheric δ13C appears to be highly sensitive to changes in the terres- trial biosphere, underlining the need to better constrain the vegetation evolution during the termination. 1 Introduction The last termination, which took place between ∼18 000 and ∼9000 years ago, is characterized by a global warm- ing (Visser et al., 2003; North Greenland Ice Core Project members, 2004; EPICA community members, 2004; Barker et al., 2009) associated to a shrinking of the ice sheets (Peltier, 1994, 2004; Svendsen et al., 2004). The climate evolved from a cold glacial state (sea surface temperature of −2 to −6◦C cooler relative to today in the Southern Ocean; MARGO Project Members, 2009) associated with large Northern Hemisphere ice sheets covering large parts of Europe and North America (Peltier, 2004) to a warmer in- terglacial state with reduced ice sheets, similar to the modern ones. In this study we use a coupled climate-carbon model of intermediate complexity to explore the role of three oceanic processes in transient simulations: the sinking of brines, stratiﬁcation-dependent diffusion and iron fertilization. Car- bonate compensation is accounted for in these simulations. We show that neither iron fertilization nor the sinking of brines alone can account for the evolution of CO2, and that only the combination of the sinking of brines and interactive diffusion can simultaneously simulate the increase in deep Southern Ocean δ13C. The scenario that agrees best with the data takes into account all mechanisms and favours a rapid cessation of the sinking of brines around 18 000 years ago, when the Antarctic ice sheet extent was at its maximum. In this scenario, we make the hypothesis that sea ice forma- tion was then shifted to the open ocean where the salty wa- ter is quickly mixed with fresher water, which prevents deep The warming in Antarctica is tightly linked to an atmo- spheric CO2 increase from ∼190 ppm at the Last Glacial Maximum (LGM, ∼21 000 years ago) to ∼260 ppm at the beginning of the Holocene (∼9000 years ago) (Monnin et al., 2001; Lourantou et al., 2010). The CO2 rise is crucial to Impact of oceanic processes on the carbon cycle during the last termination N. Bouttes1,2, D. Paillard1, D. M. Roche1,3, C. Waelbroeck1, M. Kageyama1, A. Lourantou4, E. Michel1, and L. Bopp1 1Laboratoire des Sciences du Climat et de l’Environnement, UMR8212, IPSL-CEA-CNRS-UVSQ, Centre d’Etudes de Saclay, Orme des Merisiers bat. 701, 91191 Gif Sur Yvette, France 2NCAS-Climate, Meteorology Department, University of Reading, Reading, RG66BB, UK 3Faculty of Earth and Life Sciences, Section Climate Change and Landscape dynamics, Vrije Universiteit Amsterdam, De Boelelaan, 1085, 1081 HV Amsterdam, The Netherlands 4LOCEAN, University Paris VI, Paris, France N. Bouttes et al.: Ocean processes during the last termination Moreover, if the sinking of brines is not taken into account, the mechanisms already tested are not sufﬁcient to explain the entire glacial-interglacial CO2 change in models of inter- mediate complexity (EMICs) or General Circulation Models (GCMs), as there is no physical mechanism to account for a sufﬁcient glacial deep stratiﬁcation and reduced circulation. Only box models have been able to perform a simulation of the last termination carbon cycle evolution by imposing the evolution of the oceanic circulation and mixing inferred from proxy data (K¨ohler et al., 2005a). Yet box models can be over-sensitive to changes in high latitudes (Archer et al., 2003). Additionally, because of their simplicity, the reasons for such changes in mixing and circulation that are crucial for the amplitude of the CO2 change (more than 45 ppm of the ∼90 ppm change; K¨ohler et al., 2005a) could not be tested. Furthermore, during the deglaciation the carbon content of the terrestrial biosphere globally increases as the vegetation expands on previously glaciated areas, although it decreases on areas of the continental shelf that become ﬂooded (Mon- tenegro et al., 2006). The general warming generates a mi- gration of ecosystems towards the poles while the rise of CO2 favours the uptake of carbon by plants (Kaplan et al., 2002; K¨ohler and Fischer, 2004). Carbon storage by the terrestrial biosphere from the LGM to the preindustrial is estimated by vegetation models to be between 600 GtC and 821 GtC (Kaplan et al., 2002; Brovkin et al., 2002b; K¨ohler and Fis- cher, 2004). Reconstructions based on proxy data estimate the range of possible terrestrial carbon change to be 270– 720 GtC from marine records (Bird et al., 1994), and 750– 1050 GtC from pollen based estimations (Crowley, 1995). In this study we use a climate model of intermediate com- plexity to explore the impact of two main oceanic mecha- nisms during the termination: the sinking of brines, which alters the circulation and mixing of water masses and has not yet been tested in transient simulations, and iron fertiliza- tion. Two other processes that are not independent are also considered: the ampliﬁcation of the effects of the sinking of brines by its feedback on diffusion and the ampliﬁcation of the oceanic uptake of carbon by the carbonate compensation mechanism. N. Bouttes et al.: Ocean processes during the last termination N. Bouttes et al.: Ocean processes during the last termination 150 explain the warming and shrinking of ice sheets (Berger et al., 1998; Charbit et al., 2005; Ganopolski et al., 2010), in association with the change of insolation. Yet explaining such an increase remains a challenge. change. Other mechanisms have been conﬁrmed. Enhanced marine biology by iron fertilization during the glacial period, which would then decrease during the deglaciation, is as- sumed to play a role (Martin, 1990), although of relatively small importance as it could account for a 15–20 ppm change (i.e. approximately 20 % of the ∼90 ppm total CO2 change) (Bopp et al., 2003; Tagliabue et al., 2009). Carbonate com- pensation is a recognized process that ampliﬁes the uptake or loss of carbon by the ocean (Broecker and Peng, 1987; Archer et al., 2000; Brovkin et al., 2007). Finally, changes in the oceanic circulation and mixing can have a signiﬁ- cant impact on glacial CO2 (Toggweiler, 1999; Paillard and Parrenin, 2004; K¨ohler et al., 2005a; Watson and Garabato, 2005; Bouttes et al., 2009; Skinner et al., 2010). In particular, it has been recently shown that the deep stratiﬁcation induced by enhanced brine sinking can strongly decrease CO2 during the LGM relative to the preindustrial and simultaneously in- crease the upper to deep ocean δ13C gradient, in line with data (Bouttes et al., 2010, 2011). Moreover, the isotopic composition of carbon (δ13C) both in the atmosphere and ocean also evolves during the tran- sition, providing clues and constraints on the evolution of the carbon cycle. The atmospheric δ13C (δ13Catm) presents a W-shape with two negative excursions of 0.5 % during Hein- rich event 1 (H1) and the Younger Dryas (YD) (Lourantou et al., 2010). In the ocean the vertical gradient of δ13Cocean (the gradient between the upper, −2000 m to 0 m, and the deep, −5000 m to −3000 m, ocean 1δ13Cocean = δ13Cupper− δ13Cbottom) decreases both in the South and North Atlantic. In particular, the deep South Atlantic values increase from around −0.8 ‰ at the LGM to around 0.4 ‰ in the modern ocean (Hodell et al., 2003; Curry and Oppo, 2005), and more locally from around −1 ‰ to around 0 ‰ at the location of core MD07-3076Q (44◦S, 14◦W, −3770 m) (Skinner et al., 2010; Waelbroeck et al., 2011). N. Bouttes et al.: Ocean processes during the last termination Since both the atmosphere and the terrestrial biosphere carbon contents increase during the termination, it is gen- erally concluded that the ocean, the largest of the three reser- voirs, must be the one that looses carbon. Various hypothe- ses have been proposed to explain the atmospheric CO2 in- crease based on modiﬁcations of the oceanic carbon content. Most of them focus either on changes in the dynamics of the ocean or on modiﬁcations of the marine biology (Archer et al., 2000; Sigman and Boyle, 2000; Fischer et al., 2010; Sigman et al., 2010). Yet the tight link between Antarc- tic temperature and CO2 (Cuffey and Vimeux, 2001) sug- gests a simple mechanism instead of a complex association of numerous independent processes. Besides, many of them have been discarded or only account for a small CO2 change such as the coral reef hypothesis (Berger, 1982; Broecker and Peng, 1982; Opdyke and Walker, 1992; K¨ohler et al., 2005a), modiﬁcation of winds (Toggweiler et al., 2006; Men- viel et al., 2008a), or sea ice extension (Stephens and Keel- ing, 2000; Archer et al., 2003), as they required unrealistic changes to account for most of the glacial-interglacial CO2 Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. N. Bouttes et al.: Ocean processes during the last termination combination with the concomitant sea level fall due to in- creasing ice sheets volume, it leads to a reduction of the vol- ume of water above the continental shelf. The brines rejected from the intense sea ice formation are less diluted and are re- leased closer to the shelf break. The dense water from brines can then more easily sink along the continental slope to the deep ocean. To avoid the dilution of such an effect, the sink- ing of brines to the deep ocean has been parameterized in CLIMBER-2 (Bouttes et al., 2010). The relative importance of this brine mechanism is set by the parameter frac, which is the fraction of salt rejected by sea ice formation that sinks to the bottom of the ocean. The rest of the salt (1 −frac) is diluted in the corresponding surface oceanic cell as done in the standard version. When frac = 0 no salt sinks to the abyss as it is entirely mixed in the surface oceanic cell (con- trol simulation), whereas frac = 1 is the maximum effect of the brine mechanism when all the rejected salt sinks to the bottom of the ocean. This mechanism was shown to result in a net glacial atmospheric CO2 decrease relative to the prein- dustrial as well as increased 1δ13Cocean and increased atmo- spheric δ13Catm (Bouttes et al., 2010, 2011). CLIMBER-2’s atmosphere has a coarse resolution of 10◦in latitude by 51◦in longitude, which is precise enough to take into account geographical changes, while allowing the model to be fast enough to run long simulations. The ocean is sub- divided into three zonally averaged basins with a resolution of 21 depth levels by 2.5◦latitude. It includes a carbon cycle model with the fractionation of 13C (Brovkin et al., 2002a). In addition to modules simulating the ocean, atmosphere, and continental biosphere dynamics, the model also includes a model of carbonate compensation (Brovkin et al., 2007; Archer, 1991). The coupling between CLIMBER-2 and the carbonate compensation model has been discussed in detail in Brovkin et al. (2007). Moreover, three mechanisms have been added in the present study: the sinking of brines, iron fertilization, and stratiﬁcation-dependent diffusion. 2.2.1 Iron fertilization Iron fertilization relies on the removal of the iron limita- tion in the “High Nutrient Low Chlorophyll” (HNLC) areas thanks to the supply of glacial atmospheric dust which con- tains iron (Martin, 1990; Bopp et al., 2003; Brovkin et al., 2007; Tagliabue et al., 2009). According to a proxy-based study of the change of productivity, this effect would have been restrained to the areas north of the modern-day Antarc- tic polar front (Kohfeld et al., 2005). In the model, it is simply taken into account by forcing the marine biology to use all the nutrients that would otherwise be left in the At- lantic and Indian sectors of the Sub-Antarctic surface ocean (30◦S to 50◦S) during the glacial period as previously done (Brovkin et al., 2007). This is a simple parameterization of the effect of iron fertilization as the iron cycle is not simu- lated in the model. As done in Bouttes et al. (2011), we use a parameter to vary this effect between 0 and 1. This param- eter is ﬁrst set to 1 to explore the maximum potential effect of this mechanism. In the last section, we use a combination of the three mechanisms from Bouttes et al. (2011) in which this parameter is set to 0.3. 2.2.3 Stratiﬁcation-dependent diffusion As the sinking of brines tends to modify the stratiﬁcation state of the ocean (the deep water becomes denser), it should modify the vertical diffusion. The more stratiﬁed the ocean becomes, the more energy it requires to mix water masses, implying a lower diffusion. Yet in the standard version of CLIMBER-2, the vertical diffusion coefﬁcient Kz is set by a ﬁxed proﬁle and cannot evolve. A parameterization of the vertical diffusion coefﬁcient was therefore introduced (Bouttes et al., 2010) so that vertical diffusion becomes inter- active and dependent of the stratiﬁcation state of the ocean. This allows a more physical representation of the diffusion, which can play a signiﬁcant role for the ocean circulation (Marzeion et al., 2007) and potentially inﬂuence the carbon cycle (Bouttes et al., 2009). The physical parameterization of the vertical diffusion coefﬁcient Kz was introduced depend- ing on the vertical density gradient (Marzeion et al., 2007) in the deep ocean (below 2000 m) as follows: 2.1 The Earth system model of intermediate complexity CLIMBER-2 We use the CLIMBER-2 coupled intermediate complexity model (Petoukhov et al., 2000; Ganopolski et al., 2001), which is well suited to perform the long runs of sev- eral thousands of years requested to study the deglaciation. Clim. Past, 8, 149–170, 2012 www.clim-past.net/8/149/2012/ www.clim-past.net/8/149/2012/ 151 2.2.2 Sinking of brines Kz ∝N−α (1) (1) Kz ∝N−α The version of CLIMBER-2 used here contains a parameter- ization of the sinking of brines that has been studied in LGM conditions (Bouttes et al., 2010). Brines are small pockets of very salty water rejected by sea ice formation as sea ice is mainly formed of fresh water. In the standard version of CLIMBER-2, the ﬂux of salt rejected to the ocean is mixed in the surface oceanic cell whose volume is quite large due to the coarse resolution. Yet, as brines are very dense because of their high salt content, instead of this dilution they should rapidly sink to the deep ocean where the local topography permits it. During glacial periods, the Antarctic ice sheet progressively extends and covers the continental shelves. In where α is a parameter and N = (−g ρ0 ∂ρ ∂z ) 1 2 is the local buoy- ancy frequency, with g the gravity acceleration, ρ0 a refer- ence density, and ∂ρ ∂z the vertical density gradient. The pa- rameter α controls the sensitivity of the vertical diffusivity to changes in stratiﬁcation. A previous study exploring its role during the LGM has shown that it could vary between 0.7 and 0.9 (Bouttes et al., 2011). We ﬁrst use the maximum value of 0.9 to test the maximum impact of this mechanism. In the last section we use a combination of the three mecha- nisms from Bouttes et al. (2011) where α = 0.7. The impact of the evolution of these mechanisms is ﬁrst studied in an idealized case with a ﬁxed climate (set to the www.clim-past.net/8/149/2012/ 2.3 Initial conditions The transient simulations start from initial values taken from equilibrium simulations of the Last Glacial Maximum cli- mate and carbon cycle state (Bouttes et al., 2011). The LGM conditions simultaneously imposed in all equilibrium simu- lations are the 21 kyr BP insolation (Berger, 1978), LGM ice sheets (Peltier, 2004), and atmospheric CO2 for the radia- tive code (190 ppm; Monnin et al., 2001; Lourantou et al., 2010). The atmospheric CO2 concentration is prescribed in the radiative code in order to correctly simulate the climate as previously done (Brovkin et al., 2007). The model is thus semi-coupled with respect to the climate and carbon cycle. CH4 and N2O are not explicitly considered in the model, but the CO2 level of 190 ppm can be considered as an “equivalent CO2” for a CO2 level of 167 ppm which would also consider the change of CH4 and N2O (Schneider von Deimling et al., 2006). To account for a glacial sea level fall of ∼120 m, salinity and mean nutrient concentrations are increased by 3.3 % (Brovkin et al., 2007). To ensure equilibrium for the carbon cycle, glacial simulations were run for 50 000 years. The transient simulations analysed in this study start from the equilibrium state of these 50 000 year LGM simulations. p p The sea level change is taken into account by changing the global mean salinity and nutrient concentrations based on sea level data (Waelbroeck et al., 2002). This is a global effect that does not take into account the addition of fresh wa- ter ﬂuxes in restricted areas. Indeed, although abrupt events took place during the last termination (Keigwin et al., 1991), rapid climate changes that can be triggered by the addition of fresh water ﬂuxes (Ganopolski and Rahmstorf, 2001) are beyond the scope of this study, which focuses on the general trends during the transition. The geography is thus not di- rectly changed and the closing-opening of the Bering Strait is not taken into account. The ice sheet evolution is simply imposed by interpolation between LGM and Late Holocene states based on the sea level data. First, a sea level coefﬁcient is computed: At the end of the 50 000 year simulation, the global mean surface air temperature with the preindustrial bound- ary conditions is 14.1 ◦C. With the LGM conditions, the cli- mate is 3.7 ◦C colder. 2.3 Initial conditions The effect of the dust is not taken into account in this study. According to Schneider von Deimling et al. (2006), it would cool the global climate by an additional 1.35 ± 0.35 ◦C, yielding an approximately 5.05 ± 0.35 ◦C colder climate during the LGM compared to the preindustrial. slcoeff = sl −slctrl sllgm −slctrl and 0 < slcoeff < 1 (2) (2) with sl the sea level of the considered time step, slctrl the modern sea level and sllgm the LGM sea level. Then, from slcoeff we compute the area of the Northern Hemisphere ice sheets in a given latitudinal sector as: With no additional mechanism nor carbonate compensa- tion, the obtained LGM CO2 is around 300 ppm. When car- bonate compensation is taken into account, CO2 is approxi- mately 260 ppm. In the other simulations carbonate compen- sation is always included. With iron fertilization the LGM CO2 is around 230 ppm, it falls to around 215 ppm with the sinking of brines. For the stratiﬁcation-dependent diffusion, the α parameter is ﬁrst set to its maximum value inferred from a previous study, i.e. 0.9 (Bouttes et al., 2011). The 190 ppm level is reached with either the sinking of brines combined with the stratiﬁcation-dependent diffusion or the sinking of brines with iron fertilization. The effect of the dif- ferent processes inﬂuencing the CO2 level during the LGM, including the solubility effect and the role of the additional mechanisms, has been previously studied in Bouttes et al. (2009, 2010, 2011). arealgm × sl 2 3 coeff (3) arealgm × sl 2 3 coeff (3) with arealgm the LGM ice sheets area (Peltier, 2004). The northern limit of the ice sheets is given by the CLIMBER-2 continental limit. The southern limit is computed from this area. The height of the ice sheets is calculated as: with arealgm the LGM ice sheets area (Peltier, 2004). The northern limit of the ice sheets is given by the CLIMBER-2 continental limit. The southern limit is computed from this area. The height of the ice sheets is calculated as: oroctrl + orolgm −orocrtl × sl 1 3 coeff (4) (4) with oroctrl the modern orography and orolgm the LGM orog- raphy (Peltier, 2004). This very simple ice sheet evolution formulation was initially developed for longer term simula- tions for which no information about ice sheet extent and height was precisely known. 2.4 Evolution of the forcing LGM) to assess the effect of each of the mechanisms with- out the complication of a changing climate. We then explore the evolution of the carbon cycle when the climate evolves from the LGM to the Holocene. To disentangle the effects of the ocean and vegetation, the simulations are run with either interactive or ﬁxed vegetation (“ﬁxed veg”). During deglaciation, the insolation, sea level, ice sheets, and CO2 evolved. The insolation evolution is calculated from Berger (1978). In the ﬁrst part of this study, the CO2 evolu- tion (Monnin et al., 2001; Lourantou et al., 2010) is imposed for the climate modules of the model as it is used to compute the changing radiative forcing, but it is not used for the car- bon cycle part of the model. The CO2 data are on the EDC3 age scale (Parrenin et al., 2007). Prescribing the CO2 level al- lows us to obtain a coherent climate even when the CO2 cal- culated by the carbon cycle is different from the one recorded in ice cores. Because we focus on the evolution of CO2 only, the effect of CH4 and N2O is not considered. In the second part, the model is used in an interactive mode, i.e. the CO2 calculated by the carbon cycle module is now used to com- pute the radiative scheme, no CO2 value is prescribed. www.clim-past.net/8/149/2012/ Clim. Past, 8, 149–170, 2012 N. Bouttes et al.: Ocean processes during the last termination 152 N. Bouttes et al.: Ocean processes during the last termination 153 0 2000 4000 6000 8000 10000 years 0.0 0.2 0.4 0.6 0.8 1.0 iron iron no iron or declining iron no iron Iron fertilization a B i 0 2000 4000 6000 8000 10000 190 200 210 220 230 240 250 260 CO2 (ppm) iron iron no iron or declining iron a 0 2000 4000 6000 8000 10000 190 200 210 220 230 240 250 260 CO2 (ppm) brines no brines or declining brines brines b 0 2000 4000 6000 8000 10000 years 190 200 210 220 230 240 250 260 CO2 (ppm) brines-Kz c linear decline of iron / brines ("linear") brutal halt of iron / brines ("abrupt") Fig. 2. Evolution of atmospheric CO2 with a constant climate for three mechanisms: (a) iron fertilization, (b) sinking of brines and (c) sinking of brines and interactive vertical diffusion. 0 2000 4000 6000 8000 10000 190 200 210 220 230 240 250 260 CO2 (ppm) iron iron no iron or declining iron a b 0 2000 4000 6000 8000 10000 190 200 210 220 230 240 250 260 CO2 (ppm) iron iron no iron or declining iron a 0 2000 4000 6000 8000 10000 190 200 210 220 230 240 250 260 CO2 (ppm) brines no brines or declining brines brines b c a 0 2000 4000 6000 8000 10000 years 0.0 0.2 0.4 0.6 0.8 1.0 frac brines no brines or declining brines no brines Brines b linear decline of iron / brines ("linear") brutal halt of iron / brines ("abrupt") Fig. 1. Evolution scenarios for iron fertilization and the sinking of brines with a constant climate. The two processes are active at the beginning of the simulations; then they stop. This stop can be sudden or follow a linear decline. 0 2000 4000 6000 8000 10000 years 0.0 0.2 0.4 0.6 0.8 1.0 frac brines no brines or declining brines no brines Brines b b 0 2000 4000 6000 8000 10000 years 190 200 210 220 230 240 250 260 CO2 (ppm) brines-Kz c Fig. 1. Evolution scenarios for iron fertilization and the sinking of brines with a constant climate. The two processes are active at the beginning of the simulations; then they stop. This stop can be sudden or follow a linear decline. Fig. 2. N. Bouttes et al.: Ocean processes during the last termination Evolution of atmospheric CO2 with a constant climate for three mechanisms: (a) iron fertilization, (b) sinking of brines and (c) sinking of brines and interactive vertical diffusion. In the following we test three oceanic mechanisms (iron fertilization, sinking of brines and stratiﬁcation-dependent diffusion). We explore different scenarios for their evolution in order to simulate the CO2 and atmospheric and oceanic δ13C evolution during the last deglaciation, and compare the results with proxy data to constrain the possible scenarios. 3.1.1 Scenarios for the sinking of brines and iron fertilization Two idealized scenarios are tested for the evolution of the sinking of brines and iron fertilization (Fig. 1). Both mech- anisms are active at the beginning of the simulations, and then stopped. This halt can either be instantaneous (scenario “abrupt”) or linear in time (scenario “linear”). The evolution imposed for the brines and iron mechanism is the same so that we can compare their responses in time. 3.1 Evolution of the mechanisms under a constant LGM climate: sensitivity studies We ﬁrst analyze sensitivity studies to compare the impact of the mechanisms (with different scenarios) on the evolu- tion of the carbon cycle with a constant climate and assess the role of each mechanism alone. In these idealized sim- ulations the climate is set by glacial boundary conditions. Atmospheric CO2 is prescribed to 190 ppm (Monnin et al., 2001); the northern ice sheets (Peltier, 2004) and the orbital parameter values (Berger, 1978) correspond to the situation at 21 kyr BP. 3.1.2 Impact of stopping iron fertilization The halt of iron fertilization leads to an increase of atmo- spheric CO2 as the biological pump is weakened (Fig. 2a). CO2 increases by 29 ppm (Table 1) in both scenarios. The equilibrium is rapidly reached for the abrupt halt of iron fer- tilization (Scenario “abrupt”). To compare the time needed by the system to reach a near-equilibrium state, we consider the time when the anomaly of a considered variable (deﬁned as the difference between its value and its initial value) has www.clim-past.net/8/149/2012/ 2.3 Initial conditions It provides an ice sheets evolu- tion consistent with the sea level evolution. Such a parame- terization should allow the study of a full glacial-interglacial cycle in the future. with oroctrl the modern orography and orolgm the LGM orog- raphy (Peltier, 2004). This very simple ice sheet evolution formulation was initially developed for longer term simula- tions for which no information about ice sheet extent and height was precisely known. It provides an ice sheets evolu- tion consistent with the sea level evolution. Such a parame- terization should allow the study of a full glacial-interglacial cycle in the future. Clim. Past, 8, 149–170, 2012 www.clim-past.net/8/149/2012/ www.clim-past.net/8/149/2012/ N. Bouttes et al.: Ocean processes during the last terminat 0 2000 4000 6000 8000 10000 years 0.0 0.2 0.4 0.6 0.8 1.0 iron iron no iron or declining iron no iron Iron fertilization a 0 2000 4000 6000 8000 10000 years 0.0 0.2 0.4 0.6 0.8 1.0 frac brines no brines or declining brines no brines Brines b linear decline of iron / brines ("linear") brutal halt of iron / brines ("abrupt") Fig. 1. Evolution scenarios for iron fertilization and the sinking of brines with a constant climate. The two processes are active at the beginning of the simulations; then they stop. This stop can be sudden or follow a linear decline. N. Bouttes et al.: Ocean processes during the last termination N. Bouttes et al.: Ocean processes during the last termination N. Bouttes et al.: Ocean processes during the last termination 154 N. Bouttes et al.: Ocean processes during the last termination 0 2000 4000 6000 8000 10000 12000 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 ∆δ13 Cocean(permil) iron no iron or declining iron iron a 0 2000 4000 6000 8000 10000 12000 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 ∆δ13 Cocean(permil) brines b 0 2000 4000 6000 8000 10000 12000 years 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 ∆δ13 Cocean(permil) brinesno brines or declining brines brines-Kz c linear decline of iron / brines ("linear") brutal halt of iron / brines ("abrupt") Fig. 3. Evolution of oceanic 1δ13Cocean with a constant climate for three mechanisms: (a) iron fertilization, (b) sinking of brines and (c) sinking of brines and interactive vertical diffusion. 0 2000 4000 6000 8000 10000 12000 6.9 6.8 6.7 6.6 6.5 6.4 δ13 Catm(permil) iron no iron or declining iron iron a 0 2000 4000 6000 8000 10000 12000 6.9 6.8 6.7 6.6 6.5 6.4 δ13 Catm(permil) brines b 0 2000 4000 6000 8000 10000 12000 years 6.9 6.8 6.7 6.6 6.5 6.4 δ13 Catm(permil) brinesno brines or declining brines brines-Kz c linear decline of iron / brines ("linear") brutal halt of iron / brines ("abrupt") Fig. 4. Evolution of atmospheric δ13Catm with a constant climate for three mechanisms: (a) iron fertilization, (b) sinking of brines and (c) sinking of brines and interactive vertical diffusion. Bouttes et al.: Ocean processes during the last termination 0 2000 4000 6000 8000 10000 12000 6.9 6.8 6.7 6.6 6.5 6.4 δ13 Catm(permil) iron no iron or declining iron iron a 0 2000 4000 6000 8000 10000 12000 6.9 6.8 6.7 6.6 6.5 6.4 δ13 Catm(permil) brines b 0 2000 4000 6000 8000 10000 12000 years 6.9 6.8 6.7 6.6 6.5 6.4 δ13 Catm(permil) brinesno brines or declining brines brines-Kz c linear decline of iron / brines ("linear") brutal halt of iron / brines ("abrupt") Fig. 4. N. Bouttes et al.: Ocean processes during the last termination Evolution of atmospheric δ13Catm with a constant climate f h h i ( ) i f ili i (b) i ki f b i 154 154 N 0 2000 4000 6000 8000 10000 12000 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 ∆δ13 Cocean(permil) iron no iron or declining iron iron a 0 2000 4000 6000 8000 10000 12000 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 ∆δ13 Cocean(permil) brines b 0 2000 4000 6000 8000 10000 12000 years 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 ∆δ13 Cocean(permil) brinesno brines or declining brines brines-Kz c linear decline of iron / brines ("linear") brutal halt of iron / brines ("abrupt") Fig. 3. Evolution of oceanic 1δ13Cocean with a constant climate for three mechanisms: (a) iron fertilization, (b) sinking of brines and (c) sinking of brines and interactive vertical diffusion. a c Fig. 4. Evolution of atmospheric δ13Catm with a constant climate for three mechanisms: (a) iron fertilization, (b) sinking of brines and (c) sinking of brines and interactive vertical diffusion. Fig. 3. Evolution of oceanic 1δ13Cocean with a constant climate for three mechanisms: (a) iron fertilization, (b) sinking of brines and (c) sinking of brines and interactive vertical diffusion. Table 1. Amplitude of change between the beginning and the end of the simulations under a constant glacial climate as observed on Figs. 2, 3 and 4. “Iron” corresponds to iron fertilization, “Brines” to the sinking of brines, and Brines-Kz to the sinking of brines and interactive diffusion. Table 1. Amplitude of change between the beginning and the end of the simulations under a constant glacial climate as observed on Figs. 2, 3 and 4. “Iron” corresponds to iron fertilization, “Brines” to the sinking of brines, and Brines-Kz to the sinking of brines and interactive diffusion. The impact of iron fertilization on δ13Cocean is very small (Fig. 3a). We consider the mean vertical gradient (1δ13Cocean) of δ13Cocean in the Atlantic between the upper (−2000 m to 0 m) and deep (−5000 m to −3000 m) ocean. The modiﬁcation of the gradient is only ∼0.1 ‰ (Table 1). On the other hand, the change of δ13Catm is larger (Fig. 4a) as it is decreased by 0.25 ‰ (Table 1). CO2 1δ13Cocean δ13Catm (ppm) (‰) (‰) Iron 29 −0.12 −0.25 Brines 40 −0.57 −0.25 Brines-Kz 61 −1.1 −0.34 www.clim-past.net/8/149/2012/ Clim. Past, 8, 149–170, 2012 Bouttes et al.: Ocean processes during the last termination 0 2000 4000 6000 8000 10000 12000 6.9 6.8 6.7 6.6 6.5 6.4 δ13 Catm(permil) iron no iron or declining iron iron a 0 2000 4000 6000 8000 10000 12000 6.9 6.8 6.7 6.6 6.5 6.4 δ13 Catm(permil) brines b 0 2000 4000 6000 8000 10000 12000 years 6.9 6.8 6.7 6.6 6.5 6.4 δ13 Catm(permil) brinesno brines or declining brines brines-Kz c linear decline of iron / brines ("linear") brutal halt of iron / brines ("abrupt") Fig. 4. Evolution of atmospheric δ13Catm with a constant climate for three mechanisms: (a) iron fertilization, (b) sinking of brines and (c) sinking of brines and interactive vertical diffusion. N. Bouttes et al.: Ocean processes during the last termination Halting the sinking of brines stops the transport of salt to the deep ocean. This transport of salt during the glacial period is re- sponsible for a density increase of the deep waters compared to upper waters and therefore a greater vertical stratiﬁcation relative to the preindustrial. This leads to a more isolated glacial deep water mass that can store a larger amount car- bon. When the vertical salt transport is stopped, the stratiﬁca- tion breaks down and the thermohaline circulation changes: both the North Atlantic and Southern ocean overturning cells become more vigorous (Fig. 5). Indeed, with the stratiﬁca- tion induced by the sinking of brines the maximum of the NADW export is around 15 Sv compared to around 20 Sv without it. When the stratiﬁcation breaks down, the for- mation of deep water intensiﬁes rapidly to 22 Sv in around 1500 years. It then decreases more slowly back to 20 Sv. The atmospheric CO2 follows the evolution of the oceanic circulation as the carbon stored in the abyss is progressively released when the overturning circulation increases. 4500 years (Fig. 5). Hence, the time to reach 95 % of the equilibrium value is very similar to the time in the experi- ment with iron fertilization (∼4400 years, Table 2). The amplitude of the 1δ13Cocean decrease is more signif- icant with the brine sinking mechanism than with the iron fertilization mechanism (Fig. 3), with a decrease of the ver- tical gradient of ∼0.57 ‰. The induced stratiﬁcation has a large impact on the vertical δ13Cocean gradient as it re- duces the mixing between the 13C enriched upper water and 13C depleted deep waters. As a result, it better pre- serves the vertical gradient due to biological activity (Bouttes et al., 2010). In contrast, the change in δ13Catm is simi- lar to the one induced by suppression of the iron fertiliza- tion. The sinking of brines has an important impact on both 1δ13Cocean and δ13Catm because it efﬁciently increases up- per ocean δ13C and decreases deep ocean δ13C. When the stratiﬁcation breaks down, it leads to a decrease of upper ocean δ13C and δ13Catm. The iron fertilization mechanism has a smaller effect on 1δ13Cocean partly because remineral- ization not only takes place in the deep ocean, but also above. N. Bouttes et al.: Ocean processes during the last termination 155 0 2000 4000 6000 8000 10000 time (years) 14 15 16 17 18 19 20 21 22 23 max NADW (Sv) brines no brines or declining brines a iron brines "abrupt" brines "linear" brines-Kz "abrupt" brines-Kz "linear" 0 2000 4000 6000 8000 10000 time (years) 25 20 15 10 5 min AABW (Sv) brines no brines or declining brines b Fig. 5. Evolution of the thermohaline circulation with a constant climate. (a) Evolution of the maximum value of the North Atlantic stream function (Sv) and (b) evolution of the minimum value of the Southern Ocean stream function (Sv). a Table 2. Time for the anomaly of the considered variable (deﬁned as the difference between its value and the initial value) to reach 95 % of the equilibrium anomaly value (equilibrium anomaly value taken as the difference between the value at year 12 000 of the sim- ulations and the initial value) under a constant glacial climate for (a) CO2, (b) 1δ13Cocean, and (c) δ13Catm. “Iron” corresponds to iron fertilization, “Brines” to the sinking of brines, and Brines-Kz to the sinking of brines and interactive diffusion. The two scenarios for iron fertilization and the sinking of brines (“abrupt” and “lin- ear”) are deﬁned in Fig. 1. 0 2000 4000 6000 8000 10000 time (years) 14 15 16 17 18 19 20 21 22 23 max NADW (Sv) brines no brines or declining brines a iron brines "abrupt" brines "linear" brines-Kz "abrupt" brines-Kz "linear" 5 b (a) CO2 “abrupt” scenario “linear” scenario Iron 120 years 4360 years Brines 900 years 4370 years Brines-Kz 4270 years 7440 years (b) 1δ13Cocean Iron 1250 years 6250 years Brines 1250 years 5750 years Brines-Kz 5000 years 8250 years (c) δ13Catm Iron 1270 years 5890 years Brines 1270 years 5850 years Brines-Kz 5140 years 8570 years b time (years) 0 2000 4000 6000 8000 10000 time (years) 25 20 15 10 5 min AABW (Sv) brines no brines or declining brines b Fig. 5. Evolution of the thermohaline circulation with a constant climate. (a) Evolution of the maximum value of the North Atlantic stream function (Sv) and (b) evolution of the minimum value of the Southern Ocean stream function (Sv). the brine sinking mechanism involves changes in the ther- mohaline circulation through enhanced vertical stratiﬁcation (Bouttes et al., 2010). The thermohaline circulation takes more time to equilibrate than the biological activity. 3.1.3 Impact of stopping the sinking of brines Stopping the brine sinking mechanism leads to a larger at- mospheric CO2 increase than stopping the iron fertilization (Fig. 2b) with a change of 40 ppm (Table 1). This effect of the brine sinking is not the maximum possible effect (which would be obtained for frac = 1), but corresponds to a more realistic case (frac = 1 is very idealistic as it would require no mixing at all) with frac = 0.6, which is in the middle of the range of probable values according to proxy data (Bouttes et al., 2011). The response of the system to the abrupt halt of brine sinking takes more time than in the case of the iron fertilization. In the abrupt scenario, 95 % of the equilibrium value is reached 900 years after the stop (Table 2). Indeed, reached 95 % of the anomaly of the equilibrium state (Ta- ble 2). In the “abrupt” scenario, the system has reached 95 % of the equilibrium value ∼100 years after the stop of fertil- ization, i.e. very quickly compared to the time scale of the termination (a few thousand years). In the “linear” scenario, the response of the carbon cycle follows the forcing and it takes ∼4400 years for the system to reach 95 % of the equi- librium value. Clim. Past, 8, 149–170, 2012 www.clim-past.net/8/149/2012/ www.clim-past.net/8/149/2012/ N. Bouttes et al.: Ocean processes during the last termination 156 20000 18000 16000 14000 12000 10000 years 0.0 0.2 0.4 0.6 0.8 1.0 iron Iron fertilization a 20000 18000 16000 14000 12000 10000 years 0.0 0.2 0.4 0.6 0.8 1.0 frac Brines b Scenario "abrupt" Scenario "linear" 3.1.4 Impact of stopping the sinking of brines with the stratiﬁcation-dependent diffusion included The halt of the sinking of brines follows two scenarios: a sudden halt (“abrupt”) or a linear decline (“linear”). 3.1.4 Impact of stopping the sinking of brines with the stratiﬁcation-dependent diffusion included The addition of the stratiﬁcation-dependent diffusion mainly ampliﬁes the impact of the brine sinking mechanism. Be- cause of the lower vertical diffusion induced by the enhanced vertical density gradient, the deep water mass is even more isolated at the beginning of the simulation. It yields a lower initial CO2 level (Fig. 2c), higher initial 1δ13Cocean (Fig. 3c) (Bouttes et al., 2011), and higher initial δ13Catm (Fig. 4c). When the brine sinking stops, it thus leads to a larger CO2 increase reaching 61 ppm (Table 1). g pp ( ) Moreover, the interactive diffusion induces a delay in the oceanic circulation response. After a ﬁrst rapid increase of the export of North Atlantic Deep Water similar to the one in the simulation without the interactive diffusion (it reaches 20 Sv after around 500 years), the maximum export of NADW very slowly increases of around 1 Sv in around 3500 years (Fig. 5). As the stratiﬁcation breaks down, the dif- fusion coefﬁcent which is interactively computed increases as well. Because the mixing increases, it tends to diminish the density gradient and the export of water. As a conse- quence, it leads to the same delay in the carbon evolution. Indeed, with the interactive diffusion, the diffusion coefﬁ- cient is lower at the beginning of the simulation because of the enhanced stratiﬁcation. When the stratiﬁcation collapses, the diffusion coefﬁcient progressively increases because the vertical density gradient decreases. Yet it remains smaller than in the simulation without interactive diffusion (until the vertical density gradient is the same), so that the mixing is less important and the change of circulation smaller than in the ﬁxed diffusion simulation. Because of this delay, the CO2 reaches 95 % of the equilibrium value ∼4300 years after the sudden halt of brine sinking, i.e. ∼3400 years later than with the brines mechanism alone. Similarly, in the “linear” sce- nario it takes ∼7400 years for the system to reach 95 % of the equilibrium value, i.e. ∼3100 years later compared to the brines alone. Fig. 6. Evolution scenarios for (a) iron fertilization and (b) the sink- ing of brines during the last deglaciation. The two processes are active at the beginning of the simulations then stop. The iron fer- tilization decline follows the dust transport as recorded in ice cores (Wolff et al., 2006) on the EDC3 age model (Parrenin et al., 2007). N. Bouttes et al.: Ocean processes during the last termination Even if the surface δ13Cocean is signiﬁcantly increased with iron fertilization (as well as δ13Catm), the deep (−5000 m to −3000 m) δ13Cocean is relatively less modifed (the reminer- alization also releases 12C in intermediate waters) and there- fore 1δ13Cocean changes less than with the sinking of brines. In the “linear” scenario the thermohaline circulation has more time to adapt and the evolution is smoother. The maximum export of NADW increases gradually from 15 Sv to around 20 Sv, the latter being reached after around Clim. Past, 8, 149–170, 2012 www.clim-past.net/8/149/2012/ Bouttes et al.: Ocean processes during the last termination 20000 18000 16000 14000 12000 10000 years 0.0 0.2 0.4 0.6 0.8 1.0 iron Iron fertilization a 20000 18000 16000 14000 12000 10000 years 0.0 0.2 0.4 0.6 0.8 1.0 frac Brines b Scenario "abrupt" Scenario "linear" Fig. 6. Evolution scenarios for (a) iron fertilization and (b) the sink- ing of brines during the last deglaciation. The two processes are active at the beginning of the simulations then stop. The iron fer- tilization decline follows the dust transport as recorded in ice cores (Wolff et al., 2006) on the EDC3 age model (Parrenin et al., 2007). The halt of the sinking of brines follows two scenarios: a sudden halt (“abrupt”) or a linear decline (“linear”). N. Bouttes et al.: Ocean processes during the last termination N. Bouttes et al.: Ocean processes during the last termination 157 The halt of the sinking of brines imposed in the model would, in reality, be due to the change of topography around Antarctica (Fig. 7). Indeed, the sinking of brines would de- pend on the volume of water above the shelves. It would thus depend on two variables: the evolution of sea level and the advance/retreat of the Antarctic ice sheet on the shelves. During interglacials, the volume of water above the continen- tal shelves is important and some mixing of the salt rejected by sea ice formation happens. The salty dense water can thus not easily sink to the bottom of the ocean (Fig. 7a). During the glaciation, the Antarctic ice sheet progressively increases both in volume and extension (Fig. 7b). Because of the exten- sion of the ice sheet and the sea level fall, the volume of water above the continental shelves is reduced and the salt less di- luted. Moreover, the release of salt increases as more sea ice is formed (in particular as the seasonality seems to increase; Gersonde et al., 2005). The salt rejected by sea ice formation can accumulate more and create very dense water suscepti- ble to ﬂow more easily down to the abyss. This is modelled by an increase of the fraction of salt that sinks to the deep ocean, the frac parameter. When the ice sheet reaches its maximum extent, i.e. when the continental shelves are cov- ered, a few thousand years after the Last Glacial Maximum (Ritz et al., 2001; Huybrechts, 2002), the sea ice formation is shifted to the open ocean. The absence of shelf where the brines sink, accumulate, and create very dense water suscep- tible to ﬂow down to the abyss, prevents the deep sinking of brines (Fig. 7c). In the open ocean, the dilution of the brines released by sea ice is more important and the effect of the brine-generated dense water disappears. If the conti- nental shelves are all covered simultaneously, it results in an abrupt halt of the sinking of brines. Alternatively, the halt of the sinking of brines can be linked to the sea level rise, which increases the volume of water above the continental shelf leading to more mixing and less sinking of dense water. It corresponds to a more progressive reduction of the sinking of brines, which can be ﬁrst approximated by a linear de- crease. 3.2 Evolution of the mechanisms during the last deglaciation with prescribed CO2 We now consider iron fertilization and the sinking of brines in the context of the global warming and ice sheet retreat of the last deglaciation. As described in the method section, in these simulations the three boundary conditions that are the atmospheric CO2, ice sheets, and orbital parameters vary through time. Atmospheric CO2 and ice sheets vary accord- ing to proxy data. The vegetation is either interactively cal- culated by the terrestrial biosphere model (VECODE) and therefore evolving with time according to the climate and CO2 concentration imposed or ﬁxed to the glacial distribu- tion as calculated in the glacial simulations (“ﬁxed veg”) in order to separate the impact of the ocean from the one of the vegetation. Iron fertilization and the sinking of brines follow different scenarios (Fig. 6). The evolution of iron fertiliza- tion is based on iron ﬂux records (Wolff et al., 2006) on the EDC3 age model (Parrenin et al., 2007). It is indeed mod- ulated by a parameter iron that can evolve between 0 and 1 following the iron ﬂux record. The parameter is at its maxi- mum (iron = 1) at the Last Glacial Maximum and close to 0 at the beginning of the Holocene (Fig. 6a). The evolution of the sinking of brines (which can not be directly constrained) is set by the same two idealised scenarios as in the previous part. The halt of the sinking of brines with the stratiﬁcation- dependent diffusion also leads to a decrease of both δ13Catm and 1δ13Cocean. The amplitude is slightly greater than with brines alone for δ13Catm (change of 0.34 ‰, Table 1) because of the ampliﬁcation due to the interactive diffusion. The am- plitude is, however, much higher for 1δ13Cocean with the interactive diffusion, which plays an important role in the ocean. It isolates even more the deep ocean which strongly decreases the deep ocean δ13C. The additional delay because of the progressive return to modern diffusion values is appar- ent in both cases with δ13C reaching 95 % of the equilibrium value approximately 3800–3900 years later than with brines alone. In the “linear” scenario, the ocean has more time to adapt and this delay is reduced to 2500–2700 years. Clim. Past, 8, 149–170, 2012 www.clim-past.net/8/149/2012/ www.clim-past.net/8/149/2012/ N. Bouttes et al.: Ocean processes during the last termination These two extreme scenarios (“linear” and “abrupt”) of the halt of the sinking of brines are both tested. The two scenarios explore the two extreme cases, a more probable one would lie between the two. According to proxy data, the West Antarctic ice sheet advanced to the outer shelf in most regions (Anderson et al., 2002). Because the glaciation and deglaciation are not symetrical with respect to the sea level change and advance and retreat of the Antarctic ice sheet on the continental shelves, the evolution of the sinking of brines would also not be symetrical. In particular, according to data, the Antarctic ice sheet melting starts later than the northern ones, around 14 kyr BP (Clark et al., 2009; Mackintosh et al., 2011). At that time, the sea level is already higher than at the LGM because the Northern ice sheet started to melt earlier. In association with the input of fresh water from the melt- ing of the Antarctic ice sheet, it would prevent the important sinking of the brines to happen again. It is thus not possi- katabatic wind sea ice brines katabatic wind sea ice brines interglacial sea level katabatic wind sea ice brines interglacial sea level Interglacial Glaciation Maximum Antarctic extent frac ~ 0 frac > 0 frac = 0 ice shelf Antarctic ice sheet continental slope continental shelf (a) (b) (c) Fig. 7. Schematic representation of the sinking of brines during (a) interglacial, (b) glacial and (c) deglacial periods. The main drivers of the fraction of salt sinking to the deep ocean (frac) are the Antarctic ice sheet extent on the continental shelf and sea level which govern the volume of water above the continental shelf. The less water there is, the less brines are mixed and the more they can sink into the deep ocean. When sea ice formation is shifted to the open ocean, brines are mixed and the sinking stops. katabatic wind sea ice brines Interglacial frac ~ 0 ice shelf Antarctic ice sheet continental slope continental shelf (a) katabatic wind sea ice brines interglacial sea level Glaciation frac > 0 (b) (b) katabatic wind sea ice brines interglacial sea level Maximum Antarctic extent frac = 0 (c) Fig. 7. Schematic representation of the sinking of brines during (a) interglacial, (b) glacial and (c) deglacial periods. N. Bouttes et al.: Ocean processes during the last termination The main drivers of the fraction of salt sinking to the deep ocean (frac) are the Antarctic ice sheet extent on the continental shelf and sea level which govern the volume of water above the continental shelf. The less water there is, the less brines are mixed and the more they can sink into the deep ocean. When sea ice formation is shifted to the open ocean, brines are mixed and the sinking stops. katabatic wind sea ice brines interglacial sea level Maximum Antarctic extent frac = 0 (c) Maximum Antarctic extent Maximum Antarctic extent (c) Fig. 7. Schematic representation of the sinking of brines during (a) interglacial, (b) glacial and (c) deglacial periods. The main drivers of the fraction of salt sinking to the deep ocean (frac) are the Antarctic ice sheet extent on the continental shelf and sea level which govern the volume of water above the continental shelf. The less water there is, the less brines are mixed and the more they can sink into the deep ocean. When sea ice formation is shifted to the open ocean, brines are mixed and the sinking stops. (although not at the very beginning of the deglaciation since it was inhibited by the presence of the Antarcic ice sheet on the shelves), yet it would be relatively less important. How- ever, this possibility is beyond the scope of this study, which explores the mean trend during the deglaciation, and would require further work to focus on the effect of a brief activa- tion of the sinking of brines during the deglaciation, probably in link with more rapid events. Clim. Past, 8, 149–170, 2012 www.clim-past.net/8/149/2012/ 20000 18000 16000 14000 12000 10000 180 200 220 240 260 280 300 320 340 CO2 (ppm) CTRL runs a data CTRL + fixed veg CTRL-CC + fixed veg 20000 18000 16000 14000 12000 10000 180 200 220 240 260 280 CO2 (ppm) Iron fertilization b data iron + fixed veg 20000 18000 16000 14000 12000 10000 180 200 220 240 260 280 CO2 (ppm) Brines c data brines "abrupt" + fixed veg brines "linear" + fixed veg d 3 a 20000 18000 16000 14000 12000 10000 180 200 220 240 260 280 CO2 (ppm) Iron fertilization data iron + fixed veg 20000 18000 16000 14000 12000 10000 180 200 220 240 260 280 CO2 (ppm) Brines c data brines "abrupt" + fixed veg brines "linear" + fixed veg 20000 18000 16000 14000 12000 10000 180 200 220 240 260 280 CO2 (ppm) Brines-iron d data brines-iron "abrupt" + fixed veg brines-iron "linear" + fixed veg 20000 18000 16000 14000 12000 10000 years 180 200 220 240 260 280 CO2 (ppm) Brines-Kz e data brines-Kz "abrupt" + fixed veg brines-Kz "linear" + fixed veg Fig. 8. Evolution of atmospheric CO2 during the last deglaciation with different oceanic mechanisms and comparison with data: (a) control (CTRL) runs without and with carbonate compensation (CC), all other simulations are with carbonate compensation, (b) simulations with iron fertilization, (c) simulations with the sinking of brines (two scenarios as deﬁned in Fig. 6), (d) simulations with the sinking of brines (two scenarios as deﬁned in Fig. 6) and iron fertilization, (e) simulations with the sinking of brines (two scenarios as deﬁned in Fig. 6) and interactive vertical diffusion coefﬁcient (Kz). Each simulation has either an interactive vegetation or a ﬁxed glacial vegetation (“ﬁxed veg”). The data are from Monnin et al. (2001); Lourantou et al. (2010). 20000 18000 16000 14000 12000 10000 180 200 220 240 260 280 CO2 (ppm) Brines-iron d data brines-iron "abrupt" + fixed veg brines-iron "linear" + fixed veg 20000 18000 16000 14000 12000 10000 years 180 200 220 240 260 280 CO2 (ppm) Brines-Kz e data brines-Kz "abrupt" + fixed veg brines-Kz "linear" + fixed veg Fig. 8. www.clim-past.net/8/149/2012/ N. Bouttes et al.: Ocean processes during the last termination 158 20000 18000 16000 14000 12000 10000 180 200 220 240 260 280 300 320 340 CO2 (ppm) CTRL runs a data CTRL + fixed veg CTRL-CC + fixed veg 20000 18000 16000 14000 12000 10000 180 200 220 240 260 280 CO2 (ppm) Iron fertilization b data iron + fixed veg 20000 18000 16000 14000 12000 10000 180 200 220 240 260 280 CO2 (ppm) Brines c data brines "abrupt" + fixed veg brines "linear" + fixed veg 20000 18000 16000 14000 12000 10000 180 200 220 240 260 280 CO2 (ppm) Brines-iron d data brines-iron "abrupt" + fixed veg brines-iron "linear" + fixed veg 20000 18000 16000 14000 12000 10000 years 180 200 220 240 260 280 CO2 (ppm) Brines-Kz e data brines-Kz "abrupt" + fixed veg brines-Kz "linear" + fixed veg n of atmospheric CO2 during the last deglaciation with different oceanic mechanisms and comparison with data: (a) co thout and with carbonate compensation (CC), all other simulations are with carbonate compensation, (b) simulations (c) simulations with the sinking of brines (two scenarios as deﬁned in Fig. 6), (d) simulations with the sinking of b s deﬁned in Fig. 6) and iron fertilization, (e) simulations with the sinking of brines (two scenarios as deﬁned in Fig. 6 al diffusion coefﬁcient (Kz). Each simulation has either an interactive vegetation or a ﬁxed glacial vegetation (“ﬁxed v m Monnin et al. (2001); Lourantou et al. (2010). 3.2.1 Control simulations of the evolution of the carbon cycle during the deglaciation p The evolution of δ13Cocean in the ocean is a good indica- tor of the physical processes involved and an important con- straint. One of the more striking features of the changes from the glacial to the interglacial state is the increase in the deep Southern Ocean δ13Cocean value. Hence, we focus on the evolution of δ13C at one site in the deep Southern Ocean. We compare the simulation results to the record from core MD07-3076Q (Skinner et al., 2010; Waelbroeck et al., 2011), which has a good resolution and is well dated. The simu- lated δ13Cocean at that site shows an increase of ∼0.3 ‰ only (Fig. 10a), a small amplitude compared to the measured total variation of ∼1.3 ‰. Without carbonate compensation, the initial value of atmo- spheric CO2 at −21 000 years is 298 ppm (Fig. 8a) due to the opposite effects of oceanic and vegetation changes (Brovkin et al., 2007; Bouttes et al., 2010). The effect of changing the boundary conditions to the LGM ones (orbital parame- ters, ice sheets and atmospheric CO2) is to decrease CO2 by 24 ppm, mainly because of the colder climate which results in greater solubility of CO2 in the glacial ocean compared to the preindustrial. The increase of nutrient concentrations due to the sea level fall of approximately 120 m also reduces CO2 relative to the preindustrial since the biological production is enhanced. This effect is relatively small as CO2 is reduced by 3 ppm. Yet, the sea level fall also increases the global oceanic salinity, which decreases CO2 solubility in the ocean, thus increasing CO2. The simulated CO2 increase due to the global salinity increase is of 7 ppm. Finally, the terrestrial biosphere carbon content is reduced by ∼650 GtC compared to the Holocene (Fig. 9), which releases carbon into the at- mosphere leading to an increase of CO2 of 38 ppm in the model. This effect does not take into account the increase of terrestrial biosphere carbon content on shelf areas previously ﬂooded, which would be between 182 and 417 GtC (Mon- tenegro et al., 2006; Joos et al., 2004). Although the ocean partially stores some of the released carbon, part of it remains in the atmosphere. This effect prevails and the simulated at- mospheric CO2 is 298 ppm at the LGM, a value very different from the data (∼190 ppm). N. Bouttes et al.: Ocean processes during the last termination 159 N. Bouttes et al.: Ocean processes during the last terminat 20000 18000 16000 14000 12000 10000 years 1600 1700 1800 1900 2000 2100 2200 2300 2400 Terrestrial Biosphere (GtC) LGM LGM-CC iron brines "abrupt" brines "linear" brines-iron "abrupt" brines-iron "linear" brines-Kz "abrupt" brines-Kz "linear" Fig. 9. Evolution of the carbon stock from the terrestrial bio- sphere (GtC) during the deglaciation for the simulations as deﬁned in Fig. 6, when the terrestrial biosphere is interactive. to 320 ppm when the vegetation is ﬁxed. CO2 increases be- cause of the warming and diminished global oceanic nutrient concentrations. The decrease of salinity tends to counteract the increase, but the overall evolution is still a CO2 increase when the vegetation is ﬁxed (“ﬁxed veg”). When the evolu- tion of vegetation is accounted for (interactive vegetation), it results in a CO2 decrease as the terrestrial biosphere carbon content progressively increases (Fig. 9). This effect prevails leading to the simulated decrease of atmospheric CO2. With carbonate compensation (CTRL-CC), the uptake of carbon by the ocean is ampliﬁed during the LGM, which results in a lower glacial CO2 in the initial state, around 260 ppm (Fig. 8a). When the vegetation is ﬁxed, the evo- lution of CO2 in the transient simulation is the same as in the previous simulation with a 20 ppm increase. When the vegetation is interactive, the CO2 remains roughly constant because the terrestrial biosphere carbon content increases, which acts to decrease CO2 and counteracts the CO2 increase from oceanic processes. Fig. 9. Evolution of the carbon stock from the terrestrial bio- sphere (GtC) during the deglaciation for the simulations as deﬁned in Fig. 6, when the terrestrial biosphere is interactive. www.clim-past.net/8/149/2012/ Evolution of atmospheric CO2 during the last deglaciation with different oceanic mechanisms and comparison with data: (a) control (CTRL) runs without and with carbonate compensation (CC), all other simulations are with carbonate compensation, (b) simulations with iron fertilization, (c) simulations with the sinking of brines (two scenarios as deﬁned in Fig. 6), (d) simulations with the sinking of brines (two scenarios as deﬁned in Fig. 6) and iron fertilization, (e) simulations with the sinking of brines (two scenarios as deﬁned in Fig. 6) and interactive vertical diffusion coefﬁcient (Kz). Each simulation has either an interactive vegetation or a ﬁxed glacial vegetation (“ﬁxed veg”). The data are from Monnin et al. (2001); Lourantou et al. (2010). Clim. Past, 8, 149–170, 2012 www.clim-past.net/8/149/2012/ N. Bouttes et al.: Ocean processes during the last termination 3.2.1 Control simulations of the evolution of the carbon cycle during the deglaciation For more details on these differ- ent effects we refer to Brovkin et al. (2007) and Bouttes et al. (2010). Atmospheric δ13Catm is sensitive to the evolution of the terrestrial biosphere (Fig. 11a). The difference between the simulated δ13Catm with ﬁxed vegetation (“ﬁxed veg”) and interactive vegetation gets greater with time and equals ∼0.4 ‰ at −10 000 years. With ﬁxed vegetation, the changes in δ13Catm are only due to processes taking place in the ocean. As δ13Cocean does not change much, it induces a con- stant δ13Catm value. The difference between the simulations with interactive vegetation and “ﬁxed veg” is only due to the terrestrial biosphere whose carbon content progressively in- creases (Fig. 8). Since the biosphere preferentially takes the light 12C over 13C during photosynthesis, the atmosphere be- comes enriched in 13C and δ13Catm increases (Fig. 11a). Yet, this trend disagrees with the data showing a “W” shape, sug- gesting a more complex evolution of mechanisms. Even when taking carbonate compensation into account, the computed CO2, atmospheric and deep oceanic δ13C evo- lutions are far from reproducing the evolution depicted by the data, underlining the need for additional mechanisms to ex- plain the carbon cycle evolution. In the following, we test the three additional oceanic mechanisms described before during the deglaciation and assess their impact on the carbon cycle evolution when the global climate warms. From this initial state, in the control glacial simulation without carbonate compensation (CTRL), the atmospheric CO2 slightly decreases from 298 ppm 21 000 years ago to 280 ppm 10 000 years ago when the terrestrial biosphere evo- lution is taken into account (Fig. 8a) whereas CO2 increases Clim. Past, 8, 149–170, 2012 www.clim-past.net/8/149/2012/ www.clim-past.net/8/149/2012/ N. Bouttes et al.: Ocean processes during the last termination 160 20000 18000 16000 14000 12000 10000 1.5 1.0 0.5 0.0 0.5 1.0 δ13 Cocean (permil) CTRL runs a data CTRL + fixed veg CTRL-CC + fixed veg 20000 18000 16000 14000 12000 10000 1.5 1.0 0.5 0.0 0.5 1.0 δ13 Cocean (permil) Iron fertilization b data iron + fixed veg 20000 18000 16000 14000 12000 10000 1.5 1.0 0.5 0.0 0.5 1.0 δ13 Cocean (permil) Brines c data brines "abrupt" + fixed veg brines "linear" + fixed veg 20000 18000 16000 14000 12000 10000 1.5 1.0 0.5 0.0 0.5 1.0 δ13 Cocean (permil) Brines-iron d data brines-iron "abrupt" + fixed veg brines-iron "linear" + fixed veg 20000 18000 16000 14000 12000 10000 years 1.5 1.0 0.5 0.0 0.5 1.0 δ13 Cocean (permil) Brines-Kz e data brines-Kz "abrupt" + fixed veg brines-Kz "linear" + fixed veg volution of deep Southern Ocean δ13C during the last deglaciation with different oceanic mechanisms and compa ontrol (CTRL) runs without and with carbonate compensation (CC), all other simulations are with carbonate com ons with iron fertilization, (c) simulations with the sinking of brines (two scenarios as deﬁned in Fig. 6), (d) simula of brines (two scenarios as deﬁned in Fig. 6) and iron fertilization, (e) simulations with the sinking of brines (two sc Fig. 6) and interactive vertical diffusion coefﬁcient (Kz). Each simulation has either an interactive vegetation or a ﬁx “ﬁxed veg”). The data are from Waelbroeck et al. (2011). www.clim-past.net/8/149/2012/ Bouttes et al.: Ocean processes during the last termination 161 20000 18000 16000 14000 12000 10000 7.0 6.8 6.6 6.4 6.2 δ13 Catm (permil) CTRL runs a data CTRL + fixed veg CTRL-CC + fixed ve 20000 18000 16000 14000 12000 10000 7.0 6.8 6.6 6.4 6.2 δ13 Catm (permil) Iron fertilization b data iron + fixed veg 20000 18000 16000 14000 12000 10000 7.0 6.8 6.6 6.4 6.2 δ13 Catm (permil) Brines c data brines "abrupt" + fixed veg brines "linear" + fixed veg 20000 18000 16000 14000 12000 10000 years 7.0 6.8 6.6 6.4 6.2 δ13 Catm (permil) Brines-iron d data brines-iron "abrupt" + fixed veg brines-iron "linear" + fixed veg 20000 18000 16000 14000 12000 10000 7.0 6.8 6.6 6.4 6.2 δ13 Catm (permil) Brines-Kz e data brines-Kz "abrupt" + fixed veg brines-Kz "linear" + fixed veg Evolution of atmospheric δ13Catm during the last deglaciation with different oceanic mechanisms and comparison with data: (a) con- TRL) runs without and with carbonate compensation (CC), all other simulations are with carbonate compensation, (b) simulations n fertilization, (c) simulations with the sinking of brines (two scenarios as deﬁned in Fig. 6), (d) simulations with the sinking of two scenarios as deﬁned in Fig. 6) and iron fertilization, (e) simulations with the sinking of brines (two scenarios as deﬁned in Fig. 6) ractive vertical diffusion coefﬁcient (Kz). Each simulation has either an interactive vegetation or a ﬁxed glacial vegetation (“ﬁxed The data are from Lourantou et al. (2010). 20000 18000 16000 14000 12000 10000 7.0 6.8 6.6 6.4 6.2 δ13 Catm (permil) CTRL runs a data CTRL + fixed veg CTRL-CC + fixed ve b 20000 18000 16000 14000 12000 10000 7.0 6.8 6.6 6.4 6.2 δ13 Catm (permil) CTRL runs a data CTRL + fixed veg CTRL-CC + fixed ve 20000 18000 16000 14000 12000 10000 7.0 6.8 6.6 6.4 6.2 δ13 Catm (permil) Iron fertilization b data iron + fixed veg 20000 18000 16000 14000 12000 10000 7.0 6.8 6.6 6.4 6.2 δ13 Catm (permil) Brines c data brines "abrupt" + fixed veg brines "linear" + fixed veg b y 20000 18000 16000 14000 12000 10000 7.0 6.8 6.6 6.4 6.2 δ13 Catm (permil) Brines-Kz data brines-Kz "abrupt + fixed veg brines-Kz "linear" + fixed veg Fig. 11. www.clim-past.net/8/149/2012/ 20000 18000 16000 14000 12000 10000 1.5 1.0 0.5 0.0 0.5 1.0 δ13 Cocean (permil) CTRL runs a data CTRL + fixed veg CTRL-CC + fixed veg 20000 18000 16000 14000 12000 10000 1.5 1.0 0.5 0.0 0.5 1.0 δ13 Cocean (permil) Iron fertilization b data iron + fixed veg 20000 18000 16000 14000 12000 10000 1.5 1.0 0.5 0.0 0.5 1.0 δ13 Cocean (permil) Brines c data brines "abrupt" + fixed veg brines "linear" + fixed veg d 20000 18000 16000 14000 12000 10000 1.5 1.0 0.5 0.0 0.5 1.0 δ13 Cocean (permil) Iron fertilization data iron + fixed veg 20000 18000 16000 14000 12000 10000 1.5 1.0 0.5 0.0 0.5 1.0 δ13 Cocean (permil) Brines c data brines "abrupt" + fixed veg brines "linear" + fixed veg 20000 18000 16000 14000 12000 10000 1.5 1.0 0.5 0.0 0.5 1.0 δ13 Cocean (permil) Brines-iron d data brines-iron "abrupt" + fixed veg brines-iron "linear" + fixed veg 20000 18000 16000 14000 12000 10000 years 1.5 1.0 0.5 0.0 0.5 1.0 δ13 Cocean (permil) Brines-Kz e data brines-Kz "abrupt" + fixed veg brines-Kz "linear" + fixed veg Fig. 10. Evolution of deep Southern Ocean δ13C during the last deglaciation with different oceanic mechanisms and comparison with data: (a) control (CTRL) runs without and with carbonate compensation (CC), all other simulations are with carbonate compensation, (b) simulations with iron fertilization, (c) simulations with the sinking of brines (two scenarios as deﬁned in Fig. 6), (d) simulations with the sinking of brines (two scenarios as deﬁned in Fig. 6) and iron fertilization, (e) simulations with the sinking of brines (two scenarios as deﬁned in Fig. 6) and interactive vertical diffusion coefﬁcient (Kz). Each simulation has either an interactive vegetation or a ﬁxed glacial vegetation (“ﬁxed veg”). The data are from Waelbroeck et al. (2011). Clim. Past, 8, 149–170, 2012 www.clim-past.net/8/149/2012/ N. www.clim-past.net/8/149/2012/ Evolution of atmospheric δ13Catm during the last deglaciation with different oceanic mechanisms and comparison with data: (a) con- trol (CTRL) runs without and with carbonate compensation (CC), all other simulations are with carbonate compensation, (b) simulations with iron fertilization, (c) simulations with the sinking of brines (two scenarios as deﬁned in Fig. 6), (d) simulations with the sinking of brines (two scenarios as deﬁned in Fig. 6) and iron fertilization, (e) simulations with the sinking of brines (two scenarios as deﬁned in Fig. 6) and interactive vertical diffusion coefﬁcient (Kz). Each simulation has either an interactive vegetation or a ﬁxed glacial vegetation (“ﬁxed veg”). The data are from Lourantou et al. (2010). Clim. Past, 8, 149–170, 2012 3.2.2 Impact of iron fertilization The sinking of brines has a more striking impact on δ13Catm (Fig. 11c). The abrupt halt of the sinking of brines at −18 000 years leads to a decrease of δ13Catm as the carbon from the deep ocean characterized by low δ13C is released to the atmosphere. The decrease is larger when the vegetation is ﬁxed because it only accounts for the oceanic change while in the simulation with interactive vegetation the increase of ter- restrial biosphere carbon content (which increases δ13Catm) counteracts the oceanic effect. When the halt of the sinking of brines is more progressive, the change of δ13Catm due to the ocean takes place later so that when the vegetation is in- teractive, the change due to the vegetation cancels out the one from the ocean and no decrease is simulated. The simulated atmospheric CO2 evolution improves when including the iron fertilization mechanism and rises from ∼230 ppm at −21 000 years to ∼260 ppm at −10 000 years (Fig. 8b). As the idealized experiment with a ﬁxed climate has shown, the iron fertilization reacts quickly to the imposed forcing and the response thus follows the iron availability. The amplitude of the CO2 change due to iron fertilization is probably over-estimated in this simulation as a state-of-the- art GCM including an iron cycle indicates a more probable modern to LGM CO2 decline of 15–20 ppm due to iron fer- tilization (Bopp et al., 2003; Tagliabue et al., 2009). The computed LGM deep ocean δ13C evolution also im- proves (Fig. 10b), yet its amplitude is clearly not large enough (the glacial value is ∼0 ‰ compared to ∼−1 ‰ in the data). The δ13Catm evolution changes as well (Fig. 11b). The initial glacial value is increased by 0.2 ‰ compared to the control simulation. Following the evolution of iron ﬂux, iron fertilization decreases during the deglaciation. Simi- larly to the terrestrial biosphere, marine biology preferen- tially uses 12C over 13C, which decreases δ13C in the deep ocean and increases it in the upper ocean and atmosphere. When iron fertilization becomes lower, the biological pump decreases and the deep ocean ocean δ13C increases while δ13Catm decreases. In the atmosphere, because this effect is of the same amplitude as the effect of the terrestrial bio- sphere, but with an opposite signe, the overall simulated evo- lution of δ13Catm is ﬂat. 3.2.4 Impact of the sinking of brines and iron fertilization With both the sinking of brines and iron fertilization included in the model, the entire amplitude of the CO2 rise from ∼190 ppm at −21 000 years to ∼260 ppm at −10 000 years is simulated (Fig. 8d). The “abrupt” scenario leads to an early increase of CO2, which begins in advance compared to the data (around 1000 years in advance). The “linear” scenario is more similar to the data evolution. The separate effects of the sinking of brines and iron fertilization globally reinforce each other. Indeed, the sinking of brines alters the ocean dy- namics while iron fertilization changes the marine biology. The sinking of brines induces a slight reduction of the sur- face nutrient concentration, hence a diminished effect of iron fertilization, yet the impact on atmospheric CO2 is very small (a few ppm only). Thus, the two mechanisms are almost in- dependent of each other and their effects linearly add to each other. This combined effect is responsible for the very steep increase of CO2 in the “abrupt” scenario as both mechanisms have a fast response of their own. N. Bouttes et al.: Ocean processes during the last termination 162 3.3 Evolution of the mechanisms during the last deglaciation with interactive CO2 Fig. 12. Evolution of the thermohaline circulation during the deglaciation. (a) Evolution of the maximum value of the North At- lantic stream function (Sv) and (b) evolution of the maximum value of the Southern Ocean stream function (Sv). Contrary to the previous section, here the climate and car- bon models are fully coupled and atmospheric CO2 is no longer prescribed but interactively computed. Because of this change in the version of the model, it is required to simulate new initial glacial conditions. We ﬁrst evaluate these new glacial equilibrium runs before exploring the evolution of the interactive carbon cycle during the deglaciation. 3.2.3 Impact of the sinking of brines Bouttes et al.: Ocean processes during the last termination N. Bouttes et al.: Ocean processes during the last termination 163 20000 18000 16000 14000 12000 10000 time (years) 14 16 18 20 22 24 NADW (Sv) a CTRL brines "abrupt" brines "linear" brines-Kz "abrupt" brines-Kz "linear" 20000 18000 16000 14000 12000 10000 time (years) 22 20 18 16 14 12 10 8 6 AABW (Sv) b Fig. 12. Evolution of the thermohaline circulation during the deglaciation. (a) Evolution of the maximum value of the North At- lantic stream function (Sv) and (b) evolution of the maximum value of the Southern Ocean stream function (Sv). 20000 18000 16000 14000 12000 10000 time (years) 14 16 18 20 22 24 NADW (Sv) a CTRL brines "abrupt" brines "linear" brines-Kz "abrupt" brines-Kz "linear" b vegetation while the minima are close to the values of the “ﬁxed veg” simulations. Overall the δ13Catm record seems to oscillate between these two states, which could indicate a more complex evolution of the vegetation that a simple lin- ear increase during the transition. The vegetation could begin to increase later that in the simulation, then decrease around −13 000–12 000 years and then increase again (K¨ohler et al., 2005a). It conﬁrms that the evolution of the terrestrial bio- sphere has an important impact on δ13Catm (Lourantou et al., 2010) and should be studied in more details in the future. It results that the CO2 evolution can be simulated in agree- ment with data either with the combination of brines and iron fertilisation or brines and interactive diffusion. However, the evolution of δ13Cocean better match the data with the brine and interactive diffusion combination while the δ13Catm is slightly better with the brines and iron combination. In the following we thus take the three mechanisms simultaneously into account. Because the CO2 is simulated in agreement with data, we also change the version of the model to a fully carbon-climate coupled version. The simulations are thus not forced by a CO2 record anymore. time (years) 20000 18000 16000 14000 12000 10000 time (years) 22 20 18 16 14 12 10 8 6 AABW (Sv) b b 3.2.5 Impact of the sinking of brines and stratiﬁcation-dependent diffusion With a combination of the sinking of brines and the stratiﬁcation-dependent diffusion, the amplitude of the CO2 increase is also in line with the data (Fig. 8e). As observed with a constant climate forcing, the effect of the brine in- duced stratiﬁcation is ampliﬁed, the deep water mass is fur- ther isolated and stores more carbon at the LGM. The in- teractive diffusion also generates a delay in the oceanic re- sponse to the breakdown of the stratiﬁcation and CO2 is more progressively released. Hence the scenario that best agrees with the CO2 record is now the “abrupt” scenario whereas the “linear” scenario is delayed and rises too late. 3.2.3 Impact of the sinking of brines Taking into account the halt of the sinking of brines improves the computed CO2 evolution (Fig. 8c). Atmospheric CO2 in- creases from ∼220 ppm at −21 000 years to ∼260 ppm at −10 000 years. The transition is however different for the two brine scenarios. When the sinking of brines is suddenly stopped (referred as “abrupt” in the ﬁgure captions) the CO2 rapidly increases. The degassing due to the break down of the stratiﬁcation and reorganisation of the thermohaline cir- culation (Fig. 12) takes approximately 3000 years to reach the level of the control evolution. The linear reduction of the sinking of brines (“linear”) globally follows the forcing, which results in a smoother increase of atmospheric CO2. Yet, although the CO2 evolution is slightly closer to the data when including the sinking of brines than with iron fertiliza- tion, none of them alone can account for the entire CO2 rise during the deglaciation (Fig. 8), which is mainly due to the mismatch of the initial states compared to the data. Yet, the evolution of δ13Cocean is still relatively different from the data (Fig. 10d), with a decrease of ∼0.7 ‰ com- pared to ∼1.3 ‰ in the data, mostly because of the mismatch between the initial value and the data. The sinking of brines has an important effect on δ13Cocean but the iron fertilization has a very small effect and the addition of the two cannot ac- count for the entire measured evolution during deglaciation. The combination of the sinking of brines and iron fertil- ization yields better results for the δ13Catm evolution when the vegetation is ﬁxed (Fig. 11d, dotted lines). In particu- lar, between −18 000 years and −15 000 years the halt of the sinking of brines in addition to the diminishing iron fertil- ization produces a simulated δ13Catm decrease closer to the data. Yet when the vegetation is interactive (solid lines) this effect is counteracted by the increase of δ13Catm due to the expansion of the vegetation. The evolution of the deep ocean δ13C is improved com- pared to the control simulations (Fig. 10c) as the initial LGM δ13Cocean value is closer to the data (the simulated δ13Cocean is ∼0 ‰ compared to ∼0.3 ‰ in the control simulations and ∼−1 ‰ in the data). It is nonetheless still too high compared to the data. www.clim-past.net/8/149/2012/ Clim. Past, 8, 149–170, 2012 N. 3.3.1 Initial glacial conditions We ﬁrst carry out two glacial equilibrium simulations with (“LGM-all”) and without (“LGM-ctrl”) the additional mech- anisms studied. The runs start from previous glacial equilib- rium runs (Bouttes et al., 2011) performed with prescribed CO2 values (190 ppm) for the climate model. The values of the parameters for the additional mechanisms included in the “LGM-all” simulation are based on the results from ensem- ble simulations (Bouttes et al., 2011) (frac = 0.6 for the sink- ing of brines, iron = 0.3 for iron fertilization and alpha = 0.7 for the stratiﬁcation dependand diffusion). With these three additional mechanisms (“LGM-all”), the climate at the end of the 50 000 year simulation is slightly colder compared to the standard glacial simulation (0.3 ◦C colder). The global air surface temperature during the LGM is 4 ◦C colder com- pared to the preindustrial. In the “LGM-ctrl” run, CO2 in- creases from ∼254 ppm and equilibrates close to preindus- trial level (∼284 ppm) (Fig. 13). The atmospheric CO2 level is higher than the CO2 prescribed due to the carbon-climate feedbacks. Indeed, higher CO2 leads to higher temperature, which mainly reduces ocean solubility and further increases Contrary to the simulation with the sinking of brines and iron fertilization, the computed deep ocean δ13C is signiﬁ- cantly improved (Fig. 10e) and the amplitude of the increase closer to the data. The interactive diffusion also ampliﬁes the impact of the brines on δ13Cocean, which is further decreased in the LGM and then increases to the Holocene value dur- ing the deglaciation. The scenario that best matches the data evolution is again the “abrupt” scenario, the “linear” scenario yielding too late a rising. However, the simulated δ13Catm does not really improve (Fig. 11e). It appears that the maxima of the “W” shape correspond to the values of the simulations with interactive Clim. Past, 8, 149–170, 2012 www.clim-past.net/8/149/2012/ N. Bouttes et al.: Ocean processes during the last termination N. Bouttes et al.: Ocean processes during the last termination 164 Bouttes et al.: Ocean processes during the last termination 20 18 16 14 12 10 Age (kyr BP) 0.0 0.2 0.4 0.6 0.8 1.0 parameter (iron or frac) Forcing scenarios brines declining brines no brines a iron brines: abrupt 18k brines: abrupt 17k brines: linear brines: intermediate 10 12 14 16 18 20 Age (kyr BP) 180 200 220 240 260 280 300 CO2 (ppm) Atmospheric CO2 b data CTRL without CC CTRL abrupt 18k abrupt 17k linear intermediate 10 12 14 16 18 20 Age (kyr BP) 1.0 0.5 0.0 0.5 δ13 Cocean (permil) Deep Southern Ocean δ13 C c 10 12 14 16 18 20 Age (kyr BP) 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 δ13 Catm (permil) Atmospheric δ13 C d 20 18 16 14 12 10 Age (kyr BP) 0.0 0.2 0.4 0.6 0.8 1.0 parameter (iron or frac) Forcing scenarios brines declining brines no brines a iron brines: abrupt 18k brines: abrupt 17k brines: linear brines: intermediate 10 12 14 16 18 20 Age (kyr BP) 180 200 220 240 260 280 300 CO2 (ppm) Atmospheric CO2 b data CTRL without CC CTRL abrupt 18k abrupt 17k linear intermediate 10 12 14 16 18 20 Age (kyr BP) 1.0 0.5 0.0 0.5 δ13 Cocean (permil) Deep Southern Ocean δ13 C c 10 12 14 16 18 20 Age (kyr BP) 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 δ13 Catm (permil) Atmospheric δ13 C d Fig. 14. Forcing scenarios and simulated evolution of atmospheric CO2, deep Southern Ocean δ13C and atmospheric δ13C during the deglaciation. (a) Evolution scenarios for the iron fertilization (iron parameter, grey) based on the dust transport as recorded in ice cores (Wolff et al., 2006) on the EDC3 age model (Parrenin et al., 2007), and sinking of brines (frac parameter, colors), (b) simulated evo- lution of atmospheric CO2 (ppm) for the control run (“CTRL” in brown, without carbonate compensation: dotted line and with car- bonate compensation: solid line) and with three additional mech- anisms: interactive diffusion, iron fertilization and different brine scenarios as described in (a) compared to data (black), (c) evolution of deep Southern Ocean δ13C (‰) for the same simulations and (d) of atmospheric δ13C (‰). 0 10000 20000 30000 40000 50000 years of simulation 180 200 220 240 260 280 300 CO2 (ppm) LGM-ctrl LGM-all Fig. N. Bouttes et al.: Ocean processes during the last termination 13. Equilibrium simulations with LGM boundary conditions (LGM ice sheets and orbital parameters) with carbonate compen- sation (“LGM-ctrl”) and with the combination of the three addi- tional mechanisms (“LGM-all”, sinking of brines, interactive diffu- sion and iron fertilization). atmospheric CO2 until it equilibrates. On the contrary, the “LGM-all” run is stable and the simulated CO2 remains at the glacial level of ∼190 ppm. Because of the deep stratiﬁca- tion induced by the sinking of brines, the interactive diffusion and the iron fertilization, the deep ocean contains more car- bon, hence the relatively low simulated CO2. Based on these mechanisms, because atmospheric CO2 is now interactive, it is possible to study the temporal evolution of CO2 during the deglaciation. Indeed, the ends of these equilibrium runs constitute the initial conditions for the deglacial runs. 10 12 14 16 18 20 Age (kyr BP) 10 12 14 16 18 20 Age (kyr BP) 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 δ13 Catm (permil) Atmospheric δ13 C d 3.3.2 Evolution of CO2 and δ13C during the last deglaciation with a combination of mechanisms As done previously, the ice sheet, sea level and insolation evolutions are prescribed. However, atmospheric CO2 is no longer prescribed. The CO2 concentration used to compute the radiative scheme comes from the CO2 calculated in the carbon cycle module. The evolution of iron fertilization is again prescribed following iron ﬂux data and different sce- narios are applied to the brine mechanism (Fig. 14a). The two scenarios explored before (linear decline “linear” and the sudden halt at 18 ky BP “abrupt”) are again considered, and we also study two additional ones: a sudden halt of the sink- ing of brines at 17 ky BP “abrupt 17 k” and an intermediate one “intermediate”. Fig. 14. Forcing scenarios and simulated evolution of atmospheric CO2, deep Southern Ocean δ13C and atmospheric δ13C during the deglaciation. (a) Evolution scenarios for the iron fertilization (iron parameter, grey) based on the dust transport as recorded in ice cores (Wolff et al., 2006) on the EDC3 age model (Parrenin et al., 2007), and sinking of brines (frac parameter, colors), (b) simulated evo- lution of atmospheric CO2 (ppm) for the control run (“CTRL” in brown, without carbonate compensation: dotted line and with car- bonate compensation: solid line) and with three additional mech- anisms: interactive diffusion, iron fertilization and different brine scenarios as described in (a) compared to data (black), (c) evolution of deep Southern Ocean δ13C (‰) for the same simulations and (d) of atmospheric δ13C (‰). In the control transient simulation (“CTRL”), atmospheric CO2 roughly stays around 280 ppm (Fig. 14b). This evolu- tion is due to the terrestrial biosphere, whose carbon content increase lowers atmospheric CO2. It counteracts the atmo- spheric CO2 increase due to the rising temperatures causing lower solubility. This evolution widely differs from the data, i.e. a general increase from the glacial level of ∼190 ppm until the Holocene value of ∼260 ppm. combination of iron fertilization, sinking of brines and strat- iﬁcation dependent diffusion, the obtained computed transi- tions are mainly driven by the ocean and match the global data evolution of CO2 better than the control simulation (Fig. 14b). The scenarios that best match the data are the “abrupt 17 k” and “intermediate” ones that support a rela- tively rapid halt of the sinking of brines when the Antarc- tic ice sheet is at its maximum extent. www.clim-past.net/8/149/2012/ 164 N 0 10000 20000 30000 40000 50000 years of simulation 180 200 220 240 260 280 300 CO2 (ppm) LGM-ctrl LGM-all Fig. 13. Equilibrium simulations with LGM boundary conditions (LGM ice sheets and orbital parameters) with carbonate compen- sation (“LGM-ctrl”) and with the combination of the three addi- tional mechanisms (“LGM-all”, sinking of brines, interactive diffu- sion and iron fertilization). N. Bouttes et al.: Ocean processes during the last termination Because CLIMBER-2 is an intermediate complex- ity model with a low resolution and zonally averaged ocean, it complicates the comparison with the sediment core data. Similar experiments exploring the impacts of the sinking of brines with a better resolved 3-D ocean model should lead to a better comparison. ge ( y ) 10 12 14 16 18 20 Age (kyr BP) 4000 3000 2000 1000 0 depth (m) Potential density b Fig. 15. Evolution of (a) Dissolved Inorganic Carbon (DIC, µmol kg−1) and (b) potential density anomaly (ρθ −1000 kg m−3) as a function of depth at latitude 50◦S of the Atlantic ocean sector. resulting in a positive feedback. Antarctic temperature thus reacts quicker than CO2 at the beginning of the termination, despite the same unique triggering, i.e. the halt of stratiﬁca- tion due to brines sinking. Recently, two papers studied the evolution of the carbon cycle in the context of glacial-interglacial cycles. Tschumi et al. (2011) run transient simulations testing the sensitivity of various mechanisms in a constant climate. The simula- tions support the hypothesis that a change in the ventilation of the deep ocean played an important role in the variation of CO2 during glacial interglacial cycles. In this study, the change of deep ocean ventilation was obtained by changing the windstress whereas in our study the change of deep ocean stratiﬁcation results from changing the sinking of brines. Our study also supports the important role of the change of deep ocean stratiﬁcation. Furthermore, it demonstrates that it al- lows us to simulate the evolution of CO2 during the last deglaciation in agreement with CO2 and δ13C data without prescribing CO2. In particular, the interactive carbon-climate simulation manages to capture not only the right CO2 am- plitude change, but also the right timing. With other mech- anisms, Brovkin et al. (2011) run a full glacial-interglacial evolution of CO2 and northern ice sheets. In this simula- tion the role of the carbonate compensation due to changes in the weathering rate is important. However, although most of the CO2 evolution is successfully simulated, the timing of the CO2 rise during the deglaciation lags the data indicating that a mechanism leading to an earlier CO2 change is miss- ing. N. Bouttes et al.: Ocean processes during the last termination N. Bouttes et al.: Ocean processes during the last termination 165 10 12 14 16 18 20 Age (kyr BP) 4000 3000 2000 1000 0 depth (m) DIC a simulated deep Southern Ocean δ13C transition is also im- proved, with an increase of ∼1 ‰ beginning at 15.5 kyr BP (Fig. 14c). However, if the general trend is captured by the model, the CO2 plateau during the Bølling-Allerød (from ∼14 kyr BP to ∼12 kyr BP) is not well represented, underlin- ing the lack of other processes such as abrupt AMOC varia- tions that were not considered in this study. This global behaviour for CO2 and deep ocean δ13C dur- ing the termination is due to the modiﬁcation of the ocean dynamics and reduced iron fertilization. The halt of the sink- ing of brines results in modiﬁcations of the overturning rate during the transition while iron fertilization induces changes in the biological production. During the glacial period, be- cause of the sinking of brines, both the simulated North At- lantic Deep Water (NADW) and the Antarctic Bottom Wa- ter (AABW) export rates are weaker so that the deep water enriched in carbon is less mixed with the above water con- taining less carbon. As previously studied (Bouttes et al., 2010, 2011) the deep isolated water then represents an im- portant carbon reservoir. The sinking of brines is responsi- ble for a glacial CO2 change of 39 ppm and the interactive diffusion scheme ampliﬁes it by 16 ppm. During the transi- tion the carbon trapped in the abyss is progressively released into the atmosphere because of the halt of the brines sink and break down of the stratiﬁcation (Fig. 15). In addition, the decrease in iron fertilization, which accounts for 10 ppm of the LGM CO2 change, leads to a reduced biological produc- tion and therefore to a less effective biological pump. Sim- ilarly, the evolution of deep ocean δ13C is governed by the increasing mixing between surface water with high values of δ13Cocean and deep water with low values of δ13Cocean. Be- cause of the change of oceanic circulation the deep values increase and the vertical gradient is diminished. Although the general trend of the ocean δ13C evolution is well sim- ulated with the model, the absolute values are slightly dif- ferent. 3.3.2 Evolution of CO2 and δ13C during the last deglaciation with a combination of mechanisms In these runs, the We then consider a combination of the three additional oceanic mechanisms during the deglaciation. With the Clim. Past, 8, 149–170, 2012 www.clim-past.net/8/149/2012/ n 165 10 12 14 16 18 20 Age (kyr BP) 4000 3000 2000 1000 0 depth (m) DIC a 10 12 14 16 18 20 Age (kyr BP) 4000 3000 2000 1000 0 depth (m) Potential density b 1900 2000 2100 2200 2300 2400 2500 2600 26 27 28 29 30 31 32 Fig. 15. Evolution of (a) Dissolved Inorganic Carbon (DIC, µmol kg−1) and (b) potential density anomaly (ρθ −1000 kg m−3) as a function of depth at latitude 50◦S of the Atlantic ocean sector. www.clim-past.net/8/149/2012/ N. Bouttes et al.: Ocean processes during the last termination With the change of deep stratiﬁcation from the change Although the amplitude of the glacial-interglacial Antarc- tic temperature change is underestimated in the model, it is possible to analyse the relative timing of CO2 and tempera- ture evolution (Fig. 16). The two scenarios that agree best with CO2 and δ13C data (“abrupt 17 k” and “intermediate”) result in a lead of temperature relative to CO2 as inferred from termination 3 data (Caillon et al., 2003). The halt of the sinking of brines not only breaks down the stratiﬁcation, which increases CO2 and therefore temperature, but also in- duces a decrease in sea ice formation (which is computed by a thermodynamic sea ice model) as the surface waters be- come saltier and warmer. Such an early deglacial retreat of sea ice is also indicated in the data (Shemesh et al., 2002). Because of the reduced albedo, temperature then increases, Clim. Past, 8, 149–170, 2012 www.clim-past.net/8/149/2012/ . Bouttes et al.: Ocean processes during the last termination 10 12 14 16 18 20 180 200 220 240 260 280 CO2 (ppm) CTRL 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0.5 1.0 temperature (o C) b 10 12 14 16 18 20 180 200 220 240 260 280 CO2 (ppm) abrupt 18k 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0.5 1.0 temperature (o C) d 10 12 14 16 18 20 Age (kyr BP) 180 200 220 240 260 280 CO2 (ppm) intermediate 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0.5 1.0 temperature (o C) f N. Bouttes et al.: Ocean processes during the last termination N. Bouttes et al.: Ocean processes during the last termination 166 166 N. N. Bouttes et al.: Ocean processes during the last termination Bouttes et al.: Ocean processes during the last termination 10 12 14 16 18 20 180 200 220 240 260 280 CO2 (ppm) DATA 10 8 6 4 2 0 2 temperature (o C) a 10 12 14 16 18 20 180 200 220 240 260 280 CO2 (ppm) CTRL 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0.5 1.0 temperature (o C) b 10 12 14 16 18 20 180 200 220 240 260 280 CO2 (ppm) abrupt 17k 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0.5 1.0 temperature (o C) c 10 12 14 16 18 20 180 200 220 240 260 280 CO2 (ppm) abrupt 18k 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0.5 1.0 temperature (o C) d 10 12 14 16 18 20 Age (kyr BP) 180 200 220 240 260 280 CO2 (ppm) linear 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0.5 1.0 temperature (o C) e 10 12 14 16 18 20 Age (kyr BP) 180 200 220 240 260 280 CO2 (ppm) intermediate 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0.5 1.0 temperature (o C) f Fig. 16. Evolution of CO2 (ppm) and Antarctic temperature (◦C) during the deglaciations for (a) the data (Monnin et al., 2001; Lourantou et al., 2010; Jouzel et al., 2007), (b) the CTRL simulation (with carbonate compensation), (c, d, e, f) the simulations with interactive diffusion, iron fertilization and different brines scenarios as deﬁned in Fig. 14a. The grey shading indicates the evolution of the sinking of brines according to each scenario. 10 12 14 16 18 20 180 200 220 240 260 280 CO2 (ppm) DATA 10 8 6 4 2 0 2 temperature (o C) a 10 12 14 16 18 20 180 200 220 240 260 280 CO2 (ppm) abrupt 17k 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0.5 1.0 temperature (o C) c 10 12 14 16 18 20 Age (kyr BP) 180 200 220 240 260 280 CO2 (ppm) linear 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0.5 1.0 temperature (o C) e b temperature (o C) Fig. 16. N. Bouttes et al.: Ocean processes during the last termination Evolution of CO2 (ppm) and Antarctic temperature (◦C) during the deglaciations for (a) the data (Monnin et al., 2001; Lourantou et al., 2010; Jouzel et al., 2007), (b) the CTRL simulation (with carbonate compensation), (c, d, e, f) the simulations with interactive diffusion, iron fertilization and different brines scenarios as deﬁned in Fig. 14a. The grey shading indicates the evolution of the sinking of brines according to each scenario. of sinking of brines, it allows us to get this timing right, al- though it relies on scenarios that need to be comﬁrmed. which takes more time to equilibrate than the marine biol- ogy. The combination of interactive diffusion with the sink- ing of brines induces an important delay as the vertical dif- fusion has to adjust to the evolving circulation. The carbon cycle then takes ∼4000 years to equilibrate. The impact of iron fertilization on δ13Cocean is small (−0.12 ‰). It is im- portant for δ13Catm (−0.25 ‰). The effect of the sinking of brines is important on both deep ocean δ13C (−0.57 ‰) and δ13Catm (−0.25 ‰). Adding the stratiﬁcation-dependent dif- fusion ampliﬁes the effect of the sinking of brines on CO2 (61 ppm), deep ocean δ13C (−1.1 ‰) and δ13Catm (−0.34 ‰) due to the decrease in mixing between the deep and upper waters, which makes the deep water enriched in carbon and depleted in δ13C even more isolated with more carbon and lower δ13C. References Based on the study of these mechanisms during the deglaciation and previous studies of the possible combina- tions of the mechanisms in glacial conditions (Bouttes et al., 2011), it is possible to use the model in an fully interactive carbon-climate version. The simulated evolution of CO2 is in good agreement with the data underlining the powerful im- pact of the combination of the sinking of brines, stratiﬁcation dependent diffusion and iron fertilization. Anderson, J. B., Shipp, S. S., Lowe, A. L., Wellner, J. S., and Mosola, A. B.: The Antarctic Ice Sheet during the Last Glacial Maximum and its subsequent retreat history: a review, Quater- nary Sci. Rev., 21, 49–70, doi:10.1016/S0277-3791(01)00083-X, 2002. Archer, D.: Modeling the Calcite Lysocline, J. Geophys. Res., 96, 17037–17050, 1991. Archer, D., Winguth, A., Lea, D., and Mahowald, N.: What caused the glacial/interglacial pCO2 cycles?, Rev. Geophys., 38, 159– 189, 2000. Although the computed CO2 and deep ocean δ13C are in broad agreement with proxy data, the computed δ13Catm presents important discrepancies with respect to the data both in magnitude and structure. The persisting mismatch between model results and data for δ13Catm points to the role of vegetation, which could modulate the δ13Catm sig- nal inducing low values when the vegetation is reduced and high values when the vegetation is increased. The change in the terrestrial biosphere seems to play a less important role for CO2 and deep ocean δ13C, which are mostly driven by oceanic mechanisms. This source discrimination between at- mospheric mixing and isotopic CO2 ratios has been recently explored (Lourantou et al., 2010). δ13Catm data can thus help to constrain the vegetation evolution which is poorly known. 13 Archer, D. E., Martin, P. A., Milovich, J., Brovkin, V., Plattner, G.- K., and Ashendel, C.: Model sensitivity in the effect of Antarctic sea ice and stratiﬁcation on atmospheric pCO2, Paleoceanogra- phy, 18, 1012, doi:10.1029/2002PA000760, 2003. Barker, S., Diz, P., Vautravers, M. J., Pike, J., Knorr, G., Hall, I. R., and Broecker, W. S.: Interhemispheric Atlantic seesaw response during the last deglaciation, Nature, 457, 1097–1102, doi:10.1038/nature07770, 2009. Berger, A., Loutre, M. F., and Gall´ee, H.: Sensitivity of the LLN climate model to the astronomical and CO2 forc- ings over the last 200 ky, Clim. Dynam., 14, 615–629, doi:10.1007/s003820050245, 1998. Berger, A. L.: Long-term variations of daily insolation and Quater- nary climatic changes, J. Atmos. Sci., 35, 2362–2368, 1978. The publication of this article is ﬁnanced by CNRS-INSU. The publication of this article is ﬁnanced by CNRS-INSU. 4 Conclusions To summarize, we use the intermediate complexity model CLIMBER-2 to explore the impact of three oceanic mech- anisms on the evolution of the carbon cycle during the last deglaciation: iron fertilization, sinking of brines and stratiﬁcation-dependent diffusion. The carbonate compen- sation mechanism is included in the CLIMBER-2 model, which has already been used to study the LGM carbon cy- cle (Brovkin et al., 2002b, 2007; Bouttes et al., 2009, 2010, 2011). With the varying climate of the last termination, the im- pact of the evolution of these mechanisms is modulated by other changes such as the warming and increase in the ter- restrial biosphere carbon content. In this context, either the association of the sinking of brines with iron fertilization or with interactive diffusion results in a computed atmospheric CO2 increase in agreement with the data. However, only the A ﬁrst set of simulations in a context of a constant LGM climate has allowed an evaluation of the effect of each mech- anism separately. The iron fertilization of marine biology in- duces the fastest response of the carbon cycle (∼100 years) with a rapid increase in atmospheric CO2 of ∼29 ppm. The CO2 rise due to the sinking of brines (∼40 ppm) takes longer (almost 1000 years) as it involves the oceanic circulation www.clim-past.net/8/149/2012/ Clim. Past, 8, 149–170, 2012 www.clim-past.net/8/149/2012/ 167 The publication of this article is ﬁnanced by CNRS-INSU. combination of brines with interactive diffusion also recon- ciles the simulated δ13Cocean with the recorded δ13C evo- lution in the deep Southern Ocean. In the latter case, the scenario that best matches the data is the “abrupt” scenario, i.e. a sudden halt of the sinking of brines when the Antarctic ice sheet is at its maximum extent. In such a conﬁguration, sea ice formation is shifted to the open ocean instead of the shelf. The dense water from brine rejection is then mixed with fresher water preventing it from sinking down to the abyss. N. Bouttes et al.: Ocean processes during the last termination C., Johnsen, S., Leuenberger, M., Loulergue, L., Luethi, D., Oerter, H., Parrenin, F., Raisbeck, G., Raynaud, D., Schilt, A., Schwander, J., Selmo, E., Souchez, R., Spahni, R., Stauffer, B., Steffensen, J. P., Stenni, B., Stocker, T. F., Tison, J. L., Werner, M., and Wolf, E. W.: Orbital and Mil- lennial Antarctic Climate Variability over the Past 800,000 Years, Science, 317, 793, doi:10.1126/science.1141038, 2007. Charbit, S., Kageyama, M., Roche, D., Ritz, C., and Ramstein, G.: Investigating the mechanisms leading to the deglaciation of past continental northern hemisphere ice sheets with the CLIMBER GREMLINS coupled model, Global Planet. Change, 48, 253–273, doi:10.1016/j.gloplacha.2005.01.002, 2005. G., Raynaud, D., Schilt, A., Schwander, J., Selmo, E., Souchez, R., Spahni, R., Stauffer, B., Steffensen, J. P., Stenni, B., Stocker, Clark, P. U., Dyke, A. S., Shakun, J. D., Carlson, A. E., Clark, J., Wohlfarth, B., Mitrovica, J. X., Hostetler, S. W., and Mc- Cabe, A. M.: The Last Glacial Maximum, Science, 325, 710– 714, doi:10.1126/science.1172873, 2009. Kaplan, J. O., Prentice, I. C., Knorr, W., and Valdes, P. J.: Modeling the dynamics of terrestrial carbon storage since the Last Glacial Maximum, Geophys. Res. Lett., 29, 2074, doi:10.1029/2002GL015230, 2002. Crowley, T.: Ice Age Terrestrial Carbon Changes Revisited, Global Biogeochem. Cy., 9, 377–389, 1995. Keigwin, L. D., Jones, G. A., Lehman, S. J., and Boyle, E. A.: Deglacial Meltwater Discharge, North Atlantic Deep Circula- tion, and Abrupt Climate Change, J. Geophys. Res., 96, 16811– 16826, 1991. Cuffey, K. M. and Vimeux, F.: Covariation of carbon dioxide and temperature from the Vostok ice core after deuterium-excess cor- rection, Nature, 412, 523–527, doi:10.1038/35087544, 2001. Curry, W. B. and Oppo, D. W.: Glacial water mass geometry and the distribution of δ13C of 6CO2 in the western Atlantic Ocean, Paleoceanography, 20, PA1017, doi:10.1029/2004PA001021, 2005. K¨ohler, P. and Fischer, H.: Simulating changes in the terrestrial biosphere during the last glacial/interglacial transition, Global Planet. Change, 43, 33–55, doi:10.1016/j.gloplacha.2004.02.005, 2004. EPICA community members: Eight glacial cycles from an Antarc- tic ice core, Nature, 429, 623–628, 2004. K¨ohler, P., Fischer, H., Munhoven, G., and Zeebe, R. E.: Quan- titative interpretation of atmospheric carbon records over the last glacial termination, Global Biogeochem. Cy., 19, GB4020, doi:10.1029/2004GB002345, 2005a. Fischer, H., Schmitt, J., L¨uthi, D., Stocker, T. F., Tschumi, T., Parekh, P., Joos, F., K¨ohler, P., V¨olker, C., Gersonde, R., Bar- bante, C., Floch, M. N. Bouttes et al.: Ocean processes during the last termination 168 Broecker, W. S. and Peng, T.-H.: The Role of CaCO3 Compen- sation in the Glacial to Interglacial Atmospheric CO2 Change, Global Biogeochem. Cy., 1, 15–29, 1987. Ganopolski, A., Calov, R., and Claussen, M.: Simulation of the last glacial cycle with a coupled climate ice-sheet model of interme- diate complexity, Clim. Past, 6, 229–244, doi:10.5194/cp-6-229- 2010, 2010. Brovkin, V., Bendtsen, J., Claussen, M., Ganopolski, A., Kubatzki, C., Petoukhov, V., and Andreev, A.: Carbon cycle, vegeta- tion, and climate dynamics in the Holocene: Experiments with the CLIMBER-2 model, Global Biogeochem. Cy., 16, 1139, doi:10.1029/2001GB001662, 2002a. Gersonde, R., Crosta, X., Abelmann, A., and Armand, L.: Sea- surface temperature and sea ice distribution of the South- ern Ocean at the EPILOG Last Glacial Maximum -a circum- Antarctic view based on siliceous microfossil records, Quater- nary Sci. Rev., 24, 869–896, 2005. Brovkin, V., Hofmann, M., Bendtsen, J., and Ganopolski, A.: Ocean biology could control atmospheric δ13C during glacial- interglacial cycle, Geochem. Geophys. Geosyst., 3, 1027, doi:10.1029/2001GC000270, 2002b. Hodell, D. A., Venz, K. A., Charles, C. D., and Ninnemann, U. S.: Pleistocene vertical carbon isotope and carbonate gradients in the South Atlantic sector of the Southern Ocean, Geochem. Geophy. Geosy., 4, 1–19, doi:10.1029/2002GC000367, 2003. Brovkin, V., Ganopolski, A., Archer, D., and Rahmstorf, S.: Lower- ing of glacial atmospheric CO2 in response to changes in oceanic circulation and marine biogeochemistry, Paleoceanography, 22, PA4202, doi:10.1029/2006PA001380, 2007. Huybrechts, P.: Sea-level changes at the LGM from ice-dynamic reconstructions of the Greenland and Antarctic ice sheets dur- ing the glacial cycles, Quaternary Sci. Rev., 21, 203–231, doi:10.1016/S0277-3791(01)00082-8, 2002. Brovkin, V., Ganopolski, A., Archer, D., and Munhoven, G.: Glacial CO2 cycle as a succession of key physical and bio- geochemical processes, Clim. Past Discuss., 7, 1767–1795, doi:10.5194/cpd-7-1767-2011, 2011. Joos, F., Gerber, S., Prentice, I. C., Otto-Bliesner, B. L., and Valdes, P. J.: Transient simulations of Holocene atmo- spheric carbon dioxide and terrestrial carbon since the Last Glacial Maximum, Global Biogeochem. Cy., 18, GB2002, doi:10.1029/2003GB002156, 2004. Caillon, N., Severinghaus, J. P., Jouzel, J., Barnola, J.-M., Kang, J., and Lipenkov, V. Y.: Timing of Atmospheric CO2 and Antarc- tic Temperature Changes Across Termination III, Science, 299, 1728, doi:10.1126/science.1078758, 2003. Jouzel, J., Masson-Delmotte, V., Cattani, O., Dreyfus, G., Falourd, S., Hoffmann, G., Minster, B., Nouet, J., Barnola, J. M., Chap- pellaz, J., Fischer, H., Gallet, J. References The δ13Catm mismatch can also point to the potential role of abrupt events, which were not considered in this study. Additionally, the lack of abrupt events is underlined by the absence of the CO2 plateau during the Bolling-Allerod, as this plateau could also be linked to abrupt events associated to fresh water ﬂuxes (Stocker and Wright, 1991). Indeed, fresh water ﬂuxes, which can alter the thermohaline circu- lation, have an impact on the carbon cycle (Schmittner and Galbraith, 2008; K¨ohler et al., 2005b; Menviel et al., 2008b). Taking them into account could possibly improve both the CO2 plateau and the δ13Catm evolution. Berger, W. H.: Increase of carbon dioxide in the atmosphere during Deglaciation: the coral reef hypothesis, Naturwissenschaften, 69, 87–88, 1982. Bird, M. I., Lloyd, J., and Farquhar, G. D.: Terrestrial carbon stor- age at the LGM, Nature, 371, 566, 1994. Bopp, L., Kohfeld, K. E., Qu´er´e, C. L., and Aumont, O.: Dust im- pact on marine biota and atmospheric CO2 during glacial peri- ods, Paleoceanography, 18, 1046, doi:10.1029/2002PA000810, 2003. Bouttes, N., Roche, D. M., and Paillard, D.: Impact of strong deep ocean stratiﬁcation on the carbon cycle, Paleoceanography, 24, PA3203, doi:10.1029/2008PA001707, 2009. Bouttes, N., Paillard, D., and Roche, D. M.: Impact of brine- induced stratiﬁcation on the glacial carbon cycle, Clim. Past, 6, 575–589, doi:10.5194/cp-6-575-2010, 2010. Acknowledgements. We thank Victor Brovkin, Andrey Ganopol- ski, Guy Munhoven and Emilie Capron for useful comments and discussion. We also thank the editor and two anonymous reviewers for their comments which helped improve this manuscript. Bouttes, N., Paillard, D., Roche, D. M., Brovkin, V., and Bopp, L.: Last Glacial Maximum CO2 and δ13C successfully reconciled, Geophys. Res. Lett., 38, L02705, doi:10.1029/2010GL044499, 2011. Edited by: M. Siddall Broecker, W. S. and Peng, T.-H.: Tracers in the Sea, Lamont- Doherty Geological Observatory of Columbia University, Pal- isades, New York, 1982. Clim. Past, 8, 149–170, 2012 www.clim-past.net/8/149/2012/ doi:10.1029/2009GB003545, 2010. F.: The inﬂuence of Antarctic sea ice on glacial-interglacial CO2 variations, Nature, 404, 171–174, 2000. Montenegro, A., Eby, M., Kaplan, J. O., Meissner, J., and Weaver, A. J.: Carbon storage on exposed continental shelves during the glacial-interglacial transition, Geophys. Res. Lett., 33, L08703, doi:10.1029/2005GL025480, 2006. Stocker, T. F. and Wright, D.: Rapid transitions of the ocean’s deep circulation induced by changes in the surface water ﬂuxes, Na- ture, 351, 729–732, 1991. Svendsen, J. I., Alexanderson, H., Astakhov, V. I., Demidov, I., Dowdeswell, J. A., Funder, S., Gataullin, V., Henriksen, M., Hjort, C., Houmark-Nielsen, M., Hubberten, H. W., Ing´olfsson, ´O, Jakobsson, M., Kjær, K. H., Larsen, E., Lokrantz, H., Lunkka, J. P., Lys˚a, A., Mangerud, J., Matiouchkov, A., Murray, A., M¨oller, P., Niessen, F., Nikolskaya, O., Polyak, L., Saarnisto, M., Siegert, C., Siegert, M. J., Spielhagen, R. F., and Stein, R.: Late Quaternary ice sheet history of northern Eurasia, Quaternay Sci. Rev., 23, 1229–1271, doi:10.1016/j.quascirev.2003.12.008, 2004. North Greenland Ice Core Project members: High-resolution record of Northern Hemisphere climate extending into the last inter- glacial period, Nature, 431, 147–151, doi:10.1038/nature02805, 2004. Opdyke, B. N. and Walker, J. C. G.: Return of the coral reef hy- pothesis: Basin to shelf partitioning of CaCO3 and its effect on atmospheric CO2, Geology, 20, 733–736, 1992. Paillard, D. and Parrenin, F.: The Antarctic ice sheet and the trig- gering of deglaciations, Earth Planet. Sc. Lett., 227, 263–271, 2004. Tagliabue, A., Bopp, L., Roche, D. M., Bouttes, N., Dutay, J.-C., Alkama, R., Kageyama, M., Michel, E., and Paillard, D.: Quanti- fying the roles of ocean circulation and biogeochemistry in gov- erning ocean carbon-13 and atmospheric carbon dioxide at the last glacial maximum, Clim. Past, 5, 695–706, doi:10.5194/cp- 5-695-2009, 2009. Parrenin, F., Barnola, J.-M., Beer, J., Blunier, T., Castellano, E., Chappellaz, J., Dreyfus, G., Fischer, H., Fujita, S., Jouzel, J., Kawamura, K., Lemieux-Dudon, B., Loulergue, L., Masson- Delmotte, V., Narcisi, B., Petit, J.-R., Raisbeck, G., Raynaud, D., Ruth, U., Schwander, J., Severi, M., Spahni, R., Steffensen, J. P., Svensson, A., Udisti, R., Waelbroeck, C., and Wolff, E.: The EDC3 chronology for the EPICA Dome C ice core, Clim. Past, 3, 485–497, doi:10.5194/cp-3-485-2007, 2007. Toggweiler, J. R.: Variation of atmospheric CO2 by ventilation of the ocean’s deepest water, Paleoceanography, 14, 571–588, 1999. Peltier, W. R.: Ice age paleotopography, Science, 265, 195–201, doi:10.1126/science.265.5169.195, 1994. Toggweiler, J. R., Russell, J. L., and Carson, S. doi:10.1029/2009GB003545, 2010. doi:10.1029/2009GB003545, 2010. doi:10.1029/2009GB003545, 2010. Ritz, C., Rommelaere, V., and Dumas, C.: Modeling the evolution of Antarctic ice sheet over the last 420,000 years: Implications for altitude changes in the Vostok region, J. Geophys. Res., 106, 31943–31964, 2001. Mackintosh, A., Golledge, N., Domack, E., Dunbar, R., Leven- ter, A., White, D., Pollard, D., DeConto, R., Fink, D., Zwartz, D., Gore, D., and Lavoie, C.: Retreat of the East Antarctic ice sheet during the last glacial termination, Nat. Geosci., 4, 195– 202, doi:10.1038/ngeo1061, 2011. Schmittner, A. and Galbraith, E. D.: Glacial greenhouse-gas ﬂuc- tuations controlled by ocean circulation changes, Nature, 456, 373–376, doi:10.1038/nature07531, 2008. MARGO Project Members: Constraints on the magnitude and patterns of ocean cooling at the Last Glacial Maximum, Nat. Geosci., 2, 127–132, doi:10.1038/ngeo411, 2009. Schneider von Deimling, T., Ganopolski, A., Held, H., and Rahm- storf, S.: How cold was the Last Glacial Maximum?, Geophys. Res. Lett., 33, L14709, doi:10.1029/2006GL026484, 2006. Martin, J. H.: Glacial-Interglacial CO2 change: the iron hypothesis, Paleoceanography, 5, 1–13, 1990. Shemesh, A., Hodell, D., Crosta, X., Kanfoush, S., Charles, C., and Guilderson, T.: Sequence of events during the last deglaciation in Southern Ocean sediments and Antarctic ice cores, Paleoceanog- raphy, 17, 1056, doi:10.1029/2000PA000599, 2002. Marzeion, B., Levermann, A., and Mignot, J.: The Role of Stratiﬁcation-Dependent Mixing for the Stability of the Atlantic Overturning in a Global Climate Model, J. Phys. Oceanogr., 37, 2672–2681, doi:10.1175/2007JPO3641.1, 2007. Sigman, D. M. and Boyle, E. A.: Glacial/interglacial varia- tions in atmospheric carbon dioxide, Nature, 407, 859–869, doi:10.1038/35038000, 2000. Menviel, L., Timmermann, A., Mouchet, A., and Timm, O.: Cli- mate and marine carbon cycle response to changes in the strength of the Southern Hemispheric westerlies, Paleoceanography, 23, PA4201, doi:10.1029/2008PA001604, 2008a. Sigman, D. M., Hain, M. P., and Haug, G. H.: The polar ocean and glacial cycles in atmospheric CO2 concentration, Nature, 466, 47–55, doi:10.1038/nature09149, 2010. Menviel, L., Timmermann, A., Mouchet, A., and Timm, O.: Meridional reorganizations of marine and terrestrial produc- tivity during Heinrich events, Paleoceanography, 23, PA1203, doi:10.1029/2007PA001445, 2008b. Skinner, L. C., Fallon, S., Waelbroeck, C., Michel, E., and Barker, S.: Ventilation of the Deep Southern Ocean and Deglacial CO2 Rise, Science, 328, 1147–1151, doi:10.1126/science.1183627, 2010. Monnin, E., Inderm¨uhle, A., Daellenbach, A., Flueckiger, J., Stauf- fer, B., Stocker, T. F., Raynaud, D., and Barnola, J.-M.: Atmo- spheric CO2 concentrations over the Last Glacial Termination, Science, 291, 112–114, 2001. Stephens, B. B. and Keeling, R. N. Bouttes et al.: Ocean processes during the last termination L., Raynaud, D., and Wolff, E.: The role of Southern Ocean processes on orbital and millennial CO2 variations – a synthesis, Quaternary Sci. Rev., 29, 193–205, doi:10.1016/j.quascirev.2009.06.007, 2010. K¨ohler, P., Joos, F., Gerber, S., and Knutti, R.: Simulated changes in vegetation distribution, land carbon storage, and atmospheric CO2 in response to a collapse of the North Atlantic thermohaline circulation, Clim. Dynam., 25, 689–708, doi:10.1007/s00382- 005-0058-8, 2005b. Ganopolski, A. and Rahmstorf, S.: Rapid changes of glacial cli- mate simulated in a coupled climate model, Nature, 409, 153– 158, 2001. Kohfeld, K. E., Qu´er´e, C. L., Harrison, S. P., and Anderson, R. F.: Role of Marine Biology in Glacial-Interglacial CO2 Cycles, Sci- ence, 308, 74–78, 2005. Ganopolski, A., Petoukhov, V., Rahmstorf, S., Brovkin, V., Claussen, M., Eliseev, A., and Kubatzki, C.: CLIMBER-2: A climate system model of intermediate complexity, part II: Model sensitivity, Clim. Dynam., 17, 735–751, 2001. Lourantou, A., Lavric, J. V., K¨ohler, P., Barnola, J.-M., Paillard, D., Michel, E., Raynaud, D., and Chappellaz, J.: Constraint of the CO2 rise by new atmospheric carbon isotopic measurements dur- ing the last deglaciation, Global Biogeochem. Cy., 24, GB2015, Clim. Past, 8, 149–170, 2012 www.clim-past.net/8/149/2012/ www.clim-past.net/8/149/2012/ N. Bouttes et al.: Ocean processes during the last termination 169 doi:10.1029/2009GB003545, 2010. R.: Mid- latitude westerlies, atmospheric CO2, and climate change during the ice ages, Paleoceanography, 21, PA2005, doi:10.1029/2005PA001154, 2006. Peltier, W. R.: Global glacial isostasy and the surface of the ice-age Earth: The ICE-5G (VM2) Model and GRACE, Ann. Rev. Earth Planet. Sci., 32, 111–149, doi:10.1146/annurev.earth.32.082503.144359, 2004. Tschumi, T., Joos, F., Gehlen, M., and Heinze, C.: Deep ocean ven- tilation, carbon isotopes, marine sedimentation and the deglacial CO2 rise, Clim. Past, 7, 771–800, doi:10.5194/cp-7-771-2011, 2011. Petoukhov, V., Ganopolski, A., Eliseev, A., Kubatzki, C., and Rahmstorf, S.: CLIMBER-2: A climate system model of inter- mediate complexity, part I: Model description and performance for present climate, Clim. Dynam., 16, 1–17, 2000. Clim. Past, 8, 149–170, 2012 www.clim-past.net/8/149/2012/ N. Bouttes et al.: Ocean processes during the last termination 170 Visser, K., Thunell, R., and Stott, L.: Magnitude and timing of tem- perature change in the Indo-Paciﬁc warm pool during deglacia- tion, Nature, 421, 152–155, doi:10.1038/nature01297, 2003. Watson, A. J. and Garabato, A. C. N.: The role of South- ern Ocean mixing and upwelling in glacial-interglacial atmo- spheric CO2 change, Tellus B, 58, 73–87, doi:10.1111/j.1600- 0889.2005.00167.x, 2005. Waelbroeck, C., Labeyrie, L., Michel, E., Duplessy, J. C., Mc- Manus, J. F., Lambeck, K., Balbon, E., and Labracherie, M.: Sea-level and deep water temperature changes derived from ben- thic foraminifera isotopic records, Quaternary Sci. Rev., 21, 295– 305, doi:10.1016/S0277-3791(01)00101-9, 2002. Wolff, E. W., Fischer, H., Fundel, F., Ruth, U., Twarloh, B., Littot, G. C., Mulvaney, R., R´othlisberger, R., de Angelis, M., Boutron, C. F., Hansson, M., Jonsell, U., Hutterli, M. A., Lambert, F., Kaufmann, P., Stauffer, B., Stocker, T. F., Steffensen, J. P., Bigler, M., Siggaard-Andersen, M. L., Udisti, R., Becagli, S., Castel- lano, E., Severi, M., Wagenbach, D., Barbante, C., Gabrielli, P., and Gaspari, V.: Southern Ocean sea-ice extent, productivity and iron ﬂux over the past eight glacial cycles, Nature, 440, 491–496, doi:10.1038/nature04614, 2006. Waelbroeck, C., Skinner, L. C., Labeyrie, L., Duplessy, J.-C., Michel, E., Riveiros, N. V., Gherardi, J.-M., and Dewilde, F.: The timing of deglacial circulation changes in the Atlantic, Pale- oceanography, 26, PA3213, doi:10.1029/2010PA002007, 2011. Clim. Past, 8, 149–170, 2012 www.clim-past.net/8/149/2012/
https://openalex.org/W4392879247	https://www.seer.ufal.br/index.php/revistaleitura/article/download/6537/5528	Portuguese	null	Publicidade: o linguístico e o icônico a serviço do consumo	Leitura	2,019	cc-by	2,149	trabalho retoma, com algumas modificações, uma analise de propagandas apresentada no curso de Semiótica ministrado pela professora Moníca Rector na PUC/RJ, em 1983. - 32 - - 32 - PUBLICIDADE: O LINGÜÍSTICO E O ICÔNICO A SERVIÇO DO CONSUMO PUBLICIDADE: O LINGÜÍSTICO E O ICÔNICO A SERVIÇO DO CONSUMO Marisa Bernardes Pereira Marisa Bernardes Pereira "a • ~ ^,^®Pi-raçao a status e o esnobismo, a dis- ci^iminaçao racial e sexual, o egoísmo e a carência de contato, a inveja, a cobiça, a avareza e a ausência de escrúpulos - nenhu- roa dessas atitudes e criada pela propaganda mas todas sao usadas e articuladas por ela". Baran e Sweezy public^H"^ pretendo questionar o uso da Ues na^ criaçio de comportamentos e atitjj tica do^ 1 ^ partir de uma analise semio- <fa-r>A^ ° lingüístico e do iconico em duas propa - ganaas de cigarro. de das implicações do uso da publicida- cultura capitalista, Til- d^-z que a publicidade "procura edu - lecer^ ^°^ponentes das varias classes ao estabe- em comportamentos, induzindo ate, to de °i 1® forma subliminar, um conjun- de exe^^ diretamente ligados a uma maneira adiant ^ cidadania". Fadul (1980) vai mais talism^* observar que "na fase atual do capi- a c predomínio das grandes empresas e ® ttuiçao de grandes monopÕlios, assistimos eslocaraento da concorrência do domínio dos - 33 - preços para o domínio das vendas, o que signifi ca atribuir a publicidade um papel fundamental na manutenção desse sistema, uma vez que a maior parte dos "raãdia" sao sustentados pela publicida de e pela propaganda". Observe-se, por exemplo o "merchandising" utilizado nas novelas que, ao tempo em que patrocina o programa, aproveita o ator como uma forma de dar^maior força e cre i ^ lidade ã mensagem publicitária. A escolha do produto cigarro para esta ana lise decorre do fato de que ao produzir a publi cidade, o publicitário vai encontrar, em pio, dois desafios: o de conseguir que o seu pro duto seja escolhido na concorrência com ou ra ^ marcas do mercado e o de neutralizar as campa nhas que se desenvolvem para mostrar os malefi cios que o fumo traz a saúde do homem. PUBLICIDADE: O LINGÜÍSTICO E O ICÔNICO A SERVIÇO DO CONSUMO Se a publicidade "serve de ponte entre o mo do de produção capitalista, a busca de ^cumula çio de capiLl e o escoamento dos bens de consu mo" (Tilburg, 1982), pode-se afirmar, então que o publicitã?ío, para defender os que possuem o controle dos meios de produção, tenta a escolha do produto através de uma retórica do compromisso do produto com o desejo publico Dal dizer-se que a publicidade e comandada de fo ra para dentro, isto 5, do destinatário para o autor. Dizer-se, ainda, que o que se projeta no anãncio ? a personalidade do consumidor e nao do autor. E que este procura traduzir e encarnar a vontade do consumidor. _ Dor outro lado, a partir ' Considerando-se, . ~ z: -^^oiista de Jakobson que na comu da visao f u n ci o na J. i s l ^ n — nicaçio coexistem, dentro de uma hierarqula, va rias funçSes da linguagem, na publicidade, evi- denciam-se as centradas no destinatarlo e na men sagem. AlSm disso constitui forte aliado no pre enchimento dos objetivos da publicidade o. compo- - 34 - nente estetico, em que o signo significa era si e por si. nente estetico, em que o signo significa era si e por si. Almada (1972) diz que a publicidade apreseii ta tres aspectos interdependentes: uma apresenta çao ou referencia em que o produto i oferecido a consciência, uma função predicativa que situa o objeto na cultura, no cotidiano e lhe atribui va lor de utilidade objetiva, e uma função metafori zante ou sirabolizante que faz o produto um signo, explorando-lhe as possibilidades de representa - çao. Na união desses tres aspectos e que se es tabelece a dialética do persuasorio. Mas a publi cidade vai mais alem. Como arma para sugestionar o consumidor, ela joga com apelos emocionais, so ciais, estéticos, eroticos e afetivos para rom per o muro levantado pelo consciente. CIGARROS COLOMBIA Ê inegável que toda estrutura de publicida de sustenta uma argumentação i coni co-1 i ngíl is t i ca que leva o consumidor a se persuadir consciente ou inconscientemente. Ê precisamente o relacio namento do iconico e do lingüístico em duas pro pagandas do cigarro Columbia que pretendo anali— s ar , Para Barthes (1969), na publicidade, o com ponente lingüístico realiza a ancoragem dos sig- ^ificados, Mas este componente também produz al teração na interpretação da imagem, na medida era que serve de suporte para as possíveis leituras da imagem e do discurso publicitário como um to do. Por outro lado, a publicidade utiliza a ima gem como primeira fonte de apelo. A prova disto e que esta nao so comunica o campo grafico, mas também atribui maior credibilidade quanto aos ' bons efeitos que o uso do produto anunciado cau sara ao consumidor. Considera-se, então, que na publicidade nao e o texto, tampouco a imagem que - 35 - faz criar uma necessidade no publico, mas e o seu conjunto que desempenha a função de apelo. faz criar uma necessidade no publico, mas e o seu conjunto que desempenha a função de apelo. Para efeito de analise, considero, contudo,a atualizaçao dos dois codigos separadamente como partes do plano de expressão na produção de um plano de conteúdo. O plano de expressão funciona como um sistema semiotico estruturado, visto que qualquer mudança nos seus elementos gera mudança no plano de conteúdo. Como primeira etapa deste estudo, realizei ' uma analise das imagens, enquanto portadoras de um conteúdo denotativo. Assim, nas propagandas,em anexo, encontram-se os seguintes elementos dos quais transcrevo os significados que lhes confere Aurélio Buarque de Holanda no seu Dicionário da L1ngua Portuguesa. IMAGENS DENOTAÇOES Re de Espécie de leito balançante S axo fo ne Instrumento de sopro, de metal H ornem qualquer indivíduo pertencente ã espécie animal que apresenta maior complexidade na escala evolutiva. violonceIo instrumento musical com forma de violino, mas de grandes propor - çoes . caixa arca, estojo banco assento com ou sem encosto, de formas variadas, cigarro pequena porção de tabaco, enrola do em papel ou palha de milho,pa r a f umar . Barthes (1969) diz ainda que "nao se encon - - 36 - tra nunca (pelo menos em publicidade) uma imagem literal em estado puro". CIGARROS COLOMBIA Nestas pub1icidades , i^ to pode ser perfeitamente comprovado, a partir ' dos indicadores de qualidade das imagens acima relacionadas. Nota-se, por exemplo, que os ho mens sugerem bom gosto nas roupas que usam. Tais roupas também os identificam como pertencentes a uma classe social privilegiada. Alem disso, os instrumentos musicais asso - ciados, o físico saudãvel e bem cuidado sugerem que eles nao sao músicos de qualquer orquestra , mas concertistas. Assim, as formas do saber prE tico, cultural e estético permitiram extrair de^ sas imagens os signos bom gosto, saúde, sucesso economico e cultural musical tao desejáveis á so cie a e consumista em que vivemos, á Alem dessas imagens, há nas duas publicida- der ^ °tografia do produto que se pretende ven- Um quadro escuro de onde sobressaem dois m^ ços e cigarros por si so pouco diriam, entretan marca COLOMBIA qualificados como ULTRA LiGTHq o j • • • a mensagem adquire uma signi- H^ecisa. Com isto assumimos que as imagens associadsQ n .. • , j • 1 00 • -Lsaas a um texto permitem deduzir as reais intenções das publicidades . d id i di b l ' Quando consideramos aqui o codigo verbal ' como coraplemenfPi r. o • - , , • ^ j ^ nto na criaçao do discurso como um 0 o,_es amos nos referindo ao seu poder de mobi 1 zaç ao e n qu an t o parte dos significados desse mesmo discurso. A analise dos elementos do plano de expres são nos permite inferir as seguintes conotaçoes; - 37 - PLANO DE EXPRESSÃO PLANO DE CONTEODO Lingíiis tico Iconico Conotaçoes Colúmbia maço de cigarro no quadro escuro infini to, profundidade Ultra lights sobre os maços de cigarro que parecem se aproximar e cu jos cigarros de um deles saem suavidade e reforço â idéia de leveza 0 mais baixo teor de alca •trao e nico tina homem deitado na re de tocando saxofone não prejudica a saúde, especialmente o apare lho respiratório 0 que voce quer mais? 0 homem com roupas modernas, deitado na rede ... tranqüilidade e prazer Fique afina io com o seu tempo homem com roupas mo dernas, segurando um violoncelo Pessoa atualizada,ajus tada, bem sucedida Mude para Co lumbia ultra lights homem sorrindo num banco com os pês no chão. CIGARROS COLOMBIA felicidade, segurança Fique em dia com 0 mundo ■atual, desço brindo o pra zer mesmas imagens reforça as conotaçoes anteriores através da explicitação lingüís tica PLANO DE EXPRESSÃO - 38 - Descubra o prazer su til de fu mar . . . toda a imagem do ho mem na rede mensagem explícita dos valores conotados No mínimo, o melhor maços de cigarros duas leituras: 1) no cigarro pequeno a me lhor qualidade; 2) en tre todos os cigarros este e, pelo menos, o me lhor. mensagem explícita dos valores conotados duas leituras: 1) no cigarro pequeno a me lhor qualidade; 2) en tre todos os cigarros este e, pelo menos, o me lhor. duas leituras: 1) no cigarro pequeno a me lhor qualidade; 2) en tre todos os cigarros este e, pelo menos, o me lhor. No campo semântico referente ã propaganda de cigarros, selecionamos a marca COLOMBIA. Esta marca atualiza a sua bateria de sugestões ao co^ sumldor, num campo semântico mais restrito; o da cultura musical. Na analise acima feita, nao * distinguimos duas propagandas com característi - cas diferentes porque as diferenças que se atua lizam pertencem a um mesmo paradigma. Isto nao significa que nio haja conteildos, em parte dis - tintos. A observação da imagem que apresenta o ho mem com um instrumento de sopro ê um contra-arg^ mento a tudo que se diz acerca dos efeitos noci vos do cigarro. Alias, esta intenção e complemeii tada pelo realce em que se encontra a informação de que o produto possui menos substancias toxi - cas . A outra imagem substitui o realce acima re- f®^ído, apresentando a outra opção no campo se - mantico da cultura musical: o instrumento de co£ das, pela colocação de um cigarro entre os dedos do rapaz saudável, feliz. . Resta apenas fazer uma observação acerca do item lexical COLOMBIA com que se estabelece uma referencia ã nave espacial. Daí se complementa - - 39 - rem os signos "leveza e suavidade" com a idéia de segurança sugeridos pela rede e pelo ban-co onde se encontram os homens da imagem. Estas informações, mais uma vez, confirmam a consideração do plano de expressão como um siste ma semiotico estruturado. Esta analise permite concluir que estas pu blicidades, como tantas outras, procuram desper - tar mecanismos de projeção no consumidor tais como sucesso, prazer e bem-estar. - 40 - Bibliografia ALMADA, Fernando - in: Simões, Roberto - Comuní cação Publicitária, Atlas, Sao Paulo, 1972. BARTHES, Roland, Retórica de Ia imagem, Escuela Practica de Altos Estúdios, Madrid, 1979' ECO, Humberto, A estrutura ausente. Perspectiva, Sao PauIo, 1973 FADUL, Anamaria - Meios de Comunicação de Massa e Educação, in: Revista de Cultura Vozes, n9 07, ano 74, vozes, Petropolis, setembro de 1980 JAKOBSON, Roman - Lingüística e Comunicação, Ed. Cultrix, são Pau Io TILBURG, João Luiz Van - Modismos, consumismo e o^raito da juventude, in: Juventude e domina- çao cultural, Ed. Paulinas, Sao Paulo, 1982. OMAIS BAIXO TEOR DE AIXIATRÃOE NICXynNA. O QUEV0C3Ê QUERMAIS? í)i'.-<'iibtaii praztTMiiil ilcrumiii Oikimfiia LMlni (iritiifinicipruTOfcimultrabai.vis l«irTsdi.'ulcatráu i-niailiiKi doj):u Ksiairiinciilo (adumbin Uitni Liiiht; OMAIS BAIXO TEOR DE AIXIATRÃOE NICXynNA. O QUEV0C3Ê QUERMAIS? í)i'.-<'iibtaii praztTMiiil ilcrumiii Oikimfiia LMlni (iritiifinicipruTOfcimultrabai.vis l«irTsdi.'ulcatráu i-niailiiKi doj):u í)i'.-<'iibtaii praztTMiiil ilcrumiii Oikimfiia LMlni (iritiifinicipruTOfcimultrabai.vis l«irTsdi.'ulcatráu i-niailiiKi doj):u í)i'.-<'iibtaii praztTMiiil ilcrumiii Oikimfiia LMlni (iritiifinicipruTOfcimultrabai.vis l«irTsdi.'ulcatráu i-niailiiKi doj):u í)i . < iibtaii praztTMiiil ilcrumiii Oikimfiia LMlni (iritiifinicipruTOfcimultrabai.vis l«irTsdi.'ulcatráu i-niailiiKi doj):u Ksiairiinciilo (adumbin Uitni Liiiht; FIQUE AFINADO COM O SEU TEMPO. MUDE PARA COLUMBIA ULTRA LIGHTS. Fiq^ueem dia com O mundo atual.descobrindo opni/ijr de ftunar Columbia Ultra Lights, o primeiro com ultra baixos trorcs dealcatrâo e nicotina. FIQUE AFINADO COM O SEU TEMPO. MUDE PARA COLUMBIA ULTRA LIGHTS. Fiq^ueem dia com O mundo atual.descobrindo opni/ijr de ftunar Columbia Ultra Lights, o primeiro com ultra baixos trorcs dealcatrâo e nicotina. FIQUE AFINADO COM O SEU TEMPO. MUDE PARA COLUMBIA ULTRA LIGHTS. Fiq^ueem dia com O mundo atual.descobrindo opni/ijr de ftunar Columbia Ultra Lights, o primeiro com ultra baixos trorcs dealcatrâo e nicotina. Fiq^ueem dia com O mundo atual.descobrindo opni/ijr de ftunar Columbia Ultra Lights, o primeiro com ultra baixos trorcs dealcatrâo e nicotina.
https://openalex.org/W2118019845	https://bmcmedicine.biomedcentral.com/counter/pdf/10.1186/1741-7015-10-13	English	null	Spectrum of gluten-related disorders: consensus on new nomenclature and classification	BMC medicine	2,012	cc-by	10,325	* Correspondence: afasano@mbrc.umaryland.edu 1Mucosal Biology Research Center and Center for Celiac Research, University of Maryland School of Medicine, Baltimore, MD 21201, USA Full list of author information is available at the end of the article Abstract A decade ago celiac disease was considered extremely rare outside Europe and, therefore, was almost completely ignored by health care professionals. In only 10 years, key milestones have moved celiac disease from obscurity into the popular spotlight worldwide. Now we are observing another interesting phenomenon that is generating great confusion among health care professionals. The number of individuals embracing a gluten-free diet (GFD) appears much higher than the projected number of celiac disease patients, fueling a global market of gluten-free products approaching $2.5 billion (US) in global sales in 2010. This trend is supported by the notion that, along with celiac disease, other conditions related to the ingestion of gluten have emerged as health care concerns. This review will summarize our current knowledge about the three main forms of gluten reactions: allergic (wheat allergy), autoimmune (celiac disease, dermatitis herpetiformis and gluten ataxia) and possibly immune-mediated (gluten sensitivity), and also outline pathogenic, clinical and epidemiological differences and propose new nomenclature and classifications. 10,000 years ago with the advent of agriculture, repre- sented an evolutionary challenge that created the condi- tions for human diseases related to gluten exposure, the best known of which are mediated by the adaptive immune system: wheat allergy (WA) and celiac disease (CD). In both conditions the reaction to gluten is mediated by T-cell activation in the gastrointestinal mucosa. However, in WA it is the cross-linking of immunoglobulin (Ig)E by repeat sequences in gluten peptides (for example, serine-glutamine-glutamine -glu- tamine-(glutamine-)proline-proline-phenylalanine) that triggers the release of chemical mediators, such as hista- mine, from basophils and mast cells [1]. In contrast, CD is an autoimmune disorder, as demonstrated by specific serologic autoantibodies, most notably serum anti-tissue transglutaminase (tTG) and anti-endomysial antibodies (EMA). © 2012 Sapone 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. Spectrum of gluten-related disorders: consensus on new nomenclature and classification Anna Sapone1,2, Julio C Bai3, Carolina Ciacci4, Jernej Dolinsek5, Peter HR Green6, Marios Hadjivassiliou7, Katri Kaukinen8, Kamran Rostami9, David S Sanders10, Michael Schumann11, Reiner Ullrich11, Danilo Villalta12, Umberto Volta13, Carlo Catassi1,14 and Alessio Fasano1* Sapone et al. BMC Medicine 2012, 10:13 http://www.biomedcentral.com/1741-7015/10/13 Sapone et al. BMC Medicine 2012, 10:13 http://www.biomedcentral.com/1741-7015/10/13 Sapone et al. BMC Medicine 2012, 10:13 Open Access Methods instead of CD. GS patients are unable to tolerate gluten and develop an adverse reaction when eating gluten that usually, and differently from CD, does not lead to damage in the small intestine. While the gastrointestinal symptoms in GS may resemble those associated with CD, the overall clinical picture is not accompanied by the concurrence of tTG autoantibodies or other specific celiac-related antibodies. Currently the diagnosis is made by exclusion, and an elimination diet and ‘open challenge’ (that is, the monitored reintroduction of glu- ten-containing foods) are most often used to evaluate whether health improves with the elimination of or reduction in gluten from the diet. However, this approach lacks specificity and is subject to the risk of a placebo effect of the elimination diet in improving symptoms. In order to develop a consensus on new nomenclature and classification of gluten-related disorders, a panel of 15 experts was convened in London in February 2011. Each expert was assigned a specific topic that was then collec- tively discussed and consensus was achieved when each panelist agreed on specific definitions and nomenclature. Based on the discussion and the current evidence in litera- ture, the panel generated a series of definitions and created the classifications and algorithms summarized below. Introduction Wheat, rice and maize are the most widely consumed food grains in the world. Wheat, the most widely grown crop, is immensely diverse, with more than 25,000 dif- ferent cultivars having been produced by plant breeders worldwide. Much of the world’s production of wheat is consumed after it has been processed into bread, other baked goods, pasta and noodles, and, in the Middle East and North Africa, bulgur and couscous. In addition, the wide availability of wheat flour and the functional prop- erties of gluten proteins provide the rationale for their wide use as an ingredient in food processing. Gluten is the main structural protein complex of wheat with equivalent toxic proteins found in other cer- eals, including rye and barley. The toxic protein frac- tions of gluten include gliadins and glutenins, with gliadins containing monomeric proteins and glutenins containing aggregated proteins. Possibly the introduction of gluten-containing grains, which occurred about Besides CD and WA, there are cases of gluten reac- tions in which neither allergic nor autoimmune mechan- isms are involved. These are generally defined as gluten sensitivity (GS) [2-5]. Some individuals who experience distress when eating gluten-containing products and show improvement when following a GFD may have GS Sapone et al. BMC Medicine 2012, 10:13 http://www.biomedcentral.com/1741-7015/10/13 Page 2 of 12 Page 2 of 12 Wheat allergy WA is defined as an adverse immunologic reaction to wheat proteins. Depending on the route of allergen exposure and the underlying immunologic mechanisms, WA is classified into classic food allergy affecting the skin, gastrointestinal tract or respiratory tract; wheat- dependent, exercise-induced anaphylaxis (WDEIA); occupational asthma (baker’s asthma) and rhinitis; and contact urticaria. IgE antibodies play a central role in the pathogenesis of these diseases. The diversity of gluten-induced conditions is in line with the notion that the immune system reacts to and deals with the triggering environmental factor, gliadin, in distinct ways. Here we systematically review the spec- trum of gluten-related disorders and propose new nomenclatures to fill the gaps of current classifications (Figure 1). 36 Gluten Related Disorders Pathogenesis Autoimmune Allergic Not Autoimmune Not allergic (Innate immunity?) Celiac Disease Gluten Ataxia Dermatitis herpetiformis Wheat allergy Gluten sensitivity Respiratory Allergy Food Allergy WDEIA Contact Urticaria Symptomatic Silent Potential Figure 1 Proposed new nomenclature and classification of gluten-related disorders. Gluten Related Disorders Figure 1 Proposed new nomenclature and classification of gluten-related disorders. Sapone et al. BMC Medicine 2012, 10:13 http://www.biomedcentral.com/1741-7015/10/13 Page 3 of 12 Sapone et al. BMC Medicine 2012, 10:13 http://www.biomedcentral.com/1741-7015/10/13 Epidemiology In a population-based birth cohort in Stockholm, the prevalence of sensitization to wheat in 2,336 four-year-old children was reported to be 4% [6,7], decreasing over time [8]. Another, longitudinal, study of 273 children from ages two to ten years reached contrary conclusions, showing that the preva- lence of IgE to wheat progressively increased with age, from 2% to 9% [9]. In adults in the United States, a list- assisted random-digit-dial survey by the US Food and Drug Administration found a 0.4% prevalence of initially self-reported and later on doctor-diagnosed wheat and/ or gluten allergy [10]. In a systematic review by Zuidm- eer et al. [11], two population-based studies from the UK and one from Germany reported positive wheat challenge tests in children, with a prevalence as high as 0.5%. In adults, the prevalence of sensitization to wheat (assessed by IgE) was higher (> 3% in several studies) than perceived (< 1%). With respect to heritability, an ongoing family-based food allergy cohort study in the USA revealed that the estimated heritability of food-spe- cific IgE was statistically significant for all nine tested food allergens, including wheat [12]. patients’ sera. It is clear that one group of wheat pro- teins contains the most important allergens, the a- amylase inhibitors. Wheat allergy However, a number of other pro- teins present in wheat, including germ agglutinin, per- oxidase and non-speciﬁc lipid transfer proteins (LTPs), have been reported to bind to IgE from patients with baker’s asthma [13]. It is of interest that both peroxidase and LTP have also been reported to be active in food allergy to wheat [13]. Food allergy ll Allergic responses to the ingestion of wheat can be divided into two types. WDEIA is a well-defined syn- drome that is caused by a specific type of grain protein, ω5-gliadins. Other allergic responses include atopic der- matitis, urticaria and anaphylaxis and appear to be related to a range of wheat proteins. These may vary between populations and be related to age and symp- toms. Studies with purified proteins using IgE specific assays with patients’ sera showed that 60% had IgE to a-gliadins, b-gliadins and low molecular weight subu- nits, 55% to g-gliadins, 48% to ω-gliadins, and 26% to high molecular weight subunits [13]. All patients with anaphylaxis or WDEIA and 55% of those with urticaria had IgE to ω5-gliadins [13]. Clinical presentations Much of the research on adverse allergic reactions to wheat has focused on respiratory allergy (baker’s asthma), which is one of the most preva- lent occupational allergies in many countries. Dietary allergy to wheat, which in its extreme form may lead to anaphylaxis and death, is probably less widespread in the general population. The proteins that are responsible for a dietary allergy in wheat are also less clearly defined than those contributing to baker’s asthma, but recent studies indicate that there are intriguing similarities and differences between the two conditions. Wheat-dependent exercise-induced anaphylaxis Wheat dependent exercise induced anaphylaxis Patients with WDEIA display a range of clinical symp- toms, from generalized urticaria to severe allergic reac- tions including anaphylaxis. Using synthetic peptides, scientists have identified seven epitopes (QQIPQQQ, QQLPQQQ, QQFPQQQ, QQSPEQQ, QQSPQQQ, QQYPQQQ and PYPP) within the primary sequence of ω5-gliadins as the major allergens. Four of these epi- topes were found to be dominant: QQIPQQQ, QQFPQQQ, QQSPEQQ and QQSPQQQ. Mutational analysis of the QQIPQQQ and QQFPQQQ peptides indicated that the amino acids at positions glutamine-1, proline-4, glutamine-5, glutamine-6 and glutamine-7 were critical for IgE binding [13]. Celiac disease CD is an immune-mediated enteropathy triggered by the ingestion of gluten in susceptible individuals. The onset of symptoms is usually gradual and characterized by a time lag of months or years after gluten introduction. Nevertheless, in patients on long-term treatment with a GFD, the ingestion of gluten may occasionally cause immediate symptoms, such as vomiting and abdominal pain. p Epidemiology CD is one of the most common disorders in countries predominantly populated by people of Eur- opean origin (for example, Europe, North and South America and Australia) affecting approximately 1% of the general population. Interestingly, recent studies indi- cate a trend toward a rising prevalence of CD during the last several decades for reasons that are currently unclear [15,16]. Epidemiological studies have provided evidence that this disorder is also common in other parts of the world, including North Africa, the Middle East and part of the Asian continent. CD frequency is likely to increase in many developing countries, due to the progressing ‘Westernization’ of the diet. For instance, in many Asian countries, a sharp decrease of consumption of rice per capita and a parallel increased consumption of wheat-based products is taking place. Rising income and urbanization are driving forces in the increase in wheat consumption. Whereas wheat is con- sidered an ordinary food in Western societies, in tradi- tional rice-eating Asian countries, wheat is becoming a preferred staple [15]. Because of these alimentary trends, an increasing incidence of CD in Asian countries can be anticipated in the near future. Clinical presentations and diagnosis The clinical spec- trum of CD is wide (Figure 1) and includes symptomatic cases with either classical intestinal (for example, chronic diarrhea, weight loss) or non-classical extrain- testinal (for example, anemia, osteoporosis, neurological disturbances) features and silent forms that are occa- sionally discovered because of serological screening. Additionally, potential forms of the disease, in which the auto-antibodies are detected while the autoimmune insult of the intestinal mucosa is not present, have been described (Figure 1). CD prevalence is increased in at- risk conditions, such as a family history of CD, autoim- mune diseases, IgA deficiency, some genetic syndromes (Down, Turner and William syndromes) and especially type 1 diabetes and thyroiditis. Baker’s asthma d Baker’s asthma d Recognized since the time of the Roman Empire, baker’s asthma and rhinitis are well-characterized allergic responses to the inhalation of wheat and cer- eal flours and dusts [13]. A Polish study discovered that chest respiratory symptoms ascribed to baker’s asthma were observed in 4.2% of bakery apprentices after only one year and in 8.6% after two years [14]. The corresponding values for allergic rhinitis were 8.4% and 12.5%, respectively. Diagnosis is usually based on skin prick tests and the demonstration of specific IgE antibodies (for example, anti-wheat, -bar- ley and -rye ﬂour IgE as well as anti-a-amylase IgE in serum). Little was known about the proteins responsi- ble for baker’s asthma until the application of electro- phoresis combined with immunochemistry in the 1970s. Such early studies showed that multiple aller- gens were present, with the water-soluble albumins being particularly reactive with the IgE fractions from patients’ sera [13]. More recent studies have identified individual proteins, which are recognized by IgE from Diagnosis Skin prick tests and in vitro IgE assays are first-level diagnostics for WA. However, the positive predictive value of these tests is less than 75%, particu- larly in adults due to the cross-reactivity with grass pol- lens. In addition, many commercial reagents for skin prick tests have a low sensitivity since they are mixtures of water- and salt-soluble wheat proteins that lack aller- gens from the insoluble gliadin fraction. Testing prick by prick using only raw material partially overcomes this problem, and in many cases an oral food challenge is necessary for the final diagnosis of food allergy. In recent years a large variety of wheat grain proteins have been identified and characterized as allergens. Some of them are now available for component resolved diagno- sis in WA with an increase of diagnostic accuracy of the in vitro IgE assays. There is no evidence that identifying Page 4 of 12 Sapone et al. BMC Medicine 2012, 10:13 http://www.biomedcentral.com/1741-7015/10/13 serum-IgG antibodies to wheat or gliadin indicates the presence of disease. predisposition depends on a multitude of genes, each of them adding only a modest contribution to disease development [20]. Autoimmune reactions (onset: weeks to years after gluten exposure) Gliadin-specific T-cell responses have been found to be enhanced by the action of intestinal tTG. Although there are at least 50 T-cell-stimulatory epitopes in gluten proteins, a unique 33-mer gliadin fragment (Figure 2, yellow motif) is the most immunogenic pep- tide [21]. Moreover, it is resistant to enzymatic degrada- tion by gastric, pancreatic and brush border peptidases. Altered processing by intraluminal enzymes, changes in intestinal permeability and activation of innate immunity seem to precede the activation of the adaptive immune response. Celiac disease Due to atypical features, many CD cases currently escape diagnosis and are exposed to the risk of long-term complications, for example, infertility and lymphoma, even if it is now appreciated that the prevalence of these complications is lower than previously reported. p y p Specific and sensitive serological tests are available as an initial test for CD. Measurement of IgA antibodies to tTG (anti-tTG) is recommended for initial testing for CD, while IgA anti-EMA is considered as a confirmatory test. More recently, deamidated gliadin peptides (DGP) antibodies (especially of the IgG class) have been intro- duced with sensitivity and specificity comparable to anti-tTG and anti-EMA, but with possibly a better per- formance in IgA-deficient subjects and in children younger than three years [22,23]. A high level of anti- tTG (and possibly anti-DGP) and anti-EMA antibodies (10 × or higher) is almost invariably associated with a typical celiac enteropathy found through biopsy of the small intestine [24]. Individuals with CD who are IgA deficient will not have abnormally elevated levels of IgA anti-tTG or IgA anti-EMA and need to be screened with IgG-based tests. A small intestinal biopsy is an important diagnostic investigation that should be under- taken in many patients with suspected CD. The charac- teristic histological changes include an increased Pathogenesis Genetic predisposition plays a key role in CD, and considerable progress has been made recently in identifying genes that are involved [17-19]. It is well known that CD is strongly associated with specific human leukocyte antigen (HLA) class II genes, known as HLA-DQ2 and HLA-DQ8, located on chromosome 6p21. Most CD patients (approximately 95%) express genes encoding the major histocompatibility complex (MHC) class II protein HLA-DQ2. The remaining patients are usually HLA-DQ8-positive. The HLA-DQ2 haplotype is common and is carried by approximately 30% of Caucasian individuals, implying that the presence of HLA-DQ2 and/or HLA-DQ8 is necessary for disease development but not sufficient on its own as its esti- mated risk effect is only 36% to 53%. On the other hand, non-HLA genes collectively contribute more than HLA to the CD genetic background. However, this Sapone et al. BMC Medicine 2012, 10:13 http://www.biomedcentral.com/1741-7015/10/13 Sapone et al. BMC Medicine 2012, 10:13 Page 5 of 12 Page 5 of 12 http://www.biomedcentral.com/1741-7015/10/13 Figure 2 Mapping of a-gliadin motifs. Celiac disease Those exerting cytotoxic activity are shown in red, immunomodulatory activity in yellow, zonulin release and gut permeating activity in blue, and CXCR3-dependent IL-8 release in celiac disease patients in dark green. Partially modified from [60]. Figure 2 Mapping of a-gliadin motifs. Those exerting cytotoxic activity are shown in red, immunomodulatory activity in yellow, zonulin release and gut permeating activity in blue, and CXCR3-dependent IL-8 release in celiac disease patients in dark green. Partially modified from [60]. 5. Response to a GFD 4. Celiac enteropathy found on small bowel biopsy Dermatitis herpetiformis Dermatitis herpetiformis number of intraepithelial lymphocytes (IELs) (> 25 lym- phocytes per 100 enterocytes), elongation of the crypts, partial to total villous atrophy and a decreased villous: crypt ratio [25]. Polymerase chain reaction sequence- specific oligonucleotide typing methods are now avail- able for the determination of alleles encoding HLA-DQ2 and HLA-DQ8. Since the HLA predisposing genotype is a necessary factor for disease development, the negative predictive value of HLA typing is very high, that is, the vast majority of subjects who are HLA-DQ2- and HLA- DQ8-negative will never develop CD. Dermatitis herpetiformis (DH) is a skin manifestation of CD presenting with blistering rash and pathognomonic cutaneous IgA deposits [27]. DH was named by Duhring in 1884, though his original description covered several disorders including pemphigus and erythema multiforme. Epidemiology DH occurs most commonly in individuals of European origin. The prevalence is approximately one in 10,000 in the UK and is the same in the USA among the white population of European descent, although higher rates of four and six per 10,000 have been reported from Sweden and Finland, respectively [27]. DH seldom occurs in Asians or Africans. DH can pre- sent at any age, but is rare at the extremes of life; the mean age at presentation is about 40 years. In contrast with CD, DH is more common in men than in women (1.5 to 1.9:1). Family studies indicate that 5% of first- degree relatives also will have DH and an additional 5% will have CD. Both DH and CD show the same high prevalence of HLA-DQ2 (90%) and HLA-DQ8 (5%) hap- lotypes [28]. The wide variability of CD-related findings suggests that it is difficult to conceptualize the diagnostic process into rigid algorithms that can cover the clinical com- plexity of this disease. For this reason, a quantitative approach that can be defined as the ‘four out of five rule’ was proposed recently[26]. Using this method, the diagnosis of CD is confirmed if at least four of the fol- lowing five criteria are fulfilled: 1. Typical symptoms of CD 2. Positivity of serum CD IgA class autoantibodies at high titer Pathogenesis It is not known why only some patients with CD develop DH and what factors link the bowel and skin lesions. In DH, IgA is present in the skin, and 3. HLA-DQ2 and/or HLA-DQ8 genotypes 3. Gluten ataxia Antibodies against tTG6, a primarily brain-expressed transglutaminase, have been found in patients with GA [39-41]. In GA and DH, IgA deposits seem to accumulate in the periphery of vessels where in healthy TG6 or TG3, respectively, these are not usually found [42]. Using a mouse model, it has recently been shown that serum from GA patients, as well as clonal monovalent anti-tTG Igs derived using phage display, causes ataxia when injected intraventricularly in mice [43]. These data therefore provide evidence that anti-tTG Igs (derived from patients) compromise neuronal function in selected areas of the brain, suggesting that this involves an immune-system independent mode of action. The diagnosis of DH rests on the demonstration of IgA in uninvolved skin on biopsies analyzed by immu- nofluorescence staining. The most common site is in the dermal papillae, where IgA is detected as granular or fibrillar deposits. IgA may also be laid down in a lin- ear granular fashion along the line of the basement membrane. It is important that this pattern is differen- tiated from homogeneous linear IgA deposition found in linear IgA disease, which is not gluten dependent. Diag- nosis of DH is based on skin biopsy and serological evi- dence of celiac-type autoimmunity. Since DH is the cutaneous counterpart of CD (’skin CD’), a proven diag- nosis of DH in a patient should be taken as indirect evi- dence for the presence of small bowel damage. Accordingly, a duodenal biopsy is unnecessary in DH patients [30]. Clinical presentation and diagnosis GA usually pre- sents with pure cerebellar ataxia or, rarely, ataxia in combination with myoclonus, palatal tremor or opsoclo- nus myoclonus. GA is usually of insidious onset with a mean age at onset of 53 years. Rarely, the ataxia can be rapidly progressive. Gaze-evoked nystagmus and other ocular signs of cerebellar dysfunction are seen in up to 80% of cases. All patients have gait ataxia, and the majority have limb ataxia. Less than 10% of patients with GA will have any gastrointestinal symptoms, but a third will have evidence of enteropathy on biopsy. Nevertheless, an intestinal biopsy is indicated when serum tTG2 antibodies are elevated. Up to 60% of patients have neurophysiological evidence of sensorimo- tor, length-dependent axonal neuropathy. This is usually mild and does not contribute to the ataxia. Gluten ataxia Gluten ataxia (GA) was originally defined as otherwise idiopathic sporadic ataxia with positive serological mar- kers for gluten sensitization [31]. Like CD, it is an auto- immune disease characterized by damage to the cerebellum resulting in ataxia. Epidemiology A series of 800 patients with progressive ataxia evaluated over a period of 15 years in Sheffield, UK, found that 148 out of 635 patients (23%) with sporadic ataxia had serological evidence of gluten sensi- tization (M. Hadjivassiliou, personal communication). A number of studies looking at the prevalence of anti-glia- din antibodies (AGA) in ataxias have been published [32-35]. The common theme in most of these studies is the consistently high prevalence of AGA in sporadic ataxias when compared to healthy controls. Clinical features and diagnosis The earliest skin abnormalities consist of a small erythematous macule, which rapidly develops into an urticarial papule. Small vesicles appear that may rupture, dry and form scabs. The predominant symptoms are intense itching and burning. The rash has a characteristic symmetrical dis- tribution. The elbows and upper forearms are affected in more than 90% of patients. Other sites commonly involved are the buttocks, knees, shoulders, sacrum, face, scalp, neck and trunk. The rash may be wide- spread, but can be limited to one or two sites. Once the rash appears, it is an ongoing problem in most patients, but it can run an intermittent course in 10% of cases. Only a minority of patients, about 10%, have gastrointestinal symptoms and these are usually mild. However, celiac-type villous atrophy in the upper small intestinal mucosa is found in 65% to 75% of patients with DH. Even in patients with apparently normal biopsies, subtle changes in the mucosa, such as an increased number of IELs, indicate gluten sensitization. A celiac-type pattern of autoantibodies (anti-tTG, anti- EMA and anti-DGP antibodies) is usually found in the serum of patients with DH. Likewise, DH patients may show the same array of manifestations, associated dis- orders and complications as in patients with CD (auto- immune diseases, iron-deficient anemia, osteoporosis and malignancy). Pathogenesis There is evidence to suggest that there is antibody cross-reactivity between antigenic epitopes on Purkinje cells and gluten proteins [36-38]. Widespread deposition of transglutaminase antibodies has been found around brain vessels in patients with GA. The deposition is most pronounced in the cerebellum, pons and medulla. Dermatitis herpetiformis HLA-DQ2 and/or HLA-DQ8 genotypes 4 Celiac enteropathy found on small bowel biopsy 3. HLA-DQ2 and/or HLA-DQ8 genotypes 4. Celiac enteropathy found on small bowel biopsy 4. Celiac enteropathy found on small bowel biopsy Sapone et al. BMC Medicine 2012, 10:13 http://www.biomedcentral.com/1741-7015/10/13 Page 6 of 12 potential CD), because the rash of DH is gluten sensitive. potential CD), because the rash of DH is gluten sensitive. inflammatory cells and cytokines are found in the lesions. Furthermore, anti-EMA and anti-tTG antibodies occur in the serum, and the rash is gluten-sensitive. The importance of these factors and how they interact to produce skin lesions remains unknown, though recently antibodies directed at epidermal transglutaminase (TG3) have been identified in patients with DH and this may be the dominant autoantigen in the disorder [29]. Gluten ataxia The market for gluten-free food and beverage products grew at a compound annual growth rate of 28% from 2004 to 2011, eclipsing the low carbohydrate diet and the fat-free diet in 2008, to finish with almost $1.6 billion in retail sales in 2010. By 2012 the market is expected to reach about $2.6 billion in sales. The fact that approximately three million Americans suffer from celiac disease and only a fraction of these patients have been diagnosed implies that patients suffering other forms of proven gluten reaction, including gluten sensitivity and wheat allergy, contribute to this market growth. The rest of the market is filled either by people who undertake the diet as occasional consumers (no medical necessity) or by individuals affected by maladies that have been claimed to be affected by gluten exposure, including autism spectrum disorder, attention deficit hyperactivity disorder, multiple sclerosis and irritable bowel syndrome, but for which there is no evidence of the effectiveness of the diet. The diagnosis of GA is not as straightforward as that of CD. Anti-tTG2 IgA antibodies are only present in up to 38% of patients, but often at lower titers than those seen in patients with CD. However, unlike CD, IgG class antibodies to tTG2 are more frequent than IgA. Antibodies against tTG2 and tTG6 combined can be found in 85% of patients with ataxia who are positive for AGA antibodies [41]. It is unclear at present whether combined detection of anti-tTG2 and anti-tTG6 IgA and IgG without the use of AGA identifies all patients with gluten ataxia. The current recommendation is that patients present- ing with progressive cerebellar ataxia should be screened for GS using AGA IgG and IgA, anti-tTG2 antibodies and, if available, IgG and IgA anti-tTG6 antibodies. Patients with positive anti-tTG2 antibodies should undergo a duodenal biopsy. However, irrespective of the presence of an enteropathy, patients positive for any of these antibodies with no alternative cause for their ataxia should be offered a strict GFD with regular fol- low-up to ensure that the antibodies are eliminated, which usually takes six to twelve months. Stabilization or even improvement of the ataxia after one year would be a strong indicator that the patient suffers from GA. distinguished clinically, since the symptoms experienced by GS patients are often seen in CD. Gluten ataxia We propose as a definition of GS those cases of gluten reaction in which both allergic and autoimmune mechanisms have been ruled out (diagnosis by exclusion criteria). More specifi- cally, these are cases with negative immuno-allergy tests to wheat or negative CD serology (anti-EMA and/or anti-tTG); where IgA deficiency has been ruled out; with normal duodenal histopathology; with the possible presence of biomarkers of native gluten immune-reac- tion (AGA+); with clinical symptoms that can overlap with CD or WA symptoms; and patients who show a resolution of symptoms when started on a GFD, imple- mented in a blinded fashion to avoid a possible placebo effect of the dietary intervention. Gluten ataxia Up to 60% After establishing a diagnosis of DH, GFD implemen- tation should be recommended, even when the small intestinal mucosa appears normal (as is the case in Sapone et al. BMC Medicine 2012, 10:13 http://www.biomedcentral.com/1741-7015/10/13 Page 7 of 12 Figure 3 Trend of three different diets (low carbohydrate diet, fat-free diet, and gluten-free diet), in the USA during the period 2004 to 2011. For the American general population, adopting a gluten-free diet is becoming an increasingly popular option. The market for gluten-free food and beverage products grew at a compound annual growth rate of 28% from 2004 to 2011, eclipsing the low carbohydrate diet and the fat-free diet in 2008, to finish with almost $1.6 billion in retail sales in 2010. By 2012 the market is expected to reach about $2.6 billion in sales. The fact that approximately three million Americans suffer from celiac disease and only a fraction of these patients have been diagnosed implies that patients suffering other forms of proven gluten reaction, including gluten sensitivity and wheat allergy, contribute to this market growth. The rest of the market is filled either by people who undertake the diet as occasional consumers (no medical necessity) or by individuals affected by maladies that have been claimed to be affected by gluten exposure, including autism spectrum disorder, attention deficit hyperactivity disorder, multiple sclerosis and irritable bowel syndrome, but for which there is no evidence of the effectiveness of the diet. Fi 3 T d f th diff t di t (l b h d t di t of patients with GA have evidence of cerebellar atrophy on magnetic resonance imaging. Even in those patients without cerebellar atrophy, a proton magnetic resonance spectroscopy of the cerebellum is abnormal. The response to treatment with a GFD depends on the duration of the ataxia prior to the diagnosis of GS. Loss of Purkinje cells in the cerebellum, the end result of prolonged gluten exposure in patients with GA, is irreversible and prompt treatment is more likely to result in improvement or stabilization of the ataxia. Figure 3 Trend of three different diets (low carbohydrate diet, fat-free diet, and gluten-free diet), in the USA during the period 2004 to 2011. For the American general population, period 2004 to 2011. For the American general population, adopting a gluten-free diet is becoming an increasingly popular option. Gluten sensitivity The recent rise of the gluten-free market in the USA (Figure 3), partially sustained by individuals who claim a medical necessity to undertake a GFD, raises questions about possible gluten reactions alternative to CD and WA. It is now becoming clear that, besides CD and WA, there are cases of gluten reactions in which neither allergic nor autoimmune mechanisms can be identified. These are generally defined as non-celiac GS or more simply, GS. Some individuals who experience distress when eating gluten-containing products and show improvement when following a GFD may have GS instead of CD. GS is a condition distinct from CD and is not accompanied by the concurrence of anti-tTG autoantibodies or other autoimmune comorbidities [44]. The small intestine of GS patients is usually normal [44]. However, the two conditions cannot be The symptoms in GS may resemble those associated with CD but with a prevalence of extraintestinal symp- toms, such as behavioral changes, bone or joint pain, muscle cramps, leg numbness, weight loss and chronic fatigue. Between 2004 and 2010, 5,896 patients were seen at the Center for Celiac Research, University of Maryland. The criteria for GS were fulfilled by 347 (1:17; 6%) of the patients seen. Their symptoms included abdominal pain (68%); eczema and/or rash (40%); head- ache (35%); ‘foggy mind’ (34%); fatigue (33%); diarrhea (33%); depression (22%); anemia (20%); numbness in the legs, arms or fingers 20%; and joint pain (11%). While the class II MHC haplotype HLA-DQ2 and HLA-DQ8 are present in almost all CD patients, these Sapone et al. BMC Medicine 2012, 10:13 http://www.biomedcentral.com/1741-7015/10/13 Page 8 of 12 barrier function. Tight junctions (tj) and multiple pro- teins that make up the tj strands (for example, occludin, the claudin family, zonula occludens-1 protein (ZO-1)) have a critical role in the development of intestinal immunological responsiveness. When the integrity of the intestinal tj is compromised, an immune response to environmental antigens that cross-react with host anti- gens may develop, thereby triggering the onset of CD [60,61]. Conversely, in a study conducted by Sapone et al., GS subjects showed a normal intestinal permeability and claudin-1 and ZO-1 expression compared to celiac patients, and a significantly higher expression of clau- din-4. In the same GS patients, the up-regulation of claudin-4 was associated with an increased expression of toll-like receptor-2 and a significant reduction of T-reg- ulatory cell marker FoxP3 relative to controls and CD patients. Gluten sensitivity Additionally, an increase in IELs of the classes a and b, but no increase in adaptive immunity-related gut mucosal gene expression, including interleukin (IL)- 6, IL-21 and interferon -g, was detected in GS [62]. These changes in GS could suggest an important role of the innate immune system without any involvement of the adaptive immune response [62]. In vitro studies sug- gest that wheat amylase-trypsin inhibitors (ATIs) could play a major role as triggers of the innate immune response in GS. Wheat ATIs are a family of five or more homologous small proteins highly resistant to intestinal proteolysis. They are known to be the major allergen responsible for baker’s asthma. Preliminary evi- dence suggests that the addition of 1 μg/mL to 20 μg/ mL of ATIs to monocyte-derived dendritic cells stimu- lates the release of IL-8 in a dose-dependent manner (Detlef Schuppan, unpublished data). Recently, to test the hypothesis that gluten can cause gastrointestinal symptoms in patients without CD, a double-blind, ran- domized, placebo-controlled re-challenge trial was undertaken in patients with IBS fulfilling the Rome cri- teria III, who had CD excluded by best practice methods and who reported a symptom response to a GFD [63]. Patients were randomized according to a computer-gen- erated list of random numbers held by an independent observer to either the gluten or the placebo treatment group. Over the entire study period, the severity scores of pain, satisfaction with stool consistency and tiredness were significantly higher for those consuming the gluten diet compared to the placebo group, while no evidence for intestinal inflammation or damage or for latent CD was found to offer an explanation for symptom dete- rioration caused by gluten. Therefore, this study further supports the notion that non-celiac GS is part of the t f l t l t d di d d fi d genes are present in only about 50% of patients with GS, a percentage still higher compared to the general popu- lation [45,46]. Similarly, an association of HLA-DQ2 with GS in diarrhea-predominant irritable bowel syn- drome (IBS) has been reported [47]. Therefore the involvement of an MHC-dependent, adaptive immune response in GS is currently unclear and requires further research. During the last decade, several studies have identified signs and symptoms associated with non- celiac GS, particularly concerning neuropsychiatric dis- orders. Gluten sensitivity Patients with schizophrenia have higher than expected titers of AGA, which are related to CD and GS, whereas the implementation of a GFD seems to improve the behavior of a subset of children with autism spectrum disorders (ASD) [48,49]. However, currently there are no laboratory biomarkers specific for GS. Usually the diagnosis is based on exclusion criteria; an elimination diet of gluten-containing foods followed by an open challenge is most often used to evaluate whether health improves with the elimination or reduc- tion of gluten from the patient’s diet. Pathogenesis Under physiological conditions, the first contact between food antigens and the local immune system in the gut occurs through the interaction of anti- gen-presenting cells, specifically dendritic cells that sense luminal contents and promote tolerance toward luminal food antigens, thus maintaining a disease-free state [50-57]. The maintenance of tolerance requires high differentiation and maturation of both epithelial and immune cell compartments, and even a minimal perturbation of this delicate balance may result in pathological conditions. Unfortunately, evidence-based information in this area is limited, but it is well accepted that undigested or partly digested gliadin can affect a wide range of human cell functions. Early introduction of gliadin-containing cereals was recently reported to increase the risk of islet cell autoimmunity in humans [58]. Findings from studies using non-obese diabetic mice and BioBreeding diabetic-prone (BBDP) rats have implicated wheat gliadin as a dietary diabetogenic factor. In BBDP rats, gliadin exposure is accompanied by zonu- lin-dependent increased intestinal permeability, presum- ably allowing food antigens to come in contact with the underlying lamina propria [59]. Zonulin is an important protein released by the small intestinal mucosa after sev- eral stimuli (for example, dietary antigens, including glu- ten (see Figure 2) or bacteria) and is involved in the modulation of paracellular intestinal permeability. A normal intestinal epithelium is impermeable to macro- molecules, while CD is characterized by enhanced intestinal permeability and an altered junctional struc- ture between epithelial cells, leading to compromised Page 9 of 12 Sapone et al. BMC Medicine 2012, 10:13 http://www.biomedcentral.com/1741-7015/10/13 Algorithm to differentiate the different forms of gluten- related disorders in a GFD. A wide range of attractive and palatable glu- ten-free wheat substitutes that guarantee the absence of gluten are specifically manufactured for patients with CD and other gluten-related disorders. These products are labeled by an internationally recognized mark, the crossed ear of wheat. Patients with CD not only need to be treated for life, but also require a very accurate treat- ment, as gluten traces may still be able to induce damage to their small intestinal mucosa. On the other hand, the natural history of other gluten-related disor- ders, particularly GS, is still unclear. Further studies are urgently required to clarify whether the spectrum of toxic cereals, the gluten threshold and the disease dura- tion are the same in gluten allergy and/or sensitivity as in CD. Based on a combination of clinical, biological, genetic and histological data, it is possible to differentiate the three conditions (WA, CD and GS), following the algo- rithm shown in Figure 4. In most cases, information coming from clinical presentation will be sufficient to distinguish between WA and the remaining two forms of gluten-related disorders (CD and GS). Determination of specific biomarkers for WA and CD is the proper first step in the diagnostic process including gluten chal- lenge (WA) or intestinal biopsy (CD). If these forms have been excluded and other possible causes of the symptoms experienced by patients have been ruled out, then GS should be considered. A double-placebo gluten challenge will be the final step to either confirm or rule out GS. Through the years, a GFD has been experimentally applied to schizophrenia, multiple sclerosis, ASD and dementia, to name only a few instances. The evidence that the diet is effective in any of these areas is still con- troversial, as a placebo effect of the dietary treatment is often difficult to eliminate. ‘Allergy’ is currently all the References 1. Tanabe S: Analysis of food allergen structures and development of foods for allergic patients. Biosci Biotechnol Biochem 2008, 72:649-659. 1. Tanabe S: Analysis of food allergen structures and development of foods for allergic patients. Biosci Biotechnol Biochem 2008, 72:649-659. 2. Anderson LA, McMillan SA, Watson RG, Monaghan P, Gavin AT, Fox C, Murray LJ: Malignancy and mortality in a population-based cohort of patients with coeliac disease or “gluten sensitivity”. World J Gastroenterol 2007, 13:146-151. 2. Anderson LA, McMillan SA, Watson RG, Monaghan P, Gavin AT, Fox C, Murray LJ: Malignancy and mortality in a population-based cohort of patients with coeliac disease or “gluten sensitivity”. World J Gastroenterol 2007, 13:146-151. 3. Brandtzaeg P, Halstensen TS, Kett K, Krajci P, Kvale D, Rognum TO, Scott H, Sollid LM: Immunobiology and immunopathology of human gut mucosa: humoral immunity and intraepithelial lymphocytes. Gastroenterology 1989, 97:1562-1584. 3. Brandtzaeg P, Halstensen TS, Kett K, Krajci P, Kvale D, Rognum TO, Scott H, Sollid LM: Immunobiology and immunopathology of human gut mucosa: humoral immunity and intraepithelial lymphocytes. Gastroenterology 1989, 97:1562-1584. Additionally, gluten is one of the most abundant and diffusely spread dietary components for most popula- tions, particularly those of European origin. In Europe, the mean consumption of gluten is 10 g to 20 g per day, with segments of the general population consuming as much as 50 g of daily gluten or more [66,67] All indivi- duals, even those with a low degree of risk, are therefore susceptible to some form of gluten reaction during their life span. Therefore, it is not surprising that during the past 50 years we have witnessed an ‘epidemic’ of CD [68,69] and the surging of new gluten-related disorders, including the most recently described GS [44,62]. This review provides some rationale to explain these epide- miological phenomena and expands our current knowl- edge to gain more insights into gluten-related disorders. 4. Catassi C, Fasano A: Celiac disease. Curr Opin Gastroenterol 2008, 24:687-691. 4. Catassi C, Fasano A: Celiac disease. Curr Opin Gastroenterol 2008, 24:687-691. 5. Hoffenberg EJ, MacKenzie T, Barriga KJ, Eisenbarth GS, Bao F, Haas JE, Erlich H, Bugawan Tl T, Sokol RJ, Taki I, Norris JM, Rewers M: A prospective study of the incidence of childhood celiac disease. J Pediatr 2003, 143:308-314. 5. Authors’ contributions ll h d d All authors provided input on the content of the manuscript, the classification of gluten-related disorders and the diagnostic algorithm proposed. AS, CC, and AF drafted the manuscript. All authors read and approved the final manuscript. Conclusions It is now becoming apparent that reactions to gluten are not limited to CD, rather we now appreciate the exis- tence of a spectrum of gluten-related disorders. The high frequency and wide range of adverse reactions to gluten raise the question as to why this dietary protein is toxic for so many individuals in the world. One possi- ble explanation is that the selection of wheat varieties with higher gluten content has been a continuous pro- cess during the last 10,000 years, with changes dictated more by technological rather than nutritional reasons. Wheat varieties grown for thousands of years and mostly used for human nutrition up to the Middle Ages, such as Triticum monococcum and T. dicoccum, contain less quantities of the highly toxic 33-mer gluten peptide [65]. Apparently the human organism is still largely vul- nerable to the toxic effects of this protein complex, par- ticularly due to a lack of adequate adaptation of the gastrointestinal and immunological responses. Treatment with the GFD In general, treatment of gluten-related disorders is based on excluding gluten-containing cereals from the diet. Wheat, barley and rye proteins are completely excluded HistoryandPhysicalExamͲ InitialEvaluation– ConsiderDifferentialDiagnosis WheatAllergy(WA) CeliacDisease(CD) GlutenSensitivity(GS) • Specificskinpricktests • WheatspecificserumIgE • Glutenchallenge Tests+ Challenge+ YES NO WAdiagnosisconfirmed tTG and/or dAGA+ YES NO WA ruledout • tTG IgA+/Ͳ EMA+totalIgA • Deamidated AGAIgA • AGA EGDwith biopsies Suspected GS Biopsy positive Gluten challenge+ YES GSdiagnosis confirmed NO GSruledout Considerother diagnoses YES NO PotentialCD CDdiagnosis confirmed Figure 4 Proposed algorithm for the differential diagnosis of gluten-related disorders, including celiac disease, gluten sensitivity and wheat allergy. HistoryandPhysicalExamͲ InitialEvaluation ConsiderDifferentialDiagnosis HistoryandPhysicalExamͲ InitialEvaluation– ConsiderDifferentialDiagnosis • tTG IgA+/Ͳ EMA+totalIgA • Deamidated AGAIgA • AGA • Specificskinpricktests • WheatspecificserumIgE • Glutenchallenge WA ruledout Suspected GS tTG and/or dAGA+ Tests+ Challenge+ NO YES YES GSruledout Considerother diagnoses EGDwith biopsies WAdiagnosisconfirmed Biopsy positive PotentialCD NO Figure 4 Proposed algorithm for the differential diagnosis of gluten-related disorders, including celiac disease wheat allergy. Sapone et al. BMC Medicine 2012, 10:13 http://www.biomedcentral.com/1741-7015/10/13 Page 10 of 12 Page 10 of 12 Gastroenterology and Hepatology, Royal Hallamshire Hospital and University of Sheffield Medical School, Sheffield, UK. 11Department of Gastroenterology, Rheumatology and Infectivology, Charité University Medicine, Berlin, Germany. 12Allergy and Clinical Immunology Unit, DML, AO Santa Maria degli Angeli, Pordenone, Italy. 13Department of Digestive Diseases and Internal Medicine, St Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy. 14Department of Pediatrics, Università Politecnica delle Marche, Ancona, Italy. Gastroenterology and Hepatology, Royal Hallamshire Hospital and University of Sheffield Medical School, Sheffield, UK. 11Department of Gastroenterology, Rheumatology and Infectivology, Charité University Medicine, Berlin, Germany. 12Allergy and Clinical Immunology Unit, DML, AO Santa Maria degli Angeli, Pordenone, Italy. 13Department of Digestive Diseases and Internal Medicine, St Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy. 14Department of Pediatrics, Università Politecnica delle Marche, Ancona, Italy. rage, and it is well possible that many individuals are on a GFD for no sound medical reasons. In these cases, the diagnostic algorithm described in this paper will help to select patients who really need treatment with a GFD. Acknowledgements We would like to thank Ms. Susie Flaherty at the Center for Celiac Research for assisting us in the manuscript’s editing. Competing interests UR received funding from Dr. Schär to perform a diagnostic study on celiac disease. This paper was made possible by support from Dr. Schär for traveling and lodging sponsorship for all co-authors to meet to discuss the object of this paper. Dr. Schär also covered the article-processing charge. Received: 9 October 2011 Accepted: 7 February 2012 Published: 7 February 2012 Received: 9 October 2011 Accepted: 7 February 2012 Published: 7 February 2012 Acknowledgements W ld lik h 10. Vierk KA, Koehler KM, Fein SB, Street DA: Prevalence of self-reported food allergy in American adults and use of food labels. J Allergy Clin Immunol 2007, 119:1504-1510. 11. Zuidmeer L, Goldhahn K, Rona RJ, Gislason D, Madsen C, Summers C, Sodergren E, Dahlstrom J, Lindner T, Sigurdardottir ST, McBride D, Keil T: The prevalence of plant food allergies: a systematic review. J Aller Clin Immunol 2008, 121:1210-1218. References Hoffenberg EJ, MacKenzie T, Barriga KJ, Eisenbarth GS, Bao F, Haas JE, Erlich H, Bugawan Tl T, Sokol RJ, Taki I, Norris JM, Rewers M: A prospective study of the incidence of childhood celiac disease. J Pediatr 2003, 143:308-314. 6. Ostblom E, Wickman M, van Hage M, Lilja G: Reported symptoms of food hypersensitivity and sensitization to common foods in 4-year-old children. Acta Paediatr 2008, 97:85-90. 6. Ostblom E, Wickman M, van Hage M, Lilja G: Reported symptoms of food hypersensitivity and sensitization to common foods in 4-year-old children. Acta Paediatr 2008, 97:85-90. 7. Ostblom E, Lilja G, Ahlstedt S, van Hage M, Wickman M: Patterns of quantitative food-specific IgE antibodies and reported food hypersensitivity in 4-year-old children. Allergy 2008, 63:418-424. 7. Ostblom E, Lilja G, Ahlstedt S, van Hage M, Wickman M: Patterns of quantitative food-specific IgE antibodies and reported food hypersensitivity in 4-year-old children. Allergy 2008, 63:418-424. 8. Ostblom E, Lilja G, Pershagen G, van Hage M, Wickman M: Phenotypes of food hypersensitivity and development of allergic diseases during the first 8 years of life. Clin Exp Allergy 2008, 38:1325-1332. 9. Matricardi PM, Bockelbrink A, Beyer K, Keil T, Niggemann B, Grüber C, Wahn U, Lau S: Primary versus secondary immunoglobulin E sensitization to soy and wheat in the Multi-Centre Allergy Study cohort. Clin Exp Allergy 2008, 38:493-500. Acknowledgements We would like to thank Ms. Susie Flaherty at the Center for Celiac Research for assisting us in the manuscript’s editing. 16. Catassi C, Kryszak D, Louis-Jacques O, Duerksen DR, Hill I, Crowe SE, Brown AR, Procaccini NJ, Wonderly BA, Hartley P, Moreci J, Bennett N, 10. Vierk KA, Koehler KM, Fein SB, Street DA: Prevalence of self-reported food allergy in American adults and use of food labels. J Allergy Clin Immunol 2007, 119:1504-1510. 13. Tatham AS, Shewry PR: Allergens in wheat and related cereals. Clin Experiment Aller 2008, 38:1712-1726. 12. Inomata N: Wheat allergy. Cur Opin Aller Clinic Immunol 2009, 9:238-243. Author details 1 Author details 1Mucosal Biology Research Center and Center for Celiac Research, University of Maryland School of Medicine, Baltimore, MD 21201, USA. 2Department of Internal and Experimental Medicine Magrassi-Lanzara, Second University of Naples, Naples, Italy. 3Department of Medicine, Dr Carlos Bonorino Udaondo Gastroenterology Hospital, Buenos Aires, Argentina. 4Gastroenterology Unit, University of Salerno School of Medicine, Salerno, Italy. 5University Medical Centre Maribor, Ljubljansk, Slovenia. 6Celiac Disease Center, Columbia University, New York, NY 10032, USA. 7Department of Neurology, Royal Hallamshire Hospital, Sheffield, UK. 8Department of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital and School of Medicine, University of Tampere, Finland. 9Dudley Group of Hospitals, University of Birmingham Medical School, Birmingham, UK. 10Department of 12. Inomata N: Wheat allergy. Cur Opin Aller Clinic Immunol 2009, 9:238-243. 13. Tatham AS, Shewry PR: Allergens in wheat and related cereals. Clin Experiment Aller 2008, 38:1712-1726. 14. Walusiak J, Hanke W, Górski P, Pałczyński C: Respiratory allergy in apprentice bakers: do occupational allergies follow the allergic march? Allergy 2004, 59:442-450. 15. Catassi C, Gobellis G: Coeliac disease epidemiology is alive and kicking, especially in the developing world. Dig Liver Dis 2007, 39:908-910. 16. Catassi C, Kryszak D, Louis-Jacques O, Duerksen DR, Hill I, Crowe SE, Brown AR, Procaccini NJ, Wonderly BA, Hartley P, Moreci J, Bennett N, Page 11 of 12 Sapone et al. BMC Medicine 2012, 10:13 http://www.biomedcentral.com/1741-7015/10/13 Sapone et al. BMC Medicine 2012, 10:13 http://www.biomedcentral.com/1741-7015/10/13 36. Abele M, Bürk K, Schöls L, Schwartz S, Besenthal I, Dichgans J, Zühlke C, Riess O, Klockgether T: The aetiology of sporadic adult-onset ataxia. Brain 2002, 125:961-968. Horvath K, Burk M, Fasano A: Detection of Celiac disease in primary care: a multicenter case-finding study in North America. Am J Gastroenterol 2007, 102:1454-1460. Horvath K, Burk M, Fasano A: Detection of Celiac disease in primary care: a multicenter case-finding study in North America. Am J Gastroenterol 2007, 102:1454-1460. 17. Wolters VM, Wijmenga C: Genetic background of celiac disease and its clinical implications. Am J Gastroenterol 2008, 103:190-195. 37. Hadjivassiliou M, Aeschlimann P, Strigun A, Sanders DS, Woodroofe N, Aeschlimann D: Autoantibodies in gluten ataxia recognize a novel neuronal transglutaminase. Ann Neurol 2008, 64:332-343. 18. Author details 1 Boscolo S, Lorenzon A, Sblattero D, Florian F, Stebel M, Marzari R, Not T, Aeschlimann D, Ventura A, Hadjivassiliou M, Tongiorgi E: Anti transglutaminase antibodies cause ataxia in mice. PLoS ONE 2010, 5(3): e9698. 44. Sapone A, Lammers KM, Mazzarella G, Mikhailenko I, Cartenì M, Casolaro V, Fasano A: Differential mucosal IL-17 expression in two gliadin-induced disorders: gluten sensitivity and the autoimmune enteropathy celiac disease. Int Arch Allergy Immunol 2010, 152:75-80. 21. Shan L, Molberg O, Parrot I, Hausch F, Filiz F, Gray GM, Sollid LM, Khosla C: Structural basis for gluten intolerance in celiac sprue. Science 2002, 297:2275-2279. 45. Monsuur AJ, Wijmenga C: Understanding the molecular basis of celiac disease: what genetic studies reveal. Ann Med 2006, 38:578-591. 22. Prince HE: Evaluation of the INOVA diagnostics enzyme-linked immunosorbent assay kits for measuring serum immunoglobulin G (IgG) and IgA to deamidated gliadin peptides. >Clin Vaccine Immunol 2006, 13(1):150-151. 46. Mazzarella G, Maglio M, Paparo F, Nardone G, Stefanile R, Greco L, van de Wal Y, Kooy Y, Koning F, Auricchio S, Troncone R: An immunodominant DQ8 restricted gliadinpeptide activates small intestinal immune response in in vitro cultured mucosa from HLA-DQ8 positive but not HLA-DQ8 negative coeliac patients. Gut 2003, 52:57-62. 23. Liu E, Li M, Emery L, Taki I, Barriga K, Tiberti C, Eisenbarth GS, Rewers MJ, Hoffenberg EJ: Natural history of antibodies to deamidated gliadin peptides and transglutaminase in early childhood celiac disease. J Pediatr Gastroenterol Nutr 2007, 45:293-300. 47. Wahnschaffe U, Ullrich R, Riecken EO, Schulzke JD: Celiac disease-like abnormalities in a subgroup of patients with irritable bowel syndrome. Gastroenterology 2001, 121:1329-1338. 24. Sugai E, Hwang HJ, Vázquez H, Smecuol E, Niveloni S, Mazure R, Mauriño E, Aeschlimann P, Binder W, Aeschlimann D, Bai JC: New serology assays can detect gluten sensitivity among enteropathy patients seronegative for anti-tissue transglutaminase. Clin Chem 2010, 56:661-665. 48. Cascella NG, Kryszak D, Bhatti B, Gregory P, Kelly DL, Mc Evoy JP, Fasano A, Eaton WW: Prevalence of celiac disease and gluten sensitivity in the United States clinical antipsychotic trials of intervention effectiveness study population. Schizophr Bull 2011, 37:94-100. 25. Villanacci V, Corazza GR: Coeliac disease. J Clin Pathol 2005, 58:573-574. 26. Catassi C, Fasano A: Celiac disease diagnosis: simple rules are better than complicated algorithms. Am J Med 2010, 123:691-693. 49. Author details 1 Van Heel DA, Franke L, Hunt KA, Gwilliam R, Zhemakova A, Inouye M, Wapenaar MC, Barnardo MC, Bethel G, Holmes GK, Feighery C, Jewell D, Kelleher D, Kumar P, Travis S, Walters JR, Sanders DS, Howdle P, Swift J, Playford RJ, McLaren WM, Mearin ML, Mulder CJ, McManus R, McGinnis R, Cardon LR, Deloukas P, Wijmenga C: A genome-wide association study for celiac disease identifies risk variants in the region harboring IL2 and IL21. Nat Genet 2007, 39:827-829. 38. Cooke WT, Thomas-Smith W: Neurological disorders associated with adult coeliac disease. Brain 1966, 89:683-722. 39. Hadjivassiliou M, Boscolo S, Davies-Jones GAB, Grünewald RA, Not T, Sanders DS, Simpson JE, Tongiorgi E, Williamson CA, Woodroofe NM: The humoral response in the pathogenesis of gluten ataxia. Neurology 2002, 58:1221-1226. 40. Korponay-Szabó IR, Halttunen T, Szalai Z, Laurila K, Király R, Kovács JB, Fésüs L, Mäki M: In vivo targeting of intestinal and extraintestinal transglutaminase 2 by coeliac autoantibodies. Gut 2004, 53:641-648. 19. Zhemakova A, Alizadeh BZ, Bevova M, van Leeuwen LA, Coenen MJ, Franke B, Franke L, Posthumus MD, van Heel DA, van der Steege G, Radstake TR, Barrera P, Roep BO, Koeleman BP, Wijmenga C: Novel association in chromosome 4q27 region with rheumatoid arthritis and confirmation of type 1 diabetes point to a general risk locus for autoimmune diseases. Am J Hum Genet 2007, 81:1284-1288. 41. Hadjivassiliou M, Mäki M, Sanders DS, Williamson CA, Grünewald RA, Woodroofe NM, Korponay-Szabó IR: Autoantibody targeting of brain and intestinal transglutaminase in gluten ataxia. Neurology 2006, 66:373-377. 20. Hunt KA, Zhemakowa A, Turner G, Heap GA, Franke L, Bruinenberg M, Romanos J, Dinesen LC, Ryan AW, Panesar D, Gwilliam R, Takeuchi F, McLaren WM, Holmes GK, Howdle PD, Walters JR, Sanders DS, Playford RJ, Trynka G, Mulder CJ, Mearin ML, Verbeek WH, Trimble V, Stevens FM, O’Morain C, Kennedy NP, Kelleher D, Pennington DJ, Strachan DP, McArdle WL, Mein CA, Wapenaar MC, Deloukas P, McGinnis R, McManus R, Wijmenga C, van Heel DA: Newly identified genetic risk variants for celiac disease related to the immune response. Nat Genet 2008, 40:395-402. 42. Stamnaes J, Dorum S, Fleckenstein B, Aeschlimann D, Sollid LM: Gluten T cell epitope targeting by TG3 and TG6; implications for dermatitis herpetiformis and gluten ataxia. Amino Acids 2010, 39:1183-1191. 43. Sapone et al. BMC Medicine 2012, 10:13 http://www.biomedcentral.com/1741-7015/10/13 Author details 1 de Magistris L, Familiari V, Pascotto A, Sapone A, Frolli A, Iardino P, Carteni M, De Rosa M, Francavilla R, Riegler G, Militerni R, Bravaccio C: Alterations of the intestinal barrier in patients with autism spectrum disorders and in their first-degree relatives. J Pediatr Gastroenterol Nutr 2010, 51:418-424. 27. Salmi TT, Hervonen K, Kautiainen H, Collin P, Reunala T: Prevalence and incidence of dermatitis herpetiformis: a 40-year prospective study from Finland. Brit J Dermatol 2011, 165:354-359. 28. Holmes G, Catassi C, Fasano A: Dermatitis Herpetiformis in Celiac disease Oxford: Health Press; 2009, 83-90. 50. Castellaneta A, Di Leo A, Francavilla R, Margiotta M, Barone M, Amoruso A, Troiani L, Thomson AW, Francavilla A: Functional modification of CD11c+ liver dendritic cells during liver regeneration after partial hepatectomy in mice. Hepatology 2006, 43:807-816. 29. Rose C, Armbruster FP, Ruppert J, Igl BW, Zillikens D, Shimanovich I: Autoantibodies against epidermal transglutaminase are a sensitive diagnostic marker in patients with dermatitis herpetiformis on a normal or gluten-free diet. J Am Acad Dermatol 2009, 61:39-43. 51. Castellaneta A, Abe M, Morelli AE, Thomson AW: Identification and characterization of intestinal Peyer’s patch interferon-alpha producing (plasmacytoid) dendritic cells. Hum Immunol 2004, 65(2):104-113. 30. Caproni M, Antiga E, Melani L, Fabbri P, Italian Group for Cutaneous Immunopathology: Guidelines for the diagnosis and treatment of dermatitis herpetiformis. J Eur Acad Dermatol Venereol 2009, 23:633-638. 52. Kagnoff MF: Celiac disease: pathogenesis of a model immunogenetic disease. J Clin Invest 2007, 117:41-49. 31. Hadjivassiliou M, Grunewald RA, Chattopadhyay AK, Davies-Jones GA, Gibson A, Jarratt JA, Kandler RH, Lobo A, Powell T, Smith CM: Clinical, radiological, neurophysiological and neuropathological characteristics of gluten ataxia. Lancet 1998, 352:1582-1585. 53. Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, Pulendran B, Palucka K: Immunobiology of dendritic cells. Annu Rev Immunol 2000, 18:767-811. 32. Hadjivassiliou M, Gibson A, Davies-Jones GAB, Lobo AJ, Stephenson TJ, Milford-Ward A: Does cryptic gluten sensitivity play a part in neurological illness? Lancet 1996, 347:369-371. 54. Palová-Jelínková L, Rozková D, Pecharová B, Bártová J, Sedivá A, Tlaskalová- Hogenová H, Spísek R, Tucková L: Gliadin fragments induce phenotypic and functional maturation of human dendritic cells. J Immunol 2005, 175:7038-7045. 33. Pellecchia MT, Scala R, Filla A, De Michele G, Ciacci C, Barone P: Idiopathic cerebellar ataxia associated with celiac disease: lack of distinctive neurological features. J Neurol Neurosurg Psychiatry 1999, 66:32-35. 33. Author details 1 Pellecchia MT, Scala R, Filla A, De Michele G, Ciacci C, Barone P: Idiopathic cerebellar ataxia associated with celiac disease: lack of distinctive neurological features. J Neurol Neurosurg Psychiatry 1999, 66:32-35. 34. Luostarinen LK, Collin PO, Peräaho MJ, Mäki MJ, Pirttilä TA: Coeliac disease in patients with cerebellar ataxia of unknown origin. Ann Med 2001, 33:445-449. 35. Bürk K, Bösch S, Müller CA, Melms A, Zühlke C, Stern M, Besenthal I, Skalej M, Ruck P, Ferber S, Klockgether T, Dichgans J: Sporadic cerebellar ataxia associated with gluten sensitivity. Brain 2001, 124:1013-1019. 55. Baumgart DC, Metzke D, Schmitz J, Scheffold A, Sturm A, Wiedenmann B, Dignass AU: Patients with active inflammatory bowel disease lack immature peripheral blood plasmacytoid and myeloid dendritic cells. Gut 2005, 54:228-236. 34. Luostarinen LK, Collin PO, Peräaho MJ, Mäki MJ, Pirttilä TA: Coeliac disease in patients with cerebellar ataxia of unknown origin. Ann Med 2001, 33:445-449. 56. Nikulina M, Habich C, Flohé SB, Scott FW, Kolb H: Wheat gluten causes dendritic cell maturation and chemokine secretion. J Immunol 2004, 173:1925-1933. 35. Bürk K, Bösch S, Müller CA, Melms A, Zühlke C, Stern M, Besenthal I, Skalej M, Ruck P, Ferber S, Klockgether T, Dichgans J: Sporadic cerebellar ataxia associated with gluten sensitivity. Brain 2001, 124:1013-1019. Page 12 of 12 Page 12 of 12 Sapone et al. BMC Medicine 2012, 10:13 http://www.biomedcentral.com/1741-7015/10/13 Sapone et al. BMC Medicine 2012, 10:13 http://www.biomedcentral.com/1741-7015/10/13 57. Ráki M, Tollefsen S, Molberg Ø, Lundin KE, Sollid LM, Jahnsen FL: A unique dendritic cell subset accumulates in the celiac lesion and efficiently activates gluten-reactive T cells. Gastroenterology 2006, 131:428-438. 58. Ziegler AG, Schmid S, Huber D, Hummel M, Bonifacio E: Early infant feeding and risk of developing type 1 diabetes-associated autoantibodies. JAMA 2003, 290:1721-1728. 59. Visser JT, Lammers K, Hoogendijk A, Boer MW, Brugman S, Beijer-Liefers S, Zandvoort A, Harmsen H, Welling G, Stellaard F, Bos NA, Fasano A, Rozing J: Restoration of impaired intestinal barrier function by the hydrolysed casein diet contributes to the prevention of type 1 diabetes in the diabetes-prone BioBreeding rat. Diabetologia 2010, 53:2621-2628. 60. Fasano A: Zonulin and its regulation of intestinal barrier function: the biological door to inflammation, autoimmunity, and cancer. Physiol Rev 2011, 91:151-175. 61. Author details 1 Schumann M, Günzel D, Buergel N, Richter JF, Troeger H, May C, Fromm A, Sorgenfrei D, Daum S, Bojarski C, Heyman M, Zeitz M, Fromm M, Schulzke JD: Cell polarity-determining proteins Par-3 and PP-1 are involved in epithelial tight junction defects in coeliac disease. Gut 2012, 61:220-228. 62. Sapone A, Lammers KM, Casolaro V, Cammarota M, Giuliano MT, De Rosa M, Stefanile R, Mazzarella G, Tolone C, Russo MI, Esposito P, Ferraraccio F, Cartenì M, Riegler G, de Magistris L, Fasano A: Divergence of gut permeability and mucosal immune gene expression in two gluten- associated conditions: celiac disease and gluten sensitivity. BMC Med 2011, 9:23. 63. Biesiekierski JR, Newnham ED, Irving PM, Barrett JS, Haines M, Doecke JD, Shepherd SJ, Muir JG, Gibson PR: Gluten causes gastrointestinal symptoms in subjects without celiac disease: a double-blind randomized placebo-controlled trial. Am J Gastroenterol 2011, 106:508-514. 64. Cooper BT, Holmes GK, Ferguson R, Thompson RA, Allan RN, Cooke WT: Gluten-sensitive diarrhea without evidence of celiac disease. Gastroenterology 1980, 79:801-806. 65. Molberg O, Uhlen AK, Jemsen T, Flaete NS, Fleckenstein B, Arentz-Hansen H, Raki M, Lundin KE, Sollid LM: Mapping of gluten T-cell epitopes in the bread wheat ancestors: implications for celiac disease. Gastroenterology 2005, 128:393-401. 66. Gibert A, Espadaler M, Angel Canela M, Sanchez A, Vaque C, Rafecas M: Consumption of gluten-free products: should the threshold value for trace amounts of gluten be at 20, 100 or 200 ppm? Eur J Gastroenterol Hepatol 2006, 18:1187-1195. 67. Catassi C, Fabiani E, Iacono G, D’Agate C, Francavilla R, Biagi F, Volta U, Accomando S, Picarelli A, De Vitis I, Pianelli G, Gesuita R, Carle F, Mandolesi A, Bearzi I, Fasano A: A prospective, double-blind, placebo- controlled trial to establish a safe gluten threshold for patients with celiac disease. Am J Clin Nutr 2007, 85:160-166. 68. Catassi C, Kryszak D, Bhatti B, Sturgeon C, Helzlsouer K, Clipp SL, Gelfond D, Puppa E, Sferruzza A, Fasano A: Natural history of celiac disease autoimmunity in a USA cohort followed since 1974. Ann Med 2010, 42:530-538. 69. Rubio-Tapia A, Kyle RA, Kaplan EL, Johnson DR, Page W, Erdtmann F, Brantner TL, Kim WR, Phelps TK, Lahr BD, Zinsmeister AR, Melton LJ, Murray JA: Increased prevalence and mortality in undiagnosed celiac disease. Gastroenterology 2009, 137:88-93. 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https://openalex.org/W4382045018	https://bmcnephrol.biomedcentral.com/counter/pdf/10.1186/s12882-023-03250-x	English	null	Mallory-Weiss syndrome in four hemodialysis patients: a case study	BMC nephrology	2,023	cc-by	4,646	© 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://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Mallory‑Weiss syndrome in four hemodialysis patients: a case study Yi‑Qiang Zhang5* Case presentation Conclusions We think that the mild symptoms of MWS are easily covered up by other symptoms. This may lead to delays in diagnosis and treatment. For patients with severe symptoms, gastroscopic hemostasis is still the first choice, and interventional hemostasis can also be considered. For patients with mild symptoms, drug hemostasis is the first consideration. Keywords Mallory-Weiss syndrome, Hemodialysis, Treatment Background Hemodialysis (HD) patients are prone to gastrointes- tinal bleeding, and one of the causes is Mallory-Weiss syndrome (MWS). MWS refers to non-transmural lac- erations of the esophagogastric junction caused by severe vomiting and other inducements [1]. In most cases, the disease is self-limited and benign. However, HD patients can develop MWS after mild vomiting. The early symp- toms of hematemesis are not typical, which leads to a delay in the diagnosis, and are easily complicated by seri- ous gastrointestinal bleeding and even death. This study further explored the diagnosis and treatment of MWS in HD patients by reviewing and analyzing the clinical data, gastroscopic examination results, and follow-up data after treatment. Correspondence: Yi‑Qiang Zhang yiqiangzhang@czmc.edu.cn 1 Department of Nephrology, Heji Hospital of Changzhi Medical College, Changzhi 046011, Shanxi, China 2 Graduate School of Changzhi Medical College Changzhi, Shanxi 046000, China 3 Department of Endoscopy, Heji Hospital of Changzhi Medical College, Changzhi 046011, Shanxi, China 4 Department of Radiology, Heji Hospital of Changzhi Medical College, Changzhi 046011, Shanxi, China 5 Department of Biochemistry, Changzhi Medical College, 161 JieFang East Street, Changzhi, Shanxi 046000, P.R. China Correspondence: Yi‑Qiang Zhang yiqiangzhang@czmc.edu.cn 1 Department of Nephrology, Heji Hospital of Changzhi Medical College, Changzhi 046011, Shanxi, China 2 Graduate School of Changzhi Medical College Changzhi, Shanxi 046000, China 3 Department of Endoscopy, Heji Hospital of Changzhi Medical College, Changzhi 046011, Shanxi, China 4 Department of Radiology, Heji Hospital of Changzhi Medical College, Changzhi 046011, Shanxi, China 5 Department of Biochemistry, Changzhi Medical College, 161 JieFang East Street, Changzhi, Shanxi 046000, P.R. China Abstract Background Hemodialysis patients are prone to gastrointestinal bleeding, and Mallory-Weiss syndrome (MWS) is one of the causes. Mallory-Weiss syndrome is often induced by severe vomiting, manifests as upper gastrointestinal bleeding, and is self-limited with a good prognosis. However, mild vomiting in hemodialysis patients can lead to the occurrence of MWS, and the mild early symptoms are easy to misdiagnose, leading to the aggravation of the disease. Case presentation In this paper, we report four hemodialysis patients with MWS. All patients displayed symptoms of upper gastrointestinal bleeding. The diagnosis of MWS was confirmed by gastroscopy. One patient had a history of severe vomiting; however, the other three reported histories of mild vomiting. Three patients received the conserva‑ tive hemostasis treatment, and the gastrointestinal bleeding stopped. One patient underwent the gastroscopic and interventional hemostasis treatments. The conditions of three of the patients improved. Unfortunately, one of the patients died due to the cardia insufficiency. BMC Nephrology BMC Nephrology BMC Nephrology Shi et al. BMC Nephrology (2023) 24:188 https://doi.org/10.1186/s12882-023-03250-x Open Access Open Access © 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://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Open Access T Shi et al. BMC Nephrology (2023) 24:188 Shi et al. BMC Nephrology (2023) 24:188 Page 2 of 6 Page 2 of 6 Page 2 of 6 Case presentation 2. Clinical presentation on admission 2. Clinical presentation on admission This study describes four patients receiving maintenance HD who also had MWS. The general condition of the patients, their clinical presentation on admission, gas- troscopy results, treatment histories, and prognoses are summarized below. The four patients are subsequently referred to as P1, P2, P3, and P4. The case data for each patient are as follows: P1: After eating, P1 experienced severe vomiting 30 min before dialysis. The vomit contained gastric con- tents. Hematemesis occurred two hours after the start of dialysis and was bright red with a volume of approxi- mately 200 mL. The patient complained of chest and back discomfort. P2: P2 experienced vomiting one day before admission. There was no obvious cause for the vomiting, which was dark red. The patient also had nausea and fatigue. 1. General patient condition 1. General patient condition 1. General patient condition h P3: P3 experienced nausea and vomiting one day prior to admission, and the vomit contained gastric contents. This was accompanied by abdominal pain, diarrhea, watery stools, and fever. The highest body temperature recorded for P3 was 38.2 ℃. The four patients were receiving maintenance HD treatment at our hospital. The underlying disease of P1 and P2 was primary kidney disease, and the underly- ing disease of P3 and P4 was diabetic nephropathy (see Table 1). Table 1 Summary of patients’ demographics, underlying diseases, clinical data, blood results, and hemodialysis details a Values are approximate Patient P1 P2 P3 P4 Sex Male Female Female Male Age (years) 31 65 69 61 Underlying disease IgA nephropathy Chronic glomerulonephritis Diabetic nephropathy Diabetic nephropathy Comorbid disease No No Systemic scleroderma coronary heart disease coronary heart disease Digestive system diseases No Chronic superficial gastritis, constipation Constipation Constipation Long-term oral drug admin‑ istration No No Glucocorticoids Aspirin Frequency of HD 12 h/week 8 h/week 10 h/week 12 h/week Frequency of HDF 8 h/month No 4 h/month 4 h/month Years on HD 3 2 3 1 Anticoagulants and doses used in dialysis Low-molecular-weight heparin 3000 U/time Low-molecular-weight heparin 1000 U/time Low-molecular-weight heparin 3500 U/time Low-molecular-weight heparin 5000 U/time Vascular access Arterio-venous fistula Right femoral vein catheteri‑ zation (heparin saline 1:1 tube sealing) Semi-permanent catheteriza‑ tion of right internal jugular vein (sealed with heparin only) Arterio-venous fistula Accompanied by cardiac insufficiency No No Yes Yes Accompanied by thrombo‑ cytopenia No (1231012/L) No (2561012/L) Yes (781012/L) No (2011012/L) With coagulation dysfunction No (PT 13.1 s) Yes (PT 19.3 s) No (PT 13.8 s) Yes (PT 18.1 s) Serum creatinine level before dialysis (μmol/L)a 1000 800 500 800 BUN (mmol/L) 15.4 16.78 17.2 15.3 Pre-onset Hbb levels (g/L) 115 75 107 107 Hb level at onset (g/L) 87 43 85 99 Kt/V 1.44 1.25 1.13 1.32 Shi et al. BMC Nephrology (2023) 24:188 Shi et al. BMC Nephrology (2023) 24:188 Page 3 of 6 Page 3 of 6 P4: One week before admission, P4 developed chest tightness and shortness of breath accompanied by nau- sea and vomiting. The vomit was black and occurred on three occasions. P2: The patient first underwent an interventional lower gastric artery embolization due to the large amount of bleeding after admission. The interventional effect was average. 3. Gastroscopy results (Fig. 1) P2, P3, and P4: Due to the poor coagulation function, coagulation factors and plasma were administered to stop the bleeding. P1: The gastroscopy for P1 revealed a cardia submu- cosal hematoma.hi P1: The gastroscopy for P1 revealed a cardia submu- cosal hematoma.hi P2 and P3: A 10% sodium chloride solution was used for central venous catheter sealing. P2: The first gastroscopy performed for P2 after admis- sion showed cardia mucosal hemorrhage and a poor treatment effectiveness. A second gastroscopy revealed MWS with thrombosis. 1. General patient condition Since the patient still had bleeding, three gastroscopic titanium clamps were used to stop the bleeding (Figs. 2 and 3). 3. Gastroscopy results (Fig. 1) 3. Gastroscopy results (Fig. 1) 5. Prognosis P3: The gastroscopy for P3 revealed a tear in the cardia mucosa that might have been accompanied by bleeding.hl P3: P3 did not bleed or vomit blood by the second day after admission. On the 6th day, the stool color turned yellow, and the fecal occult blood test was negative. P3 resumed eating on the 9th day. On the 12th day, the patient’s blood pressure decreased to 84/56 mmHg, and the heart rate was 100 beats/min, with a uniform sinus rhythm. After retesting results showed hemoglobin and blood potassium values of 93 g/L and 4.1 mmol/L, respectively. P3 stopped treatment and died outside the hospital. P4: The gastroscopy revealed flaky erosions at the entrance of the cardia and possible tearing. 4. Treatment After admission, the four patients were given sympto- matic and supportive treatments that included fasting, acid suppression, gastric mucosal protection, hemostasis, fluid replacement, blood transfusion (to correct anemia), and heparin-free dialysis. Fig. 1 Gastroscopy results for four patients. A The gastroscopy for P1 revealed a MWS. B The gastroscopy for P2 revealed MWS with thrombosis. C The gastroscopy for P3 revealed a tear in the cardiac mucosa that may have been accompanied by bleeding. D The gastroscopy for P4 revealed flaky erosions at the entrance of the cardia and that tearing might be possible Fig. 1 Gastroscopy results for four patients. A The gastroscopy for P1 revealed a MWS. B The gastroscopy for P2 revealed MWS with thrombosis. C The gastroscopy for P3 revealed a tear in the cardiac mucosa that may have been accompanied by bleeding. D The gastroscopy for P4 revealed flaky erosions at the entrance of the cardia and that tearing might be possible Fig. 2 Left gastric artery hemorrhage embolism diagram for patient 2. A Celiac trunk arteriography showing bleeding from the left gastric artery (black arrow). B Super selective left gastric angiography showing bleeding (black arrow). C Super selective left gastric artery embolization showing the cessation of bleeding (black arrow). D Celiac artery angiography showing the left gastric artery embolism (black arrow) Fig. 2 Left gastric artery hemorrhage embolism diagram for patient 2. A Celiac trunk arteriography showing bleeding from the left gastric artery (black arrow). B Super selective left gastric angiography showing bleeding (black arrow). C Super selective left gastric artery embolization showing the cessation of bleeding (black arrow). 5. Prognosis D Celiac artery angiography showing the left gastric artery embolism (black arrow) Shi et al. BMC Nephrology (2023) 24:188 Page 4 of 6 Fig. 3 Gastroscopic hemostasis in patient 2. A Tear and bleeding of the cardia mucosa. B Titanium clip to stop the bleeding Fig. 3 Gastroscopic hemostasis in patient 2. A Tear and bleeding of the cardia mucosa. B Titanium clip to stop the bleeding emostasis in patient 2. A Tear and bleeding of the cardia mucosa. B Titanium clip to stop the bleeding yet they developed MWS. The analysis of clinical data of these patients reveled that they had certain risk fac- tors for MWS. According to the literature, age, uremic toxins [7], anemia, certain drugs [8] (i.e., nonsteroidal anti-inflammatory drugs), vascular calcification, cardia insufficiency, related diseases such as diabetes, and vari- ous types of vasculitis are predisposing factors for MWS. Age is a factor because the strength of collagen fibers in the submucosa of the esophagus and cardia tend to grad- ually weaken and can easily be torn by external forces [9]. Various uremic toxins can cause intestinal motil- ity disorders, which can cause the patient to be more prone to bleeding and vomiting [10–13]. If the mucosa has been in a state of damage, congestion, and prolonged edema, the hemodynamic changes that occur during HD aggravate the intermittent ischemic state of the intesti- nal mucosa, leading to further aggravation and mucosal damage. Therefore, HD patients are more prone to MWS. Among the four patients in our study, two were middle- aged (61 and 65 years old), and one was an elderly female (69 years old). These three patients had a history of con- stipation, and they had received dialysis for > 1 year. Their average blood creatinine levels before dialysis reached 700 μmol/L. All patients had varying degrees of anemia, and one of them was moderately anemic. Two patients had a history of diabetes, one with a long-term use of oral hormones and the other one with aspirin. These factors most likely caused these three patients to develop MWS without severe vomiting. P1, P2, and P4: The remaining three patients recovered and were discharged after treatment. Routine stool exam- inations were carried out one month after the discharge, and the results were normal. No obvious gastrointestinal bleeding was present in the three patients. Discussion and conclusions At the same time, due to the presence of constipa- tion, examining the early stage of melena was impossible, and the first diagnosis was incorrect. Such cases serve as a warning. For those with a history of vomiting but no obvious bleeding symptoms, close observation is neces- sary. Without an obvious history of hypercoagulability, local citrate anticoagulation can be considered for the first HD after vomiting. In addition, you should observe the occult blood in the stool, and complete gastroscopy, if necessary. In addition, some conditions that can lead to MWS, such as severe cough, epilepsy, acute severe asthma, and constipation, can occur in HD patients. When these symptoms occur, we also need to closely observe the bleeding of patients and make a timely diagnosis. patient had severe bleeding. Hemostasis with a titanium clip during gastroscopy and a single interventional vascu- lar embolization were performed in another patient. The reason for the poor effect of the repeated surgery in this patient was poor coagulation and severe local bleeding. In addition to non-heparin or local citrate anticoagula- tion dialysis after a timely diagnosis, sealing the central venous catheters without heparin or with citrate is also a very important issue that is easily overlooked. There- fore, when treating HD patients with MWS, conservative treatment is not recommended, and stopping the bleed- ing using a gastroscope or interventional therapy, or even surgical treatment, is recommended. At the same time, paying attention to the coagulation function during treat- ment is very necessary.f Based on multiple database searches using different retrieval methods, case reports of patients with regular HD combined with MWS are relatively rare. Only five cases of chronic kidney disease combined with MWS have been reported in Japan, and little data are available from these cases. However, according to the diagnosis and treatment summary of four patients in this study, we believe that the clinical manifestations of mild MWS are easily hidden, which leads to delayed diagnosis. In order to ensure safe treatment, we should pay attention to the existence of coagulation function and various compli- cations. For patients with severe bleeding, endoscopic hemostasis is the first choice; Interventional methods can be provided if necessary, but necessary drug treatment is also crucial. g The current treatment of MWS is based on gastroin- testinal bleeding and tearing. Discussion and conclusions MWS refers to non-transmural lacerations at the gastroe- sophageal junction that are generally caused by severe vomiting but can result from other causes, including increased intra-abdominal pressure and a rapid increase in the intragastric pressure (i.e., vomiting, severe cough- ing, epilepsy, acute or severe asthma, constipation, or endoscopy). It was first reported by Mallory and Weiss in 1929 [2] and accounts for 1% to 15% of the causes of upper gastrointestinal bleeding in adults [3]. In most cases, MWS is a self-limiting, benign disease [4, 5]. Mild cases may be asymptomatic. In 85% of cases, the main symptom is hematemesis. The quantity of blood is vari- able. The diagnosis is based on gastroscopy, where a tear of the mucosa, submucosal hematoma, and bleeding are the diagnostic criteria. The diagnosis and cause of MWS are usually clear. HD patients tend to have gastrointestinal bleeding, and among them, the proportion with MWS is quite differ- ent. A study in the United States in 2015 found that the proportion of MWS in HD patients with upper gastro- intestinal bleeding was 2.34% [6], and a study in China examined 68 patients with upper gastrointestinal bleed- ing, of which MWS accounted for 11.8%. The reason for the large gap in the proportion of MWS reported in the literature is that there may be mild symptoms, which are not identified. The diagnosis of MWS in these clinical cases suggests that a delayed or completely missed diagnosis would not be uncommon. The main clinical symptom of MWS is upper gastrointestinal bleeding, which can present as hematemesis and melena, depending on the amount of bleeding. However, for HD patients, mild vomiting (or other reasons, as stated above) can lead to MWS. In i The four patients with HD in this article had a his- tory of vomiting with varying degrees of severity. How- ever, three of them reported a history of mild vomiting, Shi et al. BMC Nephrology (2023) 24:188 Shi et al. BMC Nephrology (2023) 24:188 Page 5 of 6 this case study, one patient had a large amount of short- term bleeding, manifesting as obvious hematemesis. The patient vomited violently, so the diagnosis and treatment were carried out as soon as possible. However, the symp- toms of hematemesis in the other three patients were not obvious and were possibly masked by other symptoms, such as chest tightness, shortness of breath, diarrhea, and fever. Funding g The present work was supported by the Natural Science Foundation of Shanxi Province of China (Grant No. 20210302124574, 20210302124328), Shanxi Scholarship Council of China (Grant No. 2022-167), Shanxi Postgraduate Innovation Project (Grant No. 2021Y744), Research Incubation Project of Heji Hospital Affiliated with Changzhi Medical College (Grant No. 202101), and National College Student Innovation and Entrepreneurship Training Program (Grant No. 20220815, D2022003). Acknowledgements g We would like to thank Editage (www.editage.cn) for English language editing. We would like to thank Editage (www.editage.cn) for English language editing. Authors’ contributions SS and ZY composed the manuscript. SL, HG, LH, and WZ provided figures. YZ and SS performed the data acquisition, analysis, or interpretation; critical revisions for important intellectual content; and final approval of the version to be published. All authors provided critical feedback and helped guide the research, analysis, and manuscript. Abbreviations Abbreviations HD Hemodialysis MWS Mallory-Weiss syndrome HD Hemodialysis MWS Mallory-Weiss syndrome Discussion and conclusions Conservative treatment approaches include acid suppression and hemostasis, and if necessary, hemostasis using gastroscopy and inter- ventional vascular embolization. Since MWS is mostly self-limited and recurrence is uncommon, a conserva- tive approach would be appropriate for most patients. However, for HD patients with MWS, if the amount of bleeding is large, long-term conservative treatment will aggravate the disease and treatment becomes more dif- ficult. These other factors include an impaired platelet function and a decreased platelet count [14, 15]. Uremic toxins, such as urea, phenol, and guanidinosuccinic acid, are also closely related to platelet dysfunction and should be monitored [16]. In addition, the regular use of antico- agulants in long-term HD patients, as well as the effects of their toxins on the bone marrow, can cause various coagulation disorders [17]. Furthermore, HD patients often have other complications such as hypertension, diabetes, and coronary heart disease. These comorbidi- ties complicate the overall disease [18, 19]. Finally, most HD patients have an abnormal calcium and phosphorus metabolism, calcification of blood vessels throughout the body, and decreased vascular elasticity [20], making them more prone to bleeding, and the bleeding is not easily stopped in such cases. In this case study, three patients were treated using a conservative approach, and the results were acceptable, but the required treatment time was longer. One patient died of heart failure, and another References 1. Laeeq SM, Tasneem AA, Hanif FM, Luck NH, Mandhwani R, Wadhva R. Upper gastrointestinal bleeding in patients with end stage renal disease: causes, characteristics and factors associated with need for endoscopic therapeutic intervention. J Transl Int Med. 2017;5:106–11. 2. Small AB, Ellis PR. Laceration of the distal esophagus due to vomiting (the Mallory-Weiss syndrome): report of a case with massive hemorrhage and recovery after repair of the laceration. N Engl J Med. 1958;258:285–6. 3. Rawla P, Devasahayam J. Mallory-Weiss Syndrome. 2023. PMDI: 30855778. 3. Rawla P, Devasahayam J. Mallory-Weiss Syndrome. 2023. PMDI: 30855778. 4. He L, Li ZB, Zhu HD, Wu XL, Tian DA, Li PY. The prediction value of scor‑ ing systems in Mallory-Weiss syndrome patients. Medicine (Baltimore). 2019;98: e15751. 4. He L, Li ZB, Zhu HD, Wu XL, Tian DA, Li PY. The prediction value of scor‑ ing systems in Mallory-Weiss syndrome patients. Medicine (Baltimore). 2019;98: e15751. 5. Cherednikov EF, Kunin AA, Cherednikov EE, Moiseeva NS. The role of etiopathogenetic aspects in prediction and prevention of discontinuous- hemorrhagic (Mallory-Weiss) syndrome. EPMA J. 2016;7:7. 5. Cherednikov EF, Kunin AA, Cherednikov EE, Moiseeva NS. The role of etiopathogenetic aspects in prediction and prevention of discontinuous- hemorrhagic (Mallory-Weiss) syndrome. EPMA J. 2016;7:7. 6. Trivedi H, Yang J, Szabo A. Gastrointestinal bleeding in patients on long- term dialysis. J Nephrol. 2015;28:235–43. 7. Kaneva K, Bansal V, Hoppensteadt D, Cunanan J, Fareed J. Variations in the circulating heparin levels during maintenance hemodialysis in patients with end-stage renal disease. Clin Appl Thromb Hemost. 2013;19:449–52. 7. Kaneva K, Bansal V, Hoppensteadt D, Cunanan J, Fareed J. Variations in the circulating heparin levels during maintenance hemodialysis in patients with end-stage renal disease. Clin Appl Thromb Hemost. 2013;19:449–52. 8. Zhang X, Ouyang J, Wieczorek R, DeSoto F. Iron medication-induced gastric mucosal injury Pathol Res Pract 2009 205 579 81 7. Kaneva K, Bansal V, Hoppensteadt D, Cunanan J, Fareed J. Variations in the circulating heparin levels during maintenance hemodialysis in patients with end-stage renal disease. Clin Appl Thromb Hemost. 2013;19:449–52. 8. Zhang X, Ouyang J, Wieczorek R, DeSoto F. Iron medication-induced gastric mucosal injury. Pathol Res Pract. 2009;205:579–81. gastric mucosal injury. Pathol Res Pract. 2009;205:579–81. 9. Chen C, Liu L, Wang CY, Choi SW, Yuen VM. A pilot study of ultrasound evaluation of gastric emptying in patients with end-stage renal failure: a comparison with healthy controls. Anaesthesia. 2017;72:714–8. 10. Consent for publication Written informed consent for publication was obtained from all participants. A copy of the consent form is available for review and can be provided on request. References Tolbert MK, Olin S, MacLane S, Gould E, Steiner JM, Vaden S, Price J. Evalu‑ ation of gastric pH and serum gastrin concentrations in cats with chronic kidney disease. J Vet Intern Med. 2017;31:1414–9. 11. Ravelli AM. Gastrointestinal function in chronic renal failure. Pediatr Nephrol. 1995;9:756–62. 11. Ravelli AM. Gastrointestinal function in chronic renal failure. Pediatr Nephrol. 1995;9:756–62. 12. Vaziri ND, Goshtasbi N, Yuan J, Jellbauer S, Moradi H, Raffatellu M, Kalantar-Zadeh K. Uremic plasma impairs barrier function and depletes the tight junction protein constituents of intestinal epithelium. Am J Nephrol. 2012;36:438–43. 12. Vaziri ND, Goshtasbi N, Yuan J, Jellbauer S, Moradi H, Raffatellu M, Kalantar-Zadeh K. Uremic plasma impairs barrier function and depletes the tight junction protein constituents of intestinal epithelium. Am J Nephrol. 2012;36:438–43. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: 13. Galbusera M, Remuzzi G, Boccardo P. Treatment of bleeding in dialysis patients. Semin Dial. 2009;22:279–86. 14. Boccardo P, Remuzzi G, Galbusera M. Platelet dysfunction in renal failure. Semin Thromb Hemost. 2004;30:579–89. 15. Lin XH, Lin CC, Wang YJ, Luo JC, Young SH, Chen PH, Hou MC, Lee FY. Risk factors of the peptic ulcer bleeding in aging uremia patients under regular hemodialysis. J Chin Med Assoc. 2018;81:1027–32. 16. Kuo CC, Kuo HW, Lee IM, Lee CT, Yang CY. The risk of upper gastrointesti‑ nal bleeding in patients treated with hemodialysis: a population-based cohort study. BMC Nephrol. 2013;14:15. 17. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. Ethics approval and consent to participate 19. Filiopoulos V, Vlassopoulos D. Hemodialysis-associated thrombocyto‑ penia: a multifactorial and idiosyncratic complication. Am J Kidney Dis. 2013;61:845. The study was approved by the Research and Ethics Committee of the Changzhi Medical College and the followed protocol was consistent with the principles of the Declaration of Helsinki. Patients were asked to sign an informed consent statement for the study. 20. Samuel R, Bilal M, Tayyem O, Guturu P. Evaluation and management of Non-variceal upper gastrointestinal bleeding. Dis Mon. 2018;64:333–43. 20. Samuel R, Bilal M, Tayyem O, Guturu P. Evaluation and management of Non-variceal upper gastrointestinal bleeding. Dis Mon. 2018;64:333–43. 20. Samuel R, Bilal M, Tayyem O, Guturu P. Evaluation and management of Non-variceal upper gastrointestinal bleeding. Dis Mon. 2018;64:333–43. Declarations 18. Pepper RJ, Gale DP, Wajed J, Bommayya G, Ashby D, McLean A, Laffan M, Maxwell PH. Inadvertent postdialysis anticoagulation due to heparin line locks. Hemodial Int. 2007;11:430–4. Competing interestsl No conflicts of interest exist related to the submission of this manuscript. Received: 18 March 2022 Accepted: 20 June 2023 Received: 18 March 2022 Accepted: 20 June 2023 Availability of data and materials All data related to this case report are within the manuscript. All data related to this case report are within the manuscript. Shi et al. BMC Nephrology (2023) 24:188 Page 6 of 6 Shi et al. BMC Nephrology • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: References Kang SJW, Madhan K. Gastrointestinal manifestations in a patient with calciphylaxis: a case report. Case Rep Nephrol Dial. 2019;9:119–25. 17. Kang SJW, Madhan K. Gastrointestinal manifestations in a patient with calciphylaxis: a case report. Case Rep Nephrol Dial. 2019;9:119–25.
https://openalex.org/W3033001087	https://newprairiepress.org/cgi/viewcontent.cgi?article=1315&context=kaesrr	English	null	Effects of Increasing Space Allowance by Removing a Pig or Gate Adjustment on Finishing Pig Growth Performance	Kansas Agricultural Experiment Station research reports	2,016	cc-by	4,680	January 2016 See next page for additional authors Part of the Other Animal Sciences Commons Kansas Agricultural Experiment Station Research Reports Kansas Agricultural Experiment Station Research Reports Volume 2 Issue 8 Swine Day Article 38 January 2016 Effects of Increasing Space Allowance by Removing a Pig or Gate Effects of Increasing Space Allowance by Removing a Pig or Gate Adjustment on Finishing Pig Growth Performance Adjustment on Finishing Pig Growth Performance C. Holder Kansas State University, Manhattan, cjholder@ksu.edu C. Carpenter Kansas State University, Manhattan, cbcarpenter@k-state.edu M. D. Tokach Kansas State University, Manhattan, mtokach@k-state.edu See next page for additional authors This report is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Kansas Agricultural Experiment Station Research Reports by an authorized administrator of New Prairie Press. Copyright 2016 the Author(s). Contents of this publication may be freely reproduced for educational purposes. All other rights reserved. Brand names appearing in this publication are for product identification purposes only. No endorsement is intended, nor is criticism implied of similar products not mentioned. K-State Follow this and additional works at: https://newprairiepress.org/kaesrr Part of the Other Animal Sciences Commons Recommended Citation Recommended Citation Holder, C.; Carpenter, C.; Tokach, M. D.; DeRouchey, J. M.; Woodworth, J. C.; Goodband, R. D.; and Dritz, S. S. (2016) "Effects of Increasing Space Allowance by Removing a Pig or Gate Adjustment on Finishing Pig Growth Performance," Kansas Agricultural Experiment Station Research Reports: Vol. 2: Iss. 8. https://doi.org/10.4148/2378-5977.1315 Kansas Agricultural Experiment Station Research Reports Kansas Agricultural Experiment Station Research Reports Volume 2 Issue 8 Swine Day Article 38 January 2016 Effects of Increasing Space Allowance by Removing a Pig or Gate Effects of Increasing Space Allowance by Removing a Pig or Gate Adjustment on Finishing Pig Growth Performance Adjustment on Finishing Pig Growth Performance C. Holder Kansas State University, Manhattan, cjholder@ksu.edu C. Carpenter Kansas State University, Manhattan, cbcarpenter@k-state.edu M. D. Tokach Kansas State University, Manhattan, mtokach@k-state.edu See next page for additional authors This report is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Kansas Agricultural Experiment Station Research Reports by an authorized administrator of New Prairie Press. Copyright 2016 the Author(s). Contents of this publication may be freely reproduced for educational purposes. All other rights reserved. Brand names appearing in this publication are for product identification purposes only. No endorsement is intended, nor is criticism implied of similar products not mentioned. K-State Follow this and additional works at: https://newprairiepress.org/kaesrr Part of the Other Animal Sciences Commons Recommended Citation Recommended Citation Holder, C.; Carpenter, C.; Tokach, M. D.; DeRouchey, J. M.; Woodworth, J. C.; Goodband, R. D.; and Dritz, S. S. (2016) "Effects of Increasing Space Allowance by Removing a Pig or Gate Adjustment on Finishing Pig Growth Performance," Kansas Agricultural Experiment Station Research Reports: Vol. 2: Iss. 8. https://doi.org/10.4148/2378-5977.1315 ansas Agricultural Experiment Station Research Reports ansas Agricultural Experiment Station Research Reports Volume 2 Issue 8 Swine Day Volume 2 Issue 8 Swine Day Article 38 This research report is available in Kansas Agricultural Experiment Station Research Reports: https://newprairiepress.org/kaesrr/vol2/iss8/38 Recommended Citation Recommended Citation Recommended Citation Recommended Citation Holder, C.; Carpenter, C.; Tokach, M. D.; DeRouchey, J. M.; Woodworth, J. C.; Goodband, R. D.; and Dritz, S. S. (2016) "Effects of Increasing Space Allowance by Removing a Pig or Gate Adjustment on Finishing Pig Growth Performance," Kansas Agricultural Experiment Station Research Reports: Vol. 2: Iss. 8. https://doi.org/10.4148/2378-5977.1315 This report is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Kansas Agricultural Experiment Station Research Reports by an authorized administrator of New Prairie Press. Copyright 2016 the Author(s). Contents of this publication may be freely reproduced for educational purposes. All other rights reserved. Brand names appearing in this publication are for product identification purposes only. No endorsement is intended, nor is criticism implied of similar products not mentioned. K-State Research and Extension is an equal opportunity provider and employer. Effects of Increasing Space Allowance by Removing a Pig or Gate Adjustment on Effects of Increasing Space Allowance by Removing a Pig or Gate Adjustment on Finishing Pig Growth Performance Finishing Pig Growth Performance Authors Authors Authors Authors C. Holder, C. Carpenter, M. D. Tokach, J. M. DeRouchey, J. C. Woodworth, R. D. Goodband, and S. S. Dritz Authors Authors C. Holder, C. Carpenter, M. D. Tokach, J. M. DeRouchey, J. C. Woodworth, R. D. Goodband, and S. S. Dritz C. Holder, C. Carpenter, M. D. Tokach, J. M. DeRouchey, J. C. Woodworth, R. D. Goodband, and S. S. Dritz Effects of Increasing Space Allowance by Removing a Pig or Gate Adjustment on Finishing Pig Growth Performance C.J. Holder, C.B. Carpenter, M.D. Tokach, J.M. DeRouchey, J.C. Woodworth, R.D. Goodband, and S.S. Dritz1 C.J. Holder, C.B. Carpenter, M.D. Tokach, J.M. DeRouchey, J.C. Woodworth, R.D. Goodband, and S.S. Dritz1 1 Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University. Summary y A total of 256 pigs (PIC 327 × 1050; initially 123.1 lb) were used in a 71 d growth study to compare the effects of increasing space allowance by removing a pig or gate adjustment, on finishing pig growth performance. At the initiation of the trial, pens of pigs were blocked by BW and allotted to 1 of 4 space allowance treatments. The 4 treatments included: 1) 9.8 ft2/pig or 2) 6.8 ft2/pig for the entire study with treat ments 3 and 4 initially providing 6.8 ft2, but either a gate was adjusted or the heaviest pig in the pen was removed to provide more space. By using the following equation, space adjustments were made to keep the pigs above their predicted minimum space re quirement before growth is impacted: space [(m2) = 0.0336 × BW (kg)0.66]. There were initially 8 pigs per pen and 8 pens per treatment. From d 0 to 28, before any gate adjustments or pig removals, ADG tended to be greater (P = 0.076) for pigs allowed 9.8 ft2 compared with pigs stocked at 6.8 ft2. Overall, d 0 to 71, pigs allowed 9.8 ft2 had greater (P = 0.001) ADG compared with pigs with all other space allowances. Removing pigs or adjusting the gating increased (P = 0.001) ADG compared to those maintained at 6.8 ft2; however, both treatments had decreased (P = 0.001) ADG compared with pigs allowed 9.8 ft2. Most of the differences in ADG can be explained by differences in ADFI. Pigs allowed 9.8 ft2 had greater (P = 0.001) ADFI compared with pigs allowed 6.8 ft2; however, intake was similar for pigs allowed increased space by gate adjustment to pigs allowed 9.8 ft2. Pigs allowed increased space by pig removal had similar ADFI to pigs allowed 6.8 ft2. Space allowance did not influ ence feed efficiency. In summary, as expected, pigs with 9.8 ft2 grew faster and consumed more feed than pigs that were restricted in space. Furthermore, either removing a pig or adjusting the gating as pigs reached the critical k value influenced growth performance similarly. Kansas State University Agricultural Experiment Station and Cooperative Extension Service Swine Day 2016 K A N S A S ST AT E U NI V E R S I T Y 20 16 Kansas State University Agricultural Experiment Station and Cooperative Extension Service 1 Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University. 2 Gonyou, H. W., M. C. Brumm, E. Bush, J. Deen, S. A. Edwards, R. Fangman, J. J. McGlone, M. Meunier-Salaun, R. B. Morrison, H. Spoolder, P. L. Sundberg, and A. K. Johnson. 2006. Application of broken-line analysis to assess floor space requirements of nursery and grower-finisher pigs expressed on an allometric basis. J. Anim. Sci. 84:229–235. 3 Flohr, J. R.; Tokach, M. D.; Patience, John F.; Gourley, G.; DeRouchey, J. M.; Dritz, S. S.; Woodworth, J. C.; and Goodband, R. D. (2015). “Re-evaluating floor space allowance and removal strategy effects on the growth of heavyweight finishing pigs.” Kansas Agricultural Experiment Station Research Reports: Vol. 1: Iss. 7. Swine Day 2016 the increased space allowance alone. As pigs grew to the minimum predicted space requirement and were subsequently allowed more space, performance was not similar compared to unrestricted pigs. This indicates the industry accepted minimum space prediction equation [m2 = 0.0336 × BW (kg)0.66] doesn’t fully explain the impacts on pig performance across multiple body weight ranges. the increased space allowance alone. As pigs grew to the minimum predicted space requirement and were subsequently allowed more space, performance was not similar compared to unrestricted pigs. This indicates the industry accepted minimum space prediction equation [m2 = 0.0336 × BW (kg)0.66] doesn’t fully explain the impacts on pig performance across multiple body weight ranges. Key words: space allowance, K-value, marketing Introduction Facility space is the second largest cost of pig production and efficiently using the space is important to maintain profitable pork production. A common allometric expression has been used to describe the relationship between floor space and pig BW, similar to that used to describe volume and surface area. Gonyou et al. (2006)2 used the allometric expression A=k*BW0.66, where A is area allowed per pig (m2), k is a coefficient, and BW is pig weight (kg), which converts BW into a 2-dimensional concept, to describe floor space allowance in order to predict productivity. Using this k value, 0.0336, the equa tion should indicate when crowding begins to limit growth. Pig growth should not be decreased until their BW reaches the point where there is inadequate space to maintain maximal growth rate, (i.e., a k coefficient less than the critical k value; Gonyou et al., 2006). A study by Flohr et al. (2015)3 suggested reductions in growth due to inadequate space allowance may start to occur before pigs reach the critical k value. They also reported that removing pigs before the entire pen is marketed increases space allowance for remaining pigs in the pen and increases pig growth performance (Flohr et al., 2015). However, it has not been evaluated whether the improvements in growth are due to the change in social dynamic from removing the heaviest pig, or simply the increased space in the pen as a result of removing the heaviest pig. Thus, the objective of our study was to determine whether the increase in growth rate that occurs when pigs are removed from pens during marketing is due to increasing space allowance by pig removal or gate adjustment during the finishing period. Kansas State University Agricultural Experiment Station and Cooperative Extension Service Summary We speculated that along with pig growth, removing the heaviest pigs could have influ enced social dynamics of the remaining pigs in the pen; however, our study indicates the performance benefit from removing the heaviest pig from the pen is primarily from Kansas State University Agricultural Experiment Station and Cooperative Extension Service 1 Swine Day 2016 Swine Day 2016 Swine Day 2016 feeder spaces and a 1-cup waterer, which provided ad libitum access to feed and water. A robotic feeding system (FeedPro; Feedlogic Corp., Wilmar, MN) was used to deliver and record daily feed additions to each individual pen. feeder spaces and a 1-cup waterer, which provided ad libitum access to feed and water. A robotic feeding system (FeedPro; Feedlogic Corp., Wilmar, MN) was used to deliver and record daily feed additions to each individual pen. A total of 256 pigs (PIC 327 × 1050; initially 123.1 lb) were used in a 71 d growth study. Pens of pigs were blocked by BW and allotted to 1 of 4 space allowance treat ments, initially with 8 pigs per pen (4 barrows and 4 gilts) and 8 pens per treatment. The 4 treatments included pens with 9.8 ft2/pig or 6.8 ft2/pig for the entire study. Two additional treatments initially provided 6.8 ft2, but either a gate was adjusted on d 28, 45, and 62 or the heaviest pig in the pen was removed from the pen on d 28 and 45 to provide more space (Table 1). The space adjustments and pig removals were made to keep the pigs above their predicted minimum space requirement [(m2) = 0.0336 × BW (kg)0.66], where 0.0336 is the k value. If a pig died or was removed from a pen during the experiment, pen size was adjusted to maintain the correct space allowance per pig. Pigs were fed a common corn-soybean-meal based diet offered in 3 phases (Table 2). Di ets were formulated to meet or exceed the pigs' nutrient requirement estimates (NRC, 2012)4 and the 3 phases were fed from approximately 123 to 185, 185 to 220, and 220 to 280 lb BW. Diets were sampled and subsamples were sent to a commercial laboratory (Ward Laboratories Inc., Kearney, NE) for analysis (DM and CP; Table 2). Pens of pigs and feeders were weighed on d 0, 14, 28, 45, 62, and 71 to calculate ADG, ADFI, and F/G. Data were analyzed as a generalized randomized block design with space allowance treatment as a fixed effect and block as a random effect using PROC MIXED in SAS (SAS Institute, Inc., Cary, NC), with pen serving as the experimental unit. Treatment means were separated using the DIFFS option from the LSMEANS statement of SAS. Swine Day 2016 Results were considered significant at P ≤ 0.05 and a tendency at P > 0.05 and P ≤ 0.10. 4 NRC. 2012. Nutrient requirements of swine. 11th rev. ed. Natl. Acad. Press, Washington, D.C. Kansas State University Agricultural Experiment Station and Cooperative Extension Service Proceduresh The Kansas State University Institutional Animal Care and Use Committee approved the protocol used in this experiment. The study was conducted at the Kansas State University Swine Teaching and Research Center in Manhattan, KS. The facility was totally enclosed and environmentally controlled, containing 32 pens. Pens were 8 × 10 ft, equipped with adjustable gates to allow different space allowances per pig, completely slatted floors and deep pits for manure storage. Each pen was equipped with a dry single-sided feeder (Farmweld, Teutopolis, IL) with two 14 × 10 in (width × depth) Kansas State University Agricultural Experiment Station and Cooperative Extension Service 2 Swine Day 2016 From d 45 to 62, ADFI was decreased (P = 0.001) for pigs provided 6.8 ft2 compared to all other treatments. During this period, increasing space allowance resulted in perfor mance similar to pigs allowed 9.8 ft2. On d 62, gates were adjusted to reach the desired k value; however, a pig was not removed from the pig removal treatment because the critical k value was reached sooner based on the actual ft2 for pigs in the gate adjustment treatment than for the pig removal treatment. From d 45 to 62, ADFI was decreased (P = 0.001) for pigs provided 6.8 ft2 compared to all other treatments. During this period, increasing space allowance resulted in perfor mance similar to pigs allowed 9.8 ft2. On d 62, gates were adjusted to reach the desired k value; however, a pig was not removed from the pig removal treatment because the critical k value was reached sooner based on the actual ft2 for pigs in the gate adjustment treatment than for the pig removal treatment. From d 62 to 71, ADG decreased (P = 0.008) when pigs were allowed 6.8 ft2 compared to all other treatments, which is likely due to the decreased (P = 0.001) ADFI, because F/G was not affected. For the cumulative period after space adjustments began (d 28 to 71), both ADG and ADFI decreased (P = 0.001) when pigs were provided 6.8 ft2 compared with pigs pro vided 9.8 ft2. Pigs provided increased space by removing pigs had similar performance to those where gates were adjusted to increase space; however, pig removal resulted in lower ADG and ADFI than pigs allowed 9.8 ft2 throughout the experiment. Overall (d 0 to 71), pigs provided 9.8 ft2 had increased (P = 0.001) ADG compared with all other treatments. Performance of pigs with gate adjustment or pig removal was similar, and both having greater ADG than pigs provided 6.8 ft2. Pigs provided 9.8 ft2 had increased (P = 0.001) ADFI compared with pigs allowed 6.8 ft2; however, intake was similar among pigs provided increased space by gate adjustment to pigs allowed 9.8 ft2. Pigs provided increased space by pig removal had similar ADFI to pigs allowed 6.8 ft2. Final BW was decreased (P = 0.001) for pigs provided 6.8 ft2 compared with those provided 9.8 ft2. Swine Day 2016 Also, final BW of pigs provided increased space by adjusting the gate was greater (P = 0.001) than pigs allowed 6.8 ft2 or increased space by pig removal, but decreased (P = 0.001) compared to pigs provided 9.8 ft2. Gonyou et al. (2006) reported that ADFI was decreased when pigs were stocked below a critical k value of 0.0336, which is also supported by our study. Reductions in per formance have been observed due to inadequate space allowance, which may start to occur before the pigs reach their critical k value. This is a similar key finding in our study where pig performance was reduced before the critical k value was reached, which confirms recent research of Flohr et al. (2015). Furthermore, our data suggest improved growth performance after pigs are removed during the finishing period may be largely due to the increased space provided to pigs remaining in the pens because performance was similar to that of pigs where space was increased by adjusting the gate (without removing the heaviest pig). In this study, pigs with greater space allowance grew faster and consumed more feed than pigs that were restricted in space. Furthermore, either removing a pig or adjusting the gating as pigs reached the critical k value influenced growth performance similarly. We speculated that along with pig growth, social dynamics of the remaining pigs in the pen could have been influenced by removing the heaviest pigs; however, our study indicates the performance benefit from removing the heaviest pig from the pen is pri marily from the increased space allowance alone. Lastly, as pigs grew to the minimum predicted space requirement and were subsequently provided more space, performance was not similar to unrestricted pigs. Increasing the space allowance by removing pigs In this study, pigs with greater space allowance grew faster and consumed more feed than pigs that were restricted in space. Furthermore, either removing a pig or adjusting the gating as pigs reached the critical k value influenced growth performance similarly. We speculated that along with pig growth, social dynamics of the remaining pigs in the pen could have been influenced by removing the heaviest pigs; however, our study indicates the performance benefit from removing the heaviest pig from the pen is pri marily from the increased space allowance alone. Results and Discussion From d 0 to 14, there was no effect of stocking density observed for ADG, ADFI, and F/G, which corresponded to a change in BW from approximately 123 to 153 lb (Table 3). From d 14 to 28, pigs provided 9.8 ft2 had increased ADFI (P = 0.041) and ADG (P = 0.002), which resulted in improved F/G (P = 0.025) and a tendency for in creased (P = 0.081) BW, compared to pigs provided 6.8 ft2. These observations suggest space restriction started to influence growth rate between 153 and 182 lb BW. Based on a k value of 0.0336, no differences in pig performance were expected before d 28 which corresponded to BW of approximately 182 lb. From d 0 to 28, before any gate adjust ments or pig removals, ADG tended to be greater (P = 0.076) for pigs allowed 9.8 ft2 compared to pigs stocked at 6.8 ft2 for the duration of the study. From d 28 to 45, pigs provided 6.8 ft2 or increasing space allowance by removal of the heaviest pig, had decreased (P = 0.025) ADFI compared to pigs provided 9.8 ft2 with pigs from pens where the gate was adjusted being intermediate. This suggests when the heaviest pig is removed from a pen, pigs did not maintain feed intake similar to pigs al lowed 9.8 ft2. 3 Kansas State University Agricultural Experiment Station and Cooperative Extension Service Swine Day 2016 Lastly, as pigs grew to the minimum predicted space requirement and were subsequently provided more space, performance was not similar to unrestricted pigs. Increasing the space allowance by removing pigs Kansas State University Agricultural Experiment Station and Cooperative Extension Service 4 Kansas State University Agricultural Experiment Station and Cooperative Extension Service Swine Day 2016 or adjusting the gating increased ADG compared to pigs provided 6.8 ft2 for the entire experiment; however, neither treatment allowed pigs to maintain ADG similar to pigs provided 9.8 ft2 throughout the study. This indicates the industry accepted minimum space prediction equation [(m2) = 0.0336 × BW (kg)0.66] doesn’t fully explain impacts on pig performance across multiple body weight ranges. Table 1. Space allowance and k value through the experiment1 Item 9.8 ft2 6.8 ft2 Gate adjustment2 Pig removal3 d 0 ft2/pig4 9.8 6.8 6.8 6.8 k value5 0.0615 0.0427 0.0427 0.0427 d 28 ft2/pig 9.8 6.8 7.8 7.8 k value Before adj. 0.0473 0.0328 0.0377 0.0377 After adj. --- --- 0.0425 0.0439 d 45 ft2/pig 9.8 6.8 8.8 9.1 k value Before adj. 0.0422 0.0293 0.0379 0.0392 After adj. --- --- 0.0422 0.0392 d 62 ft2/pig 9.8 6.8 9.8 9.1 k value Before adj. 0.0368 0.0255 0.0368 0.0342 After adj. --- --- 0.0368 0.0409 d 71 ft2/pig 9.8 6.8 9.8 9.1 k value 0.0361 0.0250 0.0361 0.0335 1A total of 256 pigs (PIC 327 × 1050, initially 123.1 lb) were used in a 71 d growth trial. Average BW on d 0, 28, 45, 62 and 71 was 123, 182, 216, 265, and 273 lb, respectively. 2Increased space by gate adjustment (d 28, 45, and 62). 3Increased space by heaviest pig removal (d 28 and 45). 4Indicates area maintained (ft2/pig) between each data collection period. 5k-value [(m2) = k × BW (kg)0.66] calculated before and after a pig was removed or gates were adjusted. Table 1. Space allowance and k value through the experiment1 Kansas State University Agricultural Experiment Station and Cooperative Extension Service Kansas State University Agricultural Experiment Station and Cooperative Extension Service 5 Swine Day 2016 Swine Day 2016 Table 2. Kansas State University Agricultural Experiment Station and Cooperative Extension Service 1Phases 1, 2, and 3 were fed d 0 to 28, 28 to 45, and 45 to 71, respectively. Swine Day 2016 Diet composition (as-fed basis) Phase1 Ingredient, % 1 2 3 Corn 71.50 78.44 82.86 Soybean meal, 47.7% CP 25.71 19.20 14.93 Monocalcium P, 21% P 0.55 0.33 0.30 Limestone 1.13 1.10 1.08 Salt 0.35 0.35 0.35 L-Lys HCl 0.31 0.25 0.22 DL-Met 0.06 0.02 --- L-Thr 0.09 0.05 0.05 Trace mineral premix 0.15 0.13 0.10 Vitamin premix 0.15 0.13 0.10 Phytase2 0.02 0.02 0.02 Total 100.00 100.00 100.00 Calculated analysis Standardized ileal digestible (SID) AA’s, % Lys 1.05 0.85 0.72 Ile:Lys 62 64 66 Met:Lys 30 29 30 Met and Cys:Lys 55 56 59 Thr:Lys 61 61 64 Trp:Lys 18.0 18.0 18.0 Val:Lys 69 73 76 Total Lys, % 1.18 0.96 0.82 ME, kcal/lb 1,494 1,501 1,504 NE, kcal/lb 1,117 1,137 1,150 SID Lys:ME, g/Mcal 3.19 2.57 2.17 CP, % 18.5 15.9 14.2 Ca, % 0.62 0.55 0.52 P, % 0.49 0.41 0.39 Available P, % 0.29 0.23 0.22 Chemical analysis3, % DM 88.32 87.25 87.41 CP 18.5 15.4 14.8 1Phases 1, 2, and 3 were fed d 0 to 28, 28 to 45, and 45 to 71, respectively. 2HiPhos (DSM Inc, Parsippany, NJ) provided 1,228,503 (FYT)/lb of product and released 0.10% available P. 3Multiple samples of each diet were collected, blended and subsampled, and analyzed (Ward Laboratories, Inc. Kearney, NE). Values are represented on an as fed basis. Table 2. Diet composition (as-fed basis) Kansas State University Agricultural Experiment Station and Cooperative Extension Service 6 Swine Day 2016 Swine Day 2016 Table 3. 7 Swine Day 2016 Effects of pig space allowance on finishing pig growth performance1,2 Item 9.8 ft2 6.8 ft2 Gate adjustment3 Pig removal4 SEM P < BW, lb d 0 123.3 123.4 123.2 122.6 0.33 0.361 d 14 152.3 152.6 153.0 152.3 0.57 0.835 d 28 185.3x 181.5y 182.1y 182.6y 1.03 0.081 d 45 221.8a 214.5b 216.5b 214.8b 1.07 0.001 d 62 261.4a 252.5c 256.8b 251.4c 1.39 0.001 d 71 280.6a 268.3c 275.4b 270.0c 1.60 0.001 d 0 to 14 ADG, lb 2.07 2.08 2.13 2.13 0.032 0.495 ADFI, lb 4.83 4.75 4.83 4.84 0.097 0.894 F/G 2.33 2.28 2.27 2.28 0.045 0.752 d 14 to 28 ADG, lb 2.32a 2.06b 2.08b 2.15b 0.045 0.002 ADFI, lb 5.70a 5.32b 5.51b 5.60b 0.091 0.041 F/G 2.45a 2.57b 2.65b 2.60b 0.047 0.025 d 0 to 28 ADG, lb 2.19x 2.07y 2.10xy 2.14xy 0.033 0.076 ADFI, lb 5.27 5.03 5.17 5.22 0.078 0.200 F/G 2.40 2.43 2.46 2.44 0.033 0.541 d 28 to 45 ADG, lb 2.15 1.94 2.02 2.06 0.061 0.143 ADFI, lb 6.32a 5.93b 6.14ab 5.90b 0.102 0.025 F/G 2.96 3.08 3.05 2.87 0.080 0.240 d 45 to 62 ADG, lb 2.33 2.24 2.38 2.32 0.049 0.260 ADFI, lb 7.06a 6.40b 6.96a 6.87a 0.100 0.001 F/G 3.03 2.87 2.94 2.98 0.056 0.237 d 62 to 71 ADG, lb 2.12a 1.75b 2.06a 2.06a 0.077 0.008 ADFI, lb 6.43a 5.88b 6.56a 6.46a 0.101 0.001 F/G 3.07 3.39 3.20 3.16 0.127 0.334 continued Table 3. Effects of pig space allowance on finishing pig growth performance1,2 Kansas State University Agricultural Experiment Station and Cooperative Extension Service 7 Swine Day 2016 Swine Day 2016 Table 3. Effects of pig space allowance on finishing pig growth performance1,2 Item 9.8 ft2 6.8 ft2 Gate adjustment3 Pig removal4 SEM P < d 28 to 71 ADG, lb 2.21a 2.01c 2.17ab 2.15b 0.029 0.001 ADFI, lb 6.64a 6.10c 6.55ab 6.37b 0.077 0.001 F/G 3.00 3.03 3.02 2.96 0.033 0.441 d 0 to 71 ADG, lb 2.21a 2.04c 2.14b 2.15b 0.021 0.001 ADFI, lb 6.09a 5.68c 6.01ab 5.85bc 0.063 0.001 F/G 2.76 2.81 2.82 2.77 0.030 0.486 1A total of 256 pigs (PIC 327 × 1050; initially 123.1 lb) were used in a 71 d growth trial with 8 replications/ treatment to determine the effects of space allowance on finishing pig growth performance. fi g g g Means within a row with different superscripts differ: abc P < 0.05, xyz P < 0.10 t 1A total of 256 pigs (PIC 327 × 1050; initially 123.1 lb) were used in a 71 d growth trial with 8 replications/fi Increased space increased gate adjustment; initially 6.8 ft / pig with gates adjusted as pigs reached the k v be non-limiting (7.8 ft2 at 180 lb (d 28), 8.8 ft2 at 220 lb (d 45), and 9.8 ft2 at 260 lb (d 62)).t f p pf Increased space = increased gate adjustment; initially 6.8 ft2/ pig with gates adjusted as pigs reached the k value, to b l ( ft2 lb (d ) ft2 lb (d 4 ) d ft2 6 lb (d 6 )) gttt 4Increased space = removal of heaviest pig; initially 6.8 ft2/ pig with a pig removed as the k value is reached to be non-limiting: 1 pig at 180 lb (d 28) and 220 lb (d 45). Kansas State University Agricultural Experiment Station and Cooperative Extension Service Swine Day 2016 2Means within a row with different superscripts differ: abc P < 0.05, xyz P < 0.10 3Increased space = increased gate adjustment; initially 6.8 ft2/ pig with gates adjusted as pigs reached the k value, to be non-limiting (7.8 ft2 at 180 lb (d 28), 8.8 ft2 at 220 lb (d 45), and 9.8 ft2 at 260 lb (d 62)). 4Increased space = removal of heaviest pig; initially 6.8 ft2/ pig with a pig removed as the k value is reached to be non-limiting: 1 pig at 180 lb (d 28) and 220 lb (d 45). Table 3. Effects of pig space allowance on finishing pig growth performance1,2 Kansas State University Agricultural Experiment Station and Cooperative Extension Service Kansas State University Agricultural Experiment Station and Cooperative Extension Service 8
https://openalex.org/W2582775304	https://europepmc.org/articles/pmc5268766?pdf=render	English	null	Evaluation of Complex Toxicity of Canbon Nanotubes and Sodium Pentachlorophenol Based on Earthworm Coelomocytes Test	PloS one	2,017	cc-by	8,595	RESEARCH ARTICLE OPEN ACCESS OPEN ACCESS Citation: Yang Y, Xiao Y, Li M, Ji F, Hu C, Cui Y (2017) Evaluation of Complex Toxicity of Canbon Nanotubes and Sodium Pentachlorophenol Based on Earthworm Coelomocytes Test. PLoS ONE 12 (1): e0170092. doi:10.1371/journal.pone.0170092 Editor: Zhen-guang Yan, Chinese Research Academy of Environmental Sciences, CHINA Received: September 26, 2016 Accepted: December 28, 2016 Published: January 26, 2017 Copyright: © 2017 Yang 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: Yang Y, Xiao Y, Li M, Ji F, Hu C, Cui Y (2017) Evaluation of Complex Toxicity of Canbon Nanotubes and Sodium Pentachlorophenol Based on Earthworm Coelomocytes Test. PLoS ONE 12 (1): e0170092. doi:10.1371/journal.pone.0170092 Editor: Zhen-guang Yan, Chinese Research Academy of Environmental Sciences, CHINA Editor: Zhen-guang Yan, Chinese Research Academy of Environmental Sciences, CHINA Copyright: © 2017 Yang 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, supporting information files, and hosted at the public repository Figshare. Please view the Figshare hosted data at the following URL: https://dx.doi.org/10.6084/m9. figshare.4542418.v2. Yang Yang1, Yao Xiao1, Mei Li1, Funian Ji1, Changwei Hu2, Yibin Cui3 1 State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, China, 2 Shandong Provincial Key Laboratory of Water and Soil Conservation and Environmental Protection, Linyi University, Linyi, China, 3 Nanjing Institute of Environmental Sciences, Ministry of Environmental Protection, Nanjing, China meili@nju.edu.cn * meili@nju.edu.cn * meili@nju.edu.cn a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Abstract As a standard testing organism in soil ecosystems, the earthworm Eisenia fetida has been used widely in toxicity studies. However, tests at the individual level are time- and animal- consuming, with limited sensitivity. Earthworm coelomocytes are important for the assimila- tion and elimination of exogenous compounds and play a key role in the processes of phagocytosis and inflammation. In this study, we explored an optimal condition to culture coelomocytes of E. fetida in vitro and investigated the cytotoxicity of multiwalled carbon nanotubes (MWCNTs) and sodium pentachlorophenol (PCP-Na) using coelomocytes via evaluating lethal toxicity, oxidative stress, membrane damage, and DNA damage. The results showed that coelomocytes can be successfully cultured in vitro in primary under the RPMI-1640 medium with 2–4×104 cells/well (1–2×105 cells/mL) in 96-well plates at 25˚C without CO2. Both MWCNTs and PCP-Na could cause oxidative damage and produce ROS, an evidence for lipid peroxidation with MDA generation and SOD and CAT activity inhibition at high stress. The two chemicals could separately damage the cell membrane structure, increasing permeability and inhibiting mitochondrial membrane potential (MMP). In addition, our results indicate that PCP-Na may be adsorbed onto MWCNTs and its toxicity on earthworm was accordingly alleviated, while a synergetic effect was revealed when PCP-Na and MWCNTs were added separately. In summary, coelomocyte toxicity in in vitro analysis is a sensitive method for detecting the adverse effects of carbon nanotubes com- bined with various pollutants. Evaluation of Complex Toxicity of Canbon Nanotubes and Sodium Pentachlorophenol Based on Earthworm Coelomocytes Test Yang Yang1, Yao Xiao1, Mei Li1, Funian Ji1, Changwei Hu2, Yibin Cui3 Provincial Natural Science Foundation of China (No. ZR2015CM023). Provincial Natural Science Foundation of China (No. ZR2015CM023). Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. Coelomocytes, which are immunocompetent cells, play an essential role in the identifica- tion and elimination of xenobiotics [7], fighting microbial infections and healing wounds [8]. Due to their sensitivity to a wide range of pollutants, they have been widely used as one of the very sensitive biomarkers of environmental stress and are frequently used to assess genotoxi- city [9, 10]. The most used approach has consisted of in vivo exposures and subsequent extru- sion of coelomocytes from earthworms for the measurement of a variety of biomarkers [11, 12]. Compared with in vivo assays, in vitro exposures of coelomocyte primary cultures of Eise- nia fetida have recently been implemented to determine toxicity profiles of different contami- nants [13, 14]. However, most of these studies have either focused on cell recognition or cell culture condition, and systematic research in the field of in vitro toxicity is scarce. Although coelomocytes are increasingly being used for metal exposure, studies on nanoparticles and pes- ticide toxicity are limited [7, 9, 15]. Carbon nanotubes (CNTs), carbon allotropes with a cylindrical nanostructure, are cur- rently attracting increased scientific interest because of their unique one-dimensional hollow structure and extraordinary mechanical, electrical, thermal, and optical characteristics [16]. However, applications using multiwalled carbon nanotubes (MWCNTs) are favored because of their lower production costs and because they do not have a critical carbon nanotube (CNT) diameter or band-gap, suggesting that the release of MWCNTs into the environment is highly possible [17, 18]. Carbon nanotubes have potential negative impacts on aquatic animals which take up nanomaterials through skin contact or orally through the gastrointestinal tract [18–20]. In addition, CNTs released into the environment will inevitably interact with other organic pollutants and influence their bioavailability as a carrier. There is still a lack of knowl- edge of the combined toxicity of CNTs and organic pollutants to terrestrial organisms. Thus, the measurement of the complex toxicity of MWCNTs and other pollutants has been an emerging issue for environmentalists. Sodium pentachlorophenate (PCP-Na), as sodium salt of pentachlorophenol (PCP), poses a higher threat to waterways and estuaries via runoff from agricultural areas than industrial wastes, due to its high solubility [21, 22]. Introduction Earthworms play a key role in nutrient mineralization, decomposition, and soil structure improvement [1]. They are considered as bioindicators of soil quality and health due to their sensitivity to various chemicals, such as nanomaterials, pesticides, and heavy metals [2, 3]. Therefore, standard methods have been established to measure the responses of individual earthworm species by determining mortality, behavior, pathological symptoms, body weight Funding: This research was supported by National Natural Science Foundation of China (No. 41571468, 21077052), the Science and Technology Support Program of Jiangsu Province (No. BE2016172, BE2012737), and the Shandong PLOS ONE \| DOI:10.1371/journal.pone.0170092 January 26, 2017 1 / 14 MWCNTs and PCP-Na Toxicity on Earthworm Coelomocytes change, and reproductive activity [4–6]. However, large quantities earthworms were demanded for the observation of the end point of their death and it was necessary to consume much more chemicals in the experiment. Moreover, it takes a long time to prepare the artificial soil, e.g., the stable time and the exposure time of soil. Consequently, there has been attempted to develop more efficient methods to evaluate earthworm responses at a cellular level. Provincial Natural Science Foundation of China (No. ZR2015CM023). Provincial Natural Science Foundation of China (No. ZR2015CM023). Although PCP-Na has been banned in China for a number of years now, its residues can still be found in the environment [2, 23]. To our knowledge, for the determination of coelomocytes in in vitro culture is still not being conducted systematically. Limited information of CNTs and PCP-Na toxicity on earth- worm coelomocytes is available. This work explored the optimal conditions to culture coelo- mocytes of E. fetida in vitro and investigated the potential effect of PCP-Na, MWCNTs, and their complexes on coelomocytes under different exposure conditions in terms of cell survival rate, oxidative stress, membrane damage, and DNA damage, to provide valuable mechanistic information for ecological risk assessment for PCP-Na and MWCNTs. Cell culture conditions We selected four different media, low-glucose DMEM (Dulbecco minimum essential medium), high-glucose DMEM, RPMI-1640 (Roswell Park Memorial Institute), and DMEM-F12 to deter- mine the culture conditions of coelomocytes. Two culture condition, 37˚C with 5% CO2 and 25˚C without CO2, were selected based on the imitation environment of common cell cultures in vitro and living earthworms, respectively. Cells were seeded into a 96-well plate and 200 μl of media were added to each well; inoculation density was 1 × 105/ml, 2 × 105/ml, and 5 × 105/ml, with four replicates. Survival rate was measured using MTT 3-(4,5-dimethyl-2-thiazolyl)- 2,5-diphenyl-2-H-tetrazolium bromide after 24, 48, and 72 h, respectively, for the optimum cul- ture conditions. Cell extraction Earthworms of the species Eisenia fetida were purchased from a farming factory in Jurong, Jiangsu Province, China. Healthy adult earthworms with a well- developed clitellum (60-days old, with a weight of 300–400 mg) were used for coelomocyte extraction after acclimation. The methods for cell extraction and toxicity measurement were based on the descriptions in Eyambe et al. (1991) with some modifications [24]. The earthworms were placed in enclosed petri dishes for 48 h on moist filter paper at 20 ± 1˚C to purge their gut contents. Subsequently, individual earthworms were rinsed in cold extrusion medium (5% ethanol, 95% normal saline, 2.5 mg/mL EDTA, 10 mg/mL guaiacol glyceryl ether, pH 7.3) for 2 min. Coelomocytes were spontaneously secreted in the medium, the extrusion medium was then centrifuged at 4˚C and 4,729 × g for 10 min) and the supernatant was removed. The coelomocytes were washed three times in phos- phate-buffered saline solution (PBS). Survival rate was assayed by using the trypan blue exclu- sion method and a coelomocyte activity of at least 95% was cultured to test acute toxicity. Materials and Methods Reagents Sodium pentachlorophenate (PCP-Na, purity > 97%) was obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 1000 mg/kg stock solutions were dissolved in distilled water to obtain the final concentrations of 0 (control), 10.67, 16, 24, 36, 54, 81, and 121.5 mg/L 2 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0170092 January 26, 2017 MWCNTs and PCP-Na Toxicity on Earthworm Coelomocytes PCP-Na in the media. The MWCNTs, produced by chemical vaporization deposition (CVD), was purchased from Shenzhen Nanotech Port Co., Led. (Shenzhen, China). PLOS ONE \| DOI:10.1371/journal.pone.0170092 January 26, 2017 Exposure treatment Abbreviation MWCNTs (mg/kg) PCP-Na (mg/kg) Control Control - - MWCNTs50 M50 50 - MWCNTs200 M200 200 - PCP-Na1/20 P1/20 - 5.22 (1/20 LC50) PCP-Na1/10 P1/10 - 10.44 (1/10 LC50) PCP-Na1/5 P1/5 - 20.88 (1/5 LC50) MWCNTs50 + PCP-Na1/20 M + P1/20 50 5.22 (1/20 LC50)a MWCNTs50 + PCP-Na1/5 M + P1/5 50 20.88 (1/5 LC50)a MWCNTs50—PCP-Na1/20 M—P1/20 50 5.22 (1/20 LC50)b MWCNTs50—PCP-Na1/5 M—P1/5 50 20.88 (1/5 LC50)b a MWCNTs and PCP-Na added separately b MWCNTs coated with PCP-Na were added. doi:10.1371/journal.pone.0170092.t001 Table 1. Dosages of MWCNTs and/or PCP-Na used for oxidative damage and membrane damage. Exposure treatment Abbreviation MWCNTs (mg/kg) PCP-Na (mg/kg) Control Control - - MWCNTs50 M50 50 - MWCNTs200 M200 200 - PCP-Na1/20 P1/20 - 5.22 (1/20 LC50) PCP-Na1/10 P1/10 - 10.44 (1/10 LC50) PCP-Na1/5 P1/5 - 20.88 (1/5 LC50) MWCNTs50 + PCP-Na1/20 M + P1/20 50 5.22 (1/20 LC50)a MWCNTs50 + PCP-Na1/5 M + P1/5 50 20.88 (1/5 LC50)a MWCNTs50—PCP-Na1/20 M—P1/20 50 5.22 (1/20 LC50)b MWCNTs50—PCP-Na1/5 M—P1/5 50 20.88 (1/5 LC50)b a MWCNTs and PCP-Na added separately b MWCNTs coated with PCP-Na were added. Table 1. Dosages of MWCNTs and/or PCP-Na used for oxidative damage and membrane damage. Oxidative damage to coelomocytes. The exposure treatments for oxidative damage to coelomocytes were conducted based on Table 1. Each treatment was performed in four repli- cates. Other steps were similar to those of the acute toxicity test. The cell suspension was re-suspended in a 96-well plate and then transferred to a 1.5 mL Eppendorf vial. The supernatant was removed after centrifugation at 1,500 rpm, 4˚C for 10 min, and the obtained pellet was washed three times and re-suspended in 1 ml PBS. After ultrasonication (0.5 s: 1 s, work: interval) for 30 s in an ice bath, the cell suspension was again centrifuged at 3,000 rpm and 4˚C for 10 min; the supernatant was transferred to a new tube for physiological and biochemical index analysis. Superoxide dismutase (SOD), catalase (CAT) activities, and malonaldehyde (MDA) were mea- sured using commercial kits (Jiancheng Co. Ltd., Nanjing, China). Reactive oxygen species (ROS) were determined using the ROS assay kit (Beyotime Institute of Biotechnology, China). Enzyme activities were standardized by protein content. Each experiment was performed in triplicate. Membrane damage. The exposure method and exposure treatments both followed the protocols for evaluation of coelomocyte oxidative damage, including cell homogenate prepara- tion. Toxicity under in vitro exposure Acute toxicity of MWCNTs and PCP-Na. A 1.5-time concentration gradient of 0 (con- trol), 10.67, 16, 24, 36, 54, 81, and 121.5 mg/L was prepared in the media to assess the LC50 of PCP-Na under the previously described optimum culture conditions. Two exposure methods were used in the combined acute toxicity examination: 1) “+”: MWCNTs and PCP-Na were separately added to the medium, and 2)“-”: MWCNTs coated with equal quantities of PCP-Na were added. Prior to the toxicity test, adsorption of PCP-Na onto MWCNTs was performed. For this, MWCNTs were dissolved in distilled water with final concentrations of 0 (control), 10.67, 16, 24, 36, 54, 81, and 121.5 mg/L PCP-Na and the mixture was covered and placed in a shaker (20˚C, 250 rpm) for 24 h to ensure adsorption. The concentration of PCP-Na was mea- sured and the amount adsorbed onto CNTs was calculated. The CNTs coated with equal quanti- ties of PCP-Na were selected and air-dried for further use [23]. Subsequently, the stock solution containing 1,000 mg/L MWCNTs was placed in the ultrasonic cleaner and ultrasonicated with 80 percent power for 60 min to completely disperse MWCNTs. We diluted 50 mg/L MWCNTs for the combined toxicity study, for the toxicity of MWCNTs was slight based on data from the literature and on preliminary tests, with PCP-Na added simultaneously with the same concen- tration before. Cells were seeded into a 96-well plate with 200 μl media per well; inoculation density was 1 × 105/ml, 2 × 105/ml, and 5 × 105/ml, with four replicates. Survival rate was detected by MTT after 24 h. We only considered data where the survival rate of the control group was at least 95%. 3 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0170092 January 26, 2017 MWCNTs and PCP-Na Toxicity on Earthworm Coelomocytes Oxidative damage to coelomocytes. The exposure treatments for oxidative damage to coelomocytes were conducted based on Table 1. Each treatment was performed in four repli- cates. Other steps were similar to those of the acute toxicity test. The cell suspension was re-suspended in a 96-well plate and then transferred to a 1.5 mL Eppendorf vial. The supernatant was removed after centrifugation at 1,500 rpm, 4˚C for 10 min, and the obtained pellet was washed three times and re-suspended in 1 ml PBS. After ultrasonication (0.5 s: 1 s, work: interval) for 30 s in an ice bath, the cell suspension was again centrifuged at 3,000 rpm and 4˚C for 10 min; the supernatant was transferred to a new tube for physiological and biochemical index analysis. Superoxide dismutase (SOD), catalase (CAT) activities, and malonaldehyde (MDA) were mea- sured using commercial kits (Jiancheng Co. Ltd., Nanjing, China). Reactive oxygen species (ROS) were determined using the ROS assay kit (Beyotime Institute of Biotechnology, China). Enzyme activities were standardized by protein content. Each experiment was performed in triplicate. Membrane damage. The exposure method and exposure treatments both followed the protocols for evaluation of coelomocyte oxidative damage, including cell homogenate prepara- tion. Lactate dehydrogenase (LDH) activity was measured using commercial kits (Jiancheng Co. Ltd., Nanjing, China) and determination of mitochondrial membrane potential (MMP) was carried out using a cell apoptosis detection kit (Rhodamine 123) (KeyGEN BioTECH, Nanjing, China). DNA damage. The exposure method followed the protocol for determining coelomocyte acute toxicity. Higher concentrations of MWCNTs, 250 and 500 mg/kg l was choosen for sin- gle treatment as to the MWCNTs toxicity was relatively low, while a lower concentration of PCP-Na1/50 (2.09 mg/L) was chosen for DNA damage, with DNA being sensitive to various environmental contaminants. Each treatment was performed in four replicates. Coelomocytes were obtained according to the non-invasive extrusion method described by Eyambe et al. [24]. The level of DNA strand breaks was determined using the method described by Singh et al. [25]. After electrophoresis, slides were washed three times with 0.5 M Tris buffer (pH 7.5) and DNA was stained with ethidium bromide. Slides were examined with a fluorescent micro- scope (BX41, Olympus, Japan). A total of five groups were examined, with three slides per Table 1. Dosages of MWCNTs and/or PCP-Na used for oxidative damage and membrane damage. PLOS ONE \| DOI:10.1371/journal.pone.0170092 January 26, 2017 4 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0170092 January 26, 2017 MWCNTs and PCP-Na Toxicity on Earthworm Coelomocytes Acute toxicity In regards to the acute toxicity test, LC50 of PCP-Na was 104.46 mg/L (Table 2). The toxicity test of PCP-Na combined with 50 mg/L CNTs was conducted when the MWCNTs and PCP-Na were added separately; LC50 of PCP-Na was 109.39 mg/L, and at the condition that PCP-Na was absorbed by CNTs before addition, LC50 of PCP-Na was 112.92 mg/L. The acute toxicity of PCP-Na was slightly reduced after combining with CNTs. Acute toxicity of CNTs was very low and we did not obtain an LC50 value, which is consistent with the acute toxicity test results on an individual level [22]. Cell culture condition After coelomocyte extraction, coelomocyte activity was over 95% in terms of survival rate mea- surement of trypan blue exclusion. Survival rates of coelomocytes under different conditions are shown in S1 and S2 Figs. The results show that the coelomocytes can be suspension-cul- tured in vitro. Cell survival rate of coelomocytes was lower than 30% after cultivation of 24 h at 37˚C with 5% CO2 (S1 Fig), cultivation at 25˚C without CO2 was showed to a more suitable method. Compared with 48 h and 72 h observation of the optimum culture condition, opti- mum exposure time was 24 h. Cell survival rate was almost 100% in 24 h for high-glucose DMEM and RPMI-1640 medium, but after 72h exposure, cell survival rate of RPMI-1640 medium was still over 80%, suggesting that RPMI-1640 is more suitable for coelomocyte culti- vation. Optimal culture conditions were RPMI-1640 medium with 2–4 × 104 cells/well (1– 2×105 cells/mL) in a 96-well plate at 25˚C, without CO2 (Fig 1). Statistics analyses Statistical differences of biological parameters between each treatment, including the control, were evaluated using one-way analysis of variance (ANOVA), followed by a Dunnett t (2-sided) test. Results were expressed as means ± standard deviation (SD). All analyses were performed using SPSS 16.0 software and the probability level for statistical significance was p < 0.05. The LC50 (median lethal concentration) values were calculated using the regression line obtained by plotting the concentration against the death percentage on a probit scale and the results were evaluated with probit analysis (SPSS 17.0), using the following equation [14]: LC50 ¼ LC100 ðab þ . . . þ abÞ=n; where LC50 is the concentration with a survival rate of 50% of the coelomocytes and LC100 is expressed as percentage values relative to the signal given by control coelomocytes (100%). The variables a, b, and n are the difference between two consecutive doses. where LC50 is the concentration with a survival rate of 50% of the coelomocytes and LC100 is expressed as percentage values relative to the signal given by control coelomocytes (100%). The variables a, b, and n are the difference between two consecutive doses. Lactate dehydrogenase (LDH) activity was measured using commercial kits (Jiancheng Co. Ltd., Nanjing, China) and determination of mitochondrial membrane potential (MMP) was carried out using a cell apoptosis detection kit (Rhodamine 123) (KeyGEN BioTECH, Nanjing, China). DNA damage. The exposure method followed the protocol for determining coelomocyte acute toxicity. Higher concentrations of MWCNTs, 250 and 500 mg/kg l was choosen for sin- gle treatment as to the MWCNTs toxicity was relatively low, while a lower concentration of PCP-Na1/50 (2.09 mg/L) was chosen for DNA damage, with DNA being sensitive to various environmental contaminants. Each treatment was performed in four replicates. Coelomocytes were obtained according to the non-invasive extrusion method described by Eyambe et al. [24]. The level of DNA strand breaks was determined using the method described by Singh et al. [25]. After electrophoresis, slides were washed three times with 0.5 M Tris buffer (pH 7.5) and DNA was stained with ethidium bromide. Slides were examined with a fluorescent micro- scope (BX41, Olympus, Japan). A total of five groups were examined, with three slides per treatment, and at least 50 cells were analyzed for each slide. Photos were taken with a digital camera (C-5050ZOOM, Olympus, Japan) and images were analyzed by CASP software accord- ing to the method of Collins et al. [26]. DNA damage. The exposure method followed the protocol for determining coelomocyte acute toxicity. Higher concentrations of MWCNTs, 250 and 500 mg/kg l was choosen for sin- gle treatment as to the MWCNTs toxicity was relatively low, while a lower concentration of PCP-Na1/50 (2.09 mg/L) was chosen for DNA damage, with DNA being sensitive to various environmental contaminants. Each treatment was performed in four replicates. Coelomocytes were obtained according to the non-invasive extrusion method described by Eyambe et al. [24]. The level of DNA strand breaks was determined using the method described by Singh et al. [25]. After electrophoresis, slides were washed three times with 0.5 M Tris buffer (pH 7.5) and DNA was stained with ethidium bromide. Slides were examined with a fluorescent micro- scope (BX41, Olympus, Japan). A total of five groups were examined, with three slides per treatment, and at least 50 cells were analyzed for each slide. Photos were taken with a digital camera (C-5050ZOOM, Olympus, Japan) and images were analyzed by CASP software accord- ing to the method of Collins et al. [26]. Membrane damage We measured LDH and MMP to assess the membrane damage caused by MWCNTs and PCP-Na (Fig 3). The results of LDH activity and MMT in coelomocytes are shown in Fig 3; LDH activity was significantly promoted after 24-h exposure to various concentrations of PCP-Na with or without MWCNTs, except for M + P1/20 (Fig 3A). The results show that the MMP level significantly (p < 0.05) decreased in treatment groups of PCP-Na combined with 50 mg/L CNTs compared with the control (Fig 3B). Moreover, sep- arate addition of MWCNTs and PCP-Na could inhibit MMP levels more efficiently after adsorption, indicating that mitochondria suffered damage under MWCNTs and PCP-Na exposure. Oxidative damage The levels of SOD, CAT, ROS, and MDA in coelomocytes were measured after 24-h exposure to characterize the changes of antioxidant defenses induced by MWCNTs and PCP-Na (Fig 2). The results show that SOD activities significantly (p < 0.05) decreased in groups with PCP-Na combined with 50 mg/L CNTs compared to the control group (Fig 2A). Moreover, adsorption of MWCNTs and PCP-Na could inhibit SOD activity more effectively compared with separate addition. The activity of CAT has no significant (p > 0.05) difference between the control group and treatment group except for the separately co-added group M + P1/20 and M + P1/5 (Fig 2B). The ROS content decreased significantly (p < 0.05) after PCP-Na exposure, but 5 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0170092 January 26, 2017 MWCNTs and PCP-Na Toxicity on Earthworm Coelomocytes Fig 1. Coelomocytes survival rate in 72 h incubated under optimal culture condition doi:10.1371/journal.pone.0170092.g001 Fig 1. Coelomocytes survival rate in 72 h incubated under optimal culture condition doi:10.1371/journal.pone.0170092.g001 increased significantly in 200 mg/L MWCNTs or M + P1/20, M—P1/20 (Fig 2C). The MDA level significantly increased after MWCNTs and PCP-Na exposure with higher adsorption val- ues for separately added substances (Fig 2D). increased significantly in 200 mg/L MWCNTs or M + P1/20, M—P1/20 (Fig 2C). The MDA level significantly increased after MWCNTs and PCP-Na exposure with higher adsorption val- ues for separately added substances (Fig 2D). MWCNTs and PCP-Na Toxicity on Earthworm Coelomocytes Table 2. Acute toxicity of PCP-Na to coelomocytes. PCP-Na PCP-Na + CNTs PCP-Na—CNTs LC50(mg/L) 104.46 109.39 112.92 95% conﬁdence interval 85.62–137.21 90.42–133.17 79.37–166.17 +): MWCNTs and PCP-Na added separately -): MWCNTs coated with PCP-Na were added. doi:10 1371/journal pone 0170092 t002 Table 2. Acute toxicity of PCP-Na to coelomocytes. +): MWCNTs and PCP-Na added separately -): MWCNTs coated with PCP-Na were added. doi:10.1371/journal.pone.0170092.t002 +): MWCNTs and PCP-Na added separately -): MWCNTs coated with PCP-Na were added. DNA damage In each treatment, earthworm coelomocyte DNA suffered evident damage compared with the control, indicating that the comet assay is sensitive enough to evaluate earthworm DNA dam- age caused by PCP-Na and CNTs (Table 3). When exposed to CNTs, there was a significant dose-response relationship between CNTs and DNA damage. In single PCP-Na groups, the higher concentration of PCP-Na had lower DNA damage. DNA damage showed more serious in higher PCP-Na exposure group (50 + 1/5 LC50) when MWCNTs and PCP-Na were added separately or MWCNTs coated with PCP Na added. MWCNTs coated with PCP-Na added, which is in accordance with other biochemical parameters, such as ROS and MDA. 6 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0170092 January 26, 2017 Discussion Cell survival rate of coelomocytes was lower than 30% after cultivation of 24 h at 37˚C with 5% CO2. Cultivation at 25˚C without CO2 was a more suitable method. A previous study has shown that the optimum temperature for earthworm growth was 20–30˚C, with a pH range between 7 and 8 [27]. The presence of 5% CO2 might decrease the pH of the medium, resulting in unsuitable conditions for cell growth. Cell survival rate decreased when inoculation density exceeded 1 × 105/ml, possibly due to increased metabolic requirements [14]. Cell survival rate Fig 2. Activities of antioxidant enzymes of the earthworm coelomocytes after exposure to different treatments of MWCNTs (M), PCP-Na (P), and their complex (A) SOD, (B) CAT, (C) ROS, and (D) MDA. Data are expressed as the mean ± standard deviation (SD) of quadruplicate analyses. means which has a significant difference from the control (CK), a means the complex group which had significant difference from M50, b means the complex group which had significant difference from corresponding P1/20 or P1/5 (p < 0.05). doi:10.1371/journal.pone.0170092.g002 Fig 2. Activities of antioxidant enzymes of the earthworm coelomocytes after exposure to different treatments of MWCNTs (M), PCP-Na (P), and their complex (A) SOD, (B) CAT, (C) ROS, and (D) MDA. Data are expressed as the mean ± standard deviation (SD) of quadruplicate analyses. * means which has a significant difference from the control (CK), a means the complex group which had significant difference from M50, b means the complex group which had significant difference from corresponding P1/20 or P1/5 (p < 0.05). doi:10 1371/journal pone 0170092 g002 doi:10.1371/journal.pone.0170092.g002 7 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0170092 January 26, 2017 NE \| DOI:10.1371/journal.pone.0170092 January 26, 20 MWCNTs and PCP-Na Toxicity on Earthworm Coelomocytes Fig 3. Activities of antioxidant enzymes of the earthworm coelomocytes after exposure to different treatments of MWCNTs (M), PCP-Na (P), and their complex (A) LDH, (B) MMP. Data are expressed as the mean ± standard deviation (SD) of quadruplicate analyses. * means which has a significant difference from the control (CK), a means the complex group which had significant difference from M50, b means the complex group which had significant difference from corresponding P1/20 or P1/5 (p < 0.05). Fig 3. Activities of antioxidant enzymes of the earthworm coelomocytes after exposure to different treatments of MWCNTs (M), PCP-Na (P), and their complex (A) LDH, (B) MMP. doi:10.1371/journal.pone.0170092.t003 a represent mean ± standard deviation (SD) of quadruplicate analyses. * indicates signiﬁcant difference from the control (CK) a complex group with signiﬁcant difference from M50, g p g 50, group with signiﬁcant difference from corresponding P1/20 or P1/5 (p < 0.05). dicates signiﬁcant difference from the control (CK) Data represent mean ± standard deviation (SD) of quadruplicate analyses. ence from M50, ence from corresponding P1/20 or P1/5 (p < 0.05). Data represent mean ± standard deviation (SD) of quadruplicate analyses. an ± standard deviation (SD) of quadruplicate analyses. g ( ) a complex group with signiﬁcant difference from M50, omplex group with signiﬁcant difference from M50, b complex group with signiﬁcant difference from corresponding P1/20 or P1/5 (p < 0.05). om the control (CK) fference from M50, d e e ce o e co o (C ) igniﬁcant difference from M50, igniﬁcant difference from corresponding P1/20 or P1/5 (p < 0.05). ant difference from the control (CK) th signiﬁcant difference from M50, th signiﬁcant difference from corresponding P or P (p < 0 05) ation (SD) of quadruplicate analyses. * indicates signiﬁcant difference from the control (CK) p with signiﬁcant difference from M50, difference from the control (CK) i iﬁ t diff f M a complex group with signiﬁcant difference from M50, MWCNTs and PCP-Na Toxicity on Earthworm Coelomocytes that the toxicity of PCP-Na for E. fetida was alleviated by the appearance of MWCNTs in the filter paper contact test and the artificial soil contact test [22, 29]; this was probably due to the fact that the acute toxicity response was not sensitive enough to evaluate earthworm damage caused by PCP-Na and CNTs in coelomocytes toxicity. Oxidative stress is a common mechanism involved in the cytotoxicity of CNTs [30, 31]. Antioxidant defenses consist of different enzymes, including SOD, CAT, and GSH-Px. Based on the results of previous studies, the biochemical parameters SOD and CAT were chosen to evaluate the responses to various environmental contaminants [32]. In this study, SOD activity decreased significantly at M200, which showed that coelomocytes still suffered from oxidative stress, although the acute toxicity of MWCNTs was quite low. The PCP-Na also caused signifi- cant inhibition over the concentration of 10.44 mg/L (1/10 LC50), both MWCNTs and PCP-Na showed a significant dose-response relationship (p < 0.05) with SOD. However, in a similar study, Zhang et al. [33] discovered that SOD activity was significantly stimulated by the highest dose (2.5 mg/kg) of spirotetramat for the entire period of exposure. In our study, MWCNTs and PCP-Na showed more serious effects on coelomocytes when they were added after adsorption, which is in contrast to the other biochemical parameters. In a study by Wei et al. [34], SOD activ- ity was elevated first as a direct response to of the increased amount of superoxide anion radicals. The underlying mechanism for this might be that the natural antioxidant defenses were not suffi- cient anymore [22]. Catalase has a sufficient capacity for decomposing excess H2O2 to protect cells from oxidative stress. Some isozymes, such as POD and GPX, perform the same function in scavenging H2O2 [35, 36]. In this study, CAT activity showed no significant change in any treat- ment group compared with the control, which might be related to the hydrogen peroxide trans- formation mechanism that POD played a significant role in deposing the excess H2O2 during this treatment. In addition, we characterized the cell response to PCP-Na and MWCNTs by examining ROS production and lipid peroxidation. Previous studies have demonstrated that ROS can be generated in earthworms exposed to stress caused by environmental contaminants [22, 37]. PLOS ONE \| DOI:10.1371/journal.pone.0170092 January 26, 2017 Discussion Data are expressed as the mean ± standard deviation (SD) of quadruplicate analyses. * means which has a significant difference from the control (CK), a means the complex group which had significant difference from M50, b means the complex group which had significant difference from corresponding P1/20 or P1/5 (p < 0.05). doi:10.1371/journal.pone.0170092.g003 was almost 100% in 24 h for high-glucose DMEM and RPMI-1640 medium, but after 72h exposure, cell survival rate of RPMI-1640 medium remained over 80%, suggesting that RPMI- 1640 was more suitable for coelomocyte cultivation. In addition, our results indicate that 24 h incubation rendered the highest and the less variable signal in overtime culture. Incubation time is critical and differs substantially between earthworm species [28]. Accordingly, the cells were thus incubated for 24 h at room temperature (21˚C) in darkness after in vivo exposure to nanoparticles [13]. Based on the 24-h exposure test for coelomocytes, the LC50 of PCP-Na was 104.46 mg/L. Acute toxicity of PCP-Na in the combined treatments with MWCNTs was lower than that of PCP-Na alone, while there was no significant difference between the group of PCP-Na alone and PCP-Na co-added with MWCNTs. This is not in agreement with previous results showing Table 3. DNA damage of earthworm coelomocytes under PCP-Na and MWCNTs exposure. Treatment(mg/kg) Tail DNA% Tail length OTM CK 8.87 ±0.67 29.22 ±7.46 4.24±0.33 50 22.62 ± 1.31* 64.26 ± 5.75* 11.09 ± 1.67* CNTs 250 24.91 ± 0.73* 79.25 ± 3.47* 12.97 ± 1.06* 500 36.93 ± 3.23* 105.47 ± 14.43* 19.01 ± 1.85* PCP-Na 1/50 LC50 33.43 ± 2.41* 84.67 ± 3.80* 19.10 ± 1.01* 1/5 LC50 24.28 ± 1.56* 76.71 ± 5.04* 15.63 ± 1.02* CNTs + PCP-Na 50+1/50 LC50 33.60 ± 1.42a 99.53 ± 5.13ab 20.56 ± 1.67a 50+1/5 LC50 39.07 ± 2.06ab 108.72 ± 12.27ab 27.16 ± 1.25ab CNTs—PCP-Na 50-1/50 LC50 36.87 ± 3.19a 89.77 ± 10.48ab 23.90 ± 4.24ab 50-1/5 LC50 29.91 ± 2.00ab 82.47 ± 5.97a 16.55 ± 1.61a Table 3. DNA damage of earthworm coelomocytes under PCP-Na and MWCNTs exposure. NA damage of earthworm coelomocytes under PCP-Na and MWCNTs exposure. PLOS ONE \| DOI:10.1371/journal.pone.0170092 January 26, 2017 8 / 14 From this, it can be deduced that MWCNTs and PCP-Na revealed a synergetic effect, inducing more serious cytomembrane dam- age when separately added, based on LDH activity. The mitochondrion is one of the most important compartments for ROS generation in cells; mitochondrial disorder is a key step during apoptosis [47]. A variety of studies have shown that apoptosis was often associated with loss of the mitochondrial inner potential, and the reduction of MMP was among the changes encountered during the early of apoptosis [44]. As shown in Fig 3, after incubation with high concentrations of PCP-Na, 50 mg/L MWCNTs, and combined compounds, the MMP showed a significant decrease, indicating that both of them can induce cell apoptosis through mitochondrial impairment. Dong et al. [48] found that PCP-induced apoptosis occurred in a dose-dependent manner with the mitochon- drial membrane potential disruption and lipid peroxidation production (MDA) increase. In our study, MMP decreased most conspicuous with 20.88 mg/L PCP-Na (1/5 LC50), and the addition of MWCNTs reduced mitochondrial damage caused by PCP-Na, which was consistent with the acute toxicity result. This finding might be explained by toxic chemical kinetics. Based on our previous studies, MWCNTs show a rapid adsorption rate and pro- mising removal efficiency for PCP-Na; PCP-Na uptake can be significantly (p < 0.05) decreased in the presence of MWCNTs, resulting in less PCP-Na accumulation in cells and thus reduced mitochondrial damage. The comet assay has been widely used for nanotoxicology research on E. fetida and is a use- ful tool to identify genotoxic effects of chemicals on invertebrates [23, 32, 49]. Tail DNA% (tDNA), olive tail moment (OTM), and tail length increased significantly (p < 0.05) under PCP-Na and MWCNTs treatment compared with control groups, demonstrating that both of them caused DNA damage to coelomocytes (Table 3). In contrast, there was a significant dose- response relationship between MWCNTs and DNA damage, while higher concentrations of PCP-Na showed lower DNA damage after exposure. This is in agreement with the findings of Hu et al. [23], who reported that high dosages (14.2 mg/kg) of PCP-Na induced significantly lower (p < 0.01) values of the parameters compared with lower dosages (1.4 mg/kg). Martı´nez et al. [50] also found no differences in the extent of DNA fragmentation related to the concen- tration of the chemical, and DNA damage was barely affected by exposure time. Our study showed that high concentrations of MWCNTs (200 mg/kg) or low concentrations of PCP-Na combined with MWCNTs (50 mg/kg) induced ROS production. Overproduction of ROS can lead to oxidative damage to macromolecules such as nucleic acids, proteins, and lipids, eventually resulting in cell damage [38]. In the present study, the MDA content in the treatment groups was higher than in the control group throughout the exposure time, indicating that more products of peroxidized unsaturated fatty acids had accumulated in tissues. Separately added MWCNTs and PCP-Na induced more serious lipid peroxidation damage compared with MWCNTs coated with PCP-Na. In a previous study, cytotoxicity was enhanced by two to three times when the biphenyl compounds were combined with SWCNTs [39]. However, several studies have demonstrated that MWCNTs or other carbon materials could reduce the bioavail- ability of chemicals or inhibit their toxicity [40–42]. For our study, the underlying mechanism for this might be that MWCNTs are effective adsorbents of organic chemicals and could adsorb PCP-Na onto the surface. When MWCNTs and PCP-Na were added separately, small detached molecules promoted higher effects on cells, thereby revealing a synergetic effect inducing more serious lipid peroxidation damage. After adsorption, PCP-Na combined preferentially with MWCNTs instead of coelomocytes, reducing bioavailability of chemicals or inhibiting their tox- icity compared with separately co-added. In response to excessive ROS, endothelial functions were impaired and even apoptosis was triggered [43]. Lactate dehydrogenase (LDH) is an important metabolic dehydrogenase which catalyzes the interconversion of pyruvate to lactate in anaerobic glycolysis [44]. It is a cytoplasmic enzyme involved in anaerobic energy production and a sensitive index of the loss of cell mem- brane integrity in nanotoxicology studies [44]. Both PCP-Na and MWCNTs (50 mg/L) could PLOS ONE \| DOI:10.1371/journal.pone.0170092 January 26, 2017 9 / 14 MWCNTs and PCP-Na Toxicity on Earthworm Coelomocytes damage the membrane structure of the cell, increasing permeability. The inhibition of LDH activ- ity in the 200 mg/L MWCNTs treatment might be due to the binding of MWCNTs or its metabo- lite(s) with the enzyme molecules or by blocking enzyme synthesis [45]. Although PCP-Na and MWCNTs showed an antagonistic effect on LDH activity for the combined groups, while the phenomenon was not significant, it might be due to the oversaturated LDH released into the media, resulting in inhibition of LDH under co-exposure [46]. PLOS ONE \| DOI:10.1371/journal.pone.0170092 January 26, 2017 Supporting Information S1 Fig. Coelomocytes survival rate after 24 h incubated in 37˚C. (TIF) S1 Fig. Coelomocytes survival rate after 24 h incubated in 37˚C. (TIF) S2 Fig. Coelomocytes survival rate after 24 h incubated in 25˚C. (TIF) S2 Fig. Coelomocytes survival rate after 24 h incubated in 25˚C. (TIF) Lower extent of DNA damage at 20.88 mg/L (1/5 LC50) may be due to the fact that most of the severely dam- aged DNA broke into small fragments and was lost during electrophoresis procedures of the comet assay, and only the slightly damaged DNA was analyzed in the higher dosage treat- ments. However, the mechanisms responsible for genotoxicity of PCP-Na to cells or animals are not clear. After 24-h exposure, the toxicity induced by MWCNTs and PCP-Na in combina- tion was enhanced more or less compared with MWCNTs (50 mg/kg) and PCP-Na (2.09 and 20.88 mg/kg) individually, especially when added separately. The adsorption of MWCNTs to PCP-Na might lessen the toxicity of PCP-Na due to the PCP-Na absorbed by MWCNTs; the smaller amount of hydrogen peroxide produced can thus be eliminated by the cell´s normal antioxidant enzymes [22]. Overall, DNA damage in each treated group was significantly differ- ent compared with the control, indicating that the comet assay has sufficient sensitivity to eval- uate earthworm DNA damage caused by PCP-Na and CNTs. 10 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0170092 January 26, 2017 MWCNTs and PCP-Na Toxicity on Earthworm Coelomocytes Conclusions Cell survival rate, oxidative stress, membrane damage, and DNA damage of coelomocytes exposed to PCP-Na, MWCNTs, and their complex in vitro were associated with exposure con- centration and methods. Overall, both CNTs and PCP-Na can cause oxidative damage and produce ROS, resulting in lipid peroxidation with MDA generation and SOD and CAT activity inhibition at high stress, which is consistent with the individual results. In comparison, higher susceptibility was obtained in in vivo cytotoxicity tests in this study, compared to traditional endpoints. Moreover, CNTs and PCP-Na could damage the membrane structure of the cells, increasing permeability and inhibiting MMP. In addition, our results indicate that the toxicity of PCP-Na may be alleviated by the appearance of MWCNTs after adsorption, while PCP-Na and MWCNTs added separately had a synergetic effect based on the cytotoxicity toxicity study. In summary, coelomocyte toxicity in vitro analysis is a new sensitive method for detect- ing the adverse effects of various pollutants on earthworms, and the cytotoxicity indexes are positively correlated with the indexes for individual levels. Earthworm cytotoxicity tests are expected to replace individual level toxicity tests and can become a fast, efficient, and sensitive method to evaluate toxicity of pollutants on soil organisms. Acknowledgments This research was supported by National Natural Science Foundation of China (No. 41571468, 21077052), the Science and Technology Support Program of Jiangsu Province (No. BE2016172, BE2012737), and the Shandong Provincial Natural Science Foundation of China (No. ZR2015CM023). This research was supported by National Natural Science Foundation of China (No. 41571468, 21077052), the Science and Technology Support Program of Jiangsu Province (No. BE2016172, BE2012737), and the Shandong Provincial Natural Science Foundation of China (No. ZR2015CM023). Funding acquisition: ML CH. Investigation: FJ. Investigation: FJ. Methodology: CH. PLOS ONE \| DOI:10.1371/journal.pone.0170092 January 26, 2017 Author Contributions Conceptualization: ML. Data curation: YY YX. Data curation: YY YX. Formal analysis: YY. Funding acquisition: ML CH. References 1. Han Y, Zhu L, Wang J, Wang J, Xie H, Zhang S. Integrated assessment of oxidative stress and dna damage in earthworms (Eisenia fetida) exposed to azoxystrobin. Ecotox Environ Safe. 2014; 107C (9): 214–219. 2. Zhou CF, Wang YJ, Li CC, Sun RJ, Yu YC, Zhou DM. Subacute toxicity of copper and glyphosate and their interaction to earthworm (Eisenia fetida). Environ Pollut. 2013; 180: 71–77. doi: 10.1016/j.envpol. 2013.05.016 PMID: 23733011 3. Heggelund LR, Diezortiz M, Lofts S, Lahive E, Jurkschat K, Wojnarowicz J, et al. Soil pH effects on the comparative toxicity of dissolved zinc, non-nano and nano ZnO to the earthworm Eisenia fetida. Nano- toxicology. 2013; 8: 559–572. doi: 10.3109/17435390.2013.809808 PMID: 23739012 4. OECD 1984 Earthworm, Acute Toxicity Tests. Guideline for testing chemicals. No. 207. OECD, Paris, France. 5. OECD 2004. Earthworm Reproduction Test. Guideline for testing chemicals. No.222. OECD, Paris, France. 6. Earthworm Subchronic Toxicity Test, Ecological Effects Test Guidelines. OCSPP 850.3100: USEPA 2012. 7. Jin IK, Kim SW, An YJ. A new and sensitive method for measuring in vivo, and in vitro, cytotoxicity in earthworm coelomocytes by flow cytometry. Environ Res. 2014; 134: 118–126. doi: 10.1016/j.envres. 2014.07.014 PMID: 25127522 8. Reinardy HC, Bodnar AG. Profiling DNA damage and repair capacity in sea urchin larvae and coelomo- cytes exposed to genotoxicants. Mutagenesis. 2015; 30: 829–839. doi: 10.1093/mutage/gev052 PMID: 26175033 9. Irizar A, Rivas C, Garcı´a-Velasco N, Cerio FGD, Etxebarria J, Marigo´mez I, et al. Establishment of toxic- ity thresholds in subpopulations of coelomocytes (Amoebocytes vs. Eleocytes) of Eisenia fetida, exposed in vitro to a variety of metals: implications for biomarker measurements. Ecotoxicology. 2015; 24: 1004–1013. doi: 10.1007/s10646-015-1441-9 PMID: 25762103 10. Ciğerci İH, Ali MM, Kaygısız ŞY, Liman R. Genotoxicity assessment of cobalt chloride in Eisenia horten- sis earthworms coelomocytes by comet assay and micronucleus test. Chemosphere. 2015; 144: 754– 757. doi: 10.1016/j.chemosphere.2015.09.053 PMID: 26408983 11. Plytycz B, Kielbasa E, Grebosz A, Duchnowski M, Morgan AJ. Riboflavin mobilization from eleocyte stores in the earthworm Dendrodrilus rubidus, inhabiting aerially-contaminated Ni smelter soil. Chemo- sphere. 2010; 81: 199–205. doi: 10.1016/j.chemosphere.2010.06.056 PMID: 20633922 12. Irizar A, Rodrı´guez MP, Izquierdo A, Cancio I, Marigo´mez I, Soto M. Effects of soil organic matter con- tent on cadmium toxicity in Eisenia fetida: implications for the use of biomarkers and standard toxicity tests. Arch Environ Con Tox. 2014b; 68: 181–192. 13. Hayashi Y, Miclaus T, Scavenius C, Kwiatkowska K, Sobota A, Engelmann P, et al. Project administration: ML YC. Resources: YY. Software: YY YX. Software: YY YX. Supervision: ML YC. Validation: ML. PLOS ONE \| DOI:10.1371/journal.pone.0170092 January 26, 2017 11 / 14 MWCNTs and PCP-Na Toxicity on Earthworm Coelomocytes Visualization: YY YX. Writing – original draft: YY FJ. Writing – review & editing: CH ML. PLOS ONE \| DOI:10.1371/journal.pone.0170092 January 26, 2017 References Singh NP, Mccoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res. 1988; 175: 184–191. PMID: 3345800 26. Collins AR, Ma AG, Duthie SJ. The kinetics of repair of oxidative DNA damage (strand breaks and oxi- dised pyrimidines) in human cells. Mutat Res/Fund Mol M. 1995; 336: 69–77. 27. Lowe C N, Butt K R. Culture techniques for soil dwelling earthworms: A review. Pedobiologia. 2005, 49 (5): 401–413. 28. Spurgeon DJ, Svendsen C, Rimmer VR, Hopkin SP, Weeks JM (2000) Relative sensitivity of life-cycle and biomarker responses in four earthworm species exposed to zinc. Environ Toxicol Chem. 19:1800– 1808. 29. Lee JW, Won EJ, Kang HM, Hwang DS, Kim DH, Kim RK, et al. Effects of multi-walled carbon nanotube (MWCNT) on antioxidant depletion, the ERK signaling pathway, and copper bioavailability in the cope- pod (Tigriopus Japonicus). Aquat Toxicol. 2015; 171: 9–19. doi: 10.1016/j.aquatox.2015.12.005 PMID: 26716406 30. Garza KM, Soto KF, Murr LE. Cytotoxicity and reactive oxygen species generation from aggregated carbon and carbonaceous nanoparticulate materials. Int J Nanomed, 2008; 3: 83–94. 31. Ursini CL, Cavallo D, Fresegna AM. Comparative cyto-genotoxicity assessment of functionalized and pristine multiwalled carbon nanotubes on human lung epithelial cells. Toxicol in Vitro. 2012; 26: 831– 840. doi: 10.1016/j.tiv.2012.05.001 PMID: 22640919 32. Yang Y, Ji FN, Cui YB, Li M. Ecotoxicological effects of earthworm following long-term dechlorane plus exposure. Chemosphere. 2015; 144: 2476–2481. doi: 10.1016/j.chemosphere.2015.11.023 PMID: 26619313 33. Zhang QM, Zhang GL, Yin PJ, Lv YZ, Yuan S, Chen JQ, et al. Toxicological effects of soil contaminated with spirotetramat to the earthworm Eisenia fetida. Chemosphere. 2015; 139: 138–145. doi: 10.1016/j. chemosphere.2015.05.091 PMID: 26081578 34. Wei Z, Kou L, Lin C, Lei C, Lin K, Fu R. A multi-biomarker risk assessment of the impact of brominated flame retardant-decabromodiphenyl ether (BDE 209) on the antioxidant system of earthworm Eisenia fetida. Environ Toxicol Phar. 2014; 38: 297–304. 35. Zhang FQ, Wang YS, Lou ZP, Dong JD. Effect of heavy metal stress on antioxidative enzymes and lipid peroxidation in leaves and roots of two mangrove plant seedlings (Kandelia candel and Bruguiera gym- norrhiza). Chemosphere. 2007; 67: 44–50. doi: 10.1016/j.chemosphere.2006.10.007 PMID: 17123580 36. Du L, Li G, Liu M, Li Y, Yin S, Zhao J. Biomarker responses in earthworms (Eisenia fetida) to soils con- taminated with di-n-butyl phthalates. Environ Sci Pollut Res. 2015; 22: 4660–4669. 37. References Species differences take shape at nanoparticles: protein corona made of the native repertoire assists cellular interaction. Environ Sci Technol. 2013; 47: 14367–14375. doi: 10.1021/es404132w PMID: 24245550 14. Irizar A, Duarte D, Guilhermino L, Marigo´mez I, Soto M. Optimization of NRU assay in primary cultures of Eisenia fetida for metal toxicity assessment. Ecotoxicology. 2014a; 23: 1326–1335. 15. Plytycz B, Klimek M, Homa J, Mazur AI, Kruk J, Morgan AJ. Species-specific sensitivity of earthworm coelomocytes to dermal metal (Cd, Cu, Ni, Pb, Zn) exposures: methodological approach. Pedobiologia. 2011; 54: S203–S210. 16. Kunzmann A, Andersson B, Thurnherr T, Krug H, Scheynius A, Fadeel B. Toxicology of engineered nanomaterials: focus on biocompatibility, biodistribution and biodegradation. Biochimica Et Biophysica Acta. 2011; 1810: 361–373. doi: 10.1016/j.bbagen.2010.04.007 PMID: 20435096 17. De Volder MF, Tawfick SH, Baughman RH, Hart AJ. Carbon nanotubes: present and future commercial applications. Science. 2013; 339: 535–539. doi: 10.1126/science.1222453 PMID: 23372006 18. Jin WL, Choi YC, Kim R, Lee SK. Multiwall carbon nanotube-induced apoptosis and antioxidant gene expression in the gills, liver, and intestine of Oryzias latipes. Aquat Toxicol. 2016; 171: 9–19. 19. Pereira MM, Mouton L, Ye´pre´mian C, Coute´ A, Lo J, Marconcini JM, et al. Ecotoxicological effects of carbon nanotubes and cellulose nanofibers in Chlorella vulgaris. J Nanobiotecg. 2014; 12: 127–131. PLOS ONE \| DOI:10.1371/journal.pone.0170092 January 26, 2017 12 / 14 MWCNTs and PCP-Na Toxicity on Earthworm Coelomocytes 20. Ali D, Yadav PG, Kumar S, Ali H, Alarifi S, Harrath AH. Sensitivity of freshwater pulmonate snail Lym- naea luteola L., to silver nanoparticles. Chemosphere. 2013; 104: 134–140. doi: 10.1016/j. chemosphere.2013.10.081 PMID: 24309155 21. Sto¨mmer P, Gebhardt C, Schultheiss KH. Adenocarcinoma of the pancreas with a predominant intra- ductal component: a special variety of ductal adenocarcinoma. Pancreas. 1990; 5: 114–118. PMID: 2152839 22. Zhang LJ, Hu CW, Wang WL, Ji FN, Cui YB, Li M. Acute toxicity of multi-walled carbon nanotubes, sodium pentachlorophenate, and their complex on earthworm Eisenia fetida. Ecotox Environ Safe. 2014; 103: 29–35. 23. Hu CW, Zhang LJ, Wang WL, Cui YB, Li M. Evaluation of the combined toxicity of multi-walled carbon nanotubes and sodium pentachlorophenate on the earthworm Eisenia fetida, using avoidance bioassay and comet assay. Soil Biol Biochem. 2014; 70: 123–130. 24. Eyambe GS, Goven AJ, Fitzpatrick LC, Venables BJ, Cooper EL. A non-invasive technique for sequen- tial collection of earthworm (Lumbricus terrestris) leukocytes during subchronic immunotoxicity studies. Lab Anim. 1991; 25: 61–67. PMID: 2010977 25. PLOS ONE \| DOI:10.1371/journal.pone.0170092 January 26, 2017 References Zhang QM, Zhu LS, Wang J, Xie H, Wang JH, Han YN, et al. Oxidative stress and lipid peroxidation in the earthworm Eisenia fetida, induced by low doses of fomesafen. Environ Sci Pollut Res. 2013; 20: 201–208. 38. Sabatini SE, Juarez AB, Eppis MR, Bianchi L, Luquet CM, et al. Oxidative stress and antioxidant defenses in two green microalgae exposed to copper. Ecotox Environ Safe. 2009; 72: 1200–1206. 39. Sun JH, Zhang LH, Wang LR, Liang XJ, Xia WU, Wang CX. Cytotoxicity of different substituted biphe- nyls and their synergy toxicity combined with single-walled carbon nanotubes. Beijing Gongye Daxue Xuebao. 2013; 39: 624–628. 40. Cui X, Wang H, Lou L, Chen Y, Yu Y, Shi J, et al. Sorption and genotoxicity of sediment-associated pen- tachlorophenol and pyrene influenced by crop residue ash. J Soil Sediment. 2009; 9: 604–612. 13 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0170092 January 26, 2017 MWCNTs and PCP-Na Toxicity on Earthworm Coelomocytes 41. Petersen EJ, Pinto RA, Landrum PF, Weber WW Jr. Influence of carbon nanotubes on pyrene bioaccu- mulation from contaminated soils by earthworms. Environ Sci Technol. 2009; 43: 4181–4187. PMID: 19569349 42. Towell MG, Browne LA, Paton GI, Semple KT. Impact of carbon nanomaterials on the behaviour of 14c- phenanthrene and 14c-benzo-[a] pyrene in soil. Environ Pollut. 2011; 159: 706–715. doi: 10.1016/j. envpol.2010.11.040 PMID: 21195517 43. Liu L, Gu L, Ma Q, Zhu D, Huang X. Resveratrol attenuates hydrogen peroxide-induced apoptosis in human umbilical vein endothelial cells. Eur Rev Med Pharmaco. 2013; 17: 88–94. 44. Chen SZ, Zhang CM, Jia G, Duan JL, Wang SX, Zhang JC. Size-dependent cytotoxicity of europium doped NaYF 4, nanoparticles in endothelial cells. Mat Sci Eng C-Mater. 2014; 43: 330–342. 45. Tripathi G, Kachhwaha N, Dabi I. Ecophysiological category based toxicological responses in metabo- lism of earthworms: impact of a pyrethroidal insecticide. Pestic Biochem Phys. 2010; 98: 333–341. 46. Li PC, Tien YC, Day CH, Pai P, Kuo WW, Chen TS, et al. Impact of LPS-induced cardiomyoblast cell apoptosis inhibited by earthworm extracts. Cardiovasc Toxicol. 2015; 15: 172–179. doi: 10.1007/ s12012-014-9281-z PMID: 25249212 47. Chang JC, Kou SJ, Liu CS. Regulatory role of mitochondria in oxidative stress and atherosclerosis. World J Cardiol. 2010; 2(6): 150–159. doi: 10.4330/wjc.v2.i6.150 PMID: 21160733 48. Dong YL, Zhou PJ, Jiang SY, Pan XW, Xiao HZ. Induction of oxidative stress and apoptosis by penta- chlorophenol in primary cultures of Carassius carassius hepatocytes. Comp Biochem Phys C. PLOS ONE \| DOI:10.1371/journal.pone.0170092 January 26, 2017 References 2009; 150: 179–185. 49. Saez G, Aye M, Meo MD, Aime´ A, Bestel I, Barthe´le´my P, et al. Genotoxic and oxidative responses in coelomocytes of Eisenia fetida, and hediste diversicolor, exposed to lipid-coated cdse/zns quantum dots and CdCl2. Environ Toxicol. 2014; 30: 918–926. doi: 10.1002/tox.21966 PMID: 24500942 50. Martı´nez-Paz P, Morales M, Martı´nez-Guitarte JL, Morcillo G. Genotoxic effects of environmental endo- crine disruptors on the aquatic insect Chironomus riparius, evaluated using the comet assay. Mutat Res/Gen Tox Environ M. 2013; 758: 41–47. 14 / 14
https://openalex.org/W3144814345	https://www.research.ed.ac.uk/files/204890126/journal.pgen.1009428.pdf	English	null	Sex-stratified genome-wide association study of multisite chronic pain in UK Biobank	PLOS genetics	2,021	cc-by	16,837	Sex-stratified genome-wide association study of multisite chronic pain in UK Biobank Citation for published version: Johnston, KJA, Ward, J, Ray, PR, Adams, MJ, Mcintosh, AM, Smith, BH, Strawbridge, RJ, Price, TJ, Smith, DJ, Nicholl, BI, Bailey, MES & Epstein, MP (ed.) 2021, 'Sex-stratified genome-wide association study of multisite chronic pain in UK Biobank', PLoS Genetics, vol. 17, no. 4, pp. e1009428. https://doi.org/10.1371/journal.pgen.1009428 Citation for published version: Johnston, KJA, Ward, J, Ray, PR, Adams, MJ, Mcintosh, AM, Smith, BH, Strawbridge, RJ, Price, TJ, Smith, DJ, Nicholl, BI, Bailey, MES & Epstein, MP (ed.) 2021, 'Sex-stratified genome-wide association study of multisite chronic pain in UK Biobank', PLoS Genetics, vol. 17, no. 4, pp. e1009428. https://doi.org/10.1371/journal.pgen.1009428 Link: Link to publication record in Edinburgh Research Explorer Document Version: Publisher's PDF, also known as Version of record Edinburgh Research Explorer General rights C i h f h General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Take down policy Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 RESEARCH ARTICLE Sex-stratified genome-wide association study of multisite chronic pain in UK Biobank Keira J. A. JohnstonID1,2,3, Joey WardID1, Pradipta R. Ray4, Mark J. AdamsID2, Andrew M. McIntoshID2, Blair H. SmithID5, Rona J. StrawbridgeID1,6, Theodore J. PriceID4, Daniel J. SmithID1, Barbara I. NichollID1, Mark E. S. BaileyID3 Keira J. A. JohnstonID1,2,3, Joey WardID1, Pradipta R. Ray4, Mark J. AdamsID2, Andrew M. McIntoshID2, Blair H. SmithID5, Rona J. StrawbridgeID1,6, Theodore J. PriceID4, Daniel J. SmithID1, Barbara I. NichollID1, Mark E. S. BaileyID3 1 Institute of Health and Wellbeing, University of Glasgow, Glasgow, Scotland, United Kingdom, 2 Division of Psychiatry, University of Edinburgh, Edinburgh, Scotland, United Kingdom, 3 School of Life Sciences, College of Medical, Veterinary & Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom, 4 School of Behavioral and Brain Sciences, The University of Texas at Dallas, Richardson, Texas, United States of America, 5 Division of Population Health Sciences, University of Dundee, Ninewells Hospital and Medical School, Dundee, Scotland, United Kingdom, 6 Department of Medicine Solna, Karolinska Institute, Stockholm, Sweden a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * k.johnston.2@research.gla.ac.uk OPEN ACCESS Chronic pain is highly prevalent worldwide and imparts a significant socioeconomic and pub- lic health burden. Factors influencing susceptibility to, and mechanisms of, chronic pain development, are not fully understood, but sex is thought to play a significant role, and chronic pain is more prevalent in women than in men. To investigate sex differences in chronic pain, we carried out a sex-stratified genome-wide association study of Multisite Chronic Pain (MCP), a derived chronic pain phenotype, in UK Biobank on 178,556 men and 209,093 women, as well as investigating sex-specific genetic correlations with a range of psychiatric, autoimmune and anthropometric phenotypes and the relationship between sex- specific polygenic risk scores for MCP and chronic widespread pain. We also assessed whether MCP-associated genes showed expression pattern enrichment across tissues. A total of 123 SNPs at five independent loci were significantly associated with MCP in men. In women, a total of 286 genome-wide significant SNPs at ten independent loci were discov- ered. Meta-analysis of sex-stratified GWAS outputs revealed a further 87 independent asso- ciated SNPs. Gene-level analyses revealed sex-specific MCP associations, with 31 genes significantly associated in females, 37 genes associated in males, and a single gene, DCC, associated in both sexes. We found evidence for sex-specific pleiotropy and risk for MCP was found to be associated with chronic widespread pain in a sex-differential manner. Male and female MCP were highly genetically correlated, but at an rg of significantly less than 1 (0.92). All 37 male MCP-associated genes and all but one of 31 female MCP-associated genes were found to be expressed in the dorsal root ganglion, and there was a degree of enrichment for expression in sex-specific tissues. Overall, the findings indicate that sex dif- ferences in chronic pain exist at the SNP, gene and transcript abundance level, and highlight possible sex-specific pleiotropy for MCP. Results support the proposition of a strong central nervous-system component to chronic pain in both sexes, additionally highlighting a poten- tial role for the DRG and nociception. Citation: Johnston KJA, Ward J, Ray PR, Adams MJ, McIntosh AM, Smith BH, et al. (2021) Sex- stratified genome-wide association study of multisite chronic pain in UK Biobank. PLoS Genet 17(4): e1009428. https://doi.org/10.1371/journal. pgen.1009428 Editor: Michael P. Epstein, Emory University, UNITED STATES * k.johnston.2@research.gla.ac.uk Editor: Michael P. Epstein, Emory University, UNITED STATES Received: August 20, 2020 Accepted: February 16, 2021 Published: April 8, 2021 Copyright: © 2021 Johnston 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: August 20, 2020 Accepted: February 16, 2021 Published: April 8, 2021 Received: August 20, 2020 Accepted: February 16, 2021 Published: April 8, 2021 Copyright: © 2021 Johnston 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: UK Biobank Data is available upon application to UK Biobank. Summary statistics of the sex-stratified and GWAS meta-analyses are available for download from the University of Glasgow Enlighten system http://dx. doi.org/10.5525/gla.researchdata.1050. p pp Summary statistics of the sex-stratified and GWAS meta-analyses are available for download from the University of Glasgow Enlighten system http://dx. doi.org/10.5525/gla.researchdata.1050. Funding: RJS is supported by a UKRI Innovation- HDR-UK Fellowship (MR/S003061/1). JW is supported by the JMAS Sim Fellowship for depression research from the Royal College of Physicians of Edinburgh (173558). KJAJ is 1 / 27 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 PLOS GENETICS Sex-stratified multisite chronic pain GWAS supported by an MRC Doctoral Training Programme Studentship at the Universities of Glasgow and Edinburgh (MR/N013166/1). DJS acknowledges the support of a Lister Prize Fellowship (173096) and the MRC Mental Health Data Pathfinder Award (MC_PC_17217). M.J.A. and A.M.M. are supported by Pathfinder Award MC_PC_17209 and by Wellcome Trust Grant 104036/Z/14/Z. TJP and PRR are supported by a National Institute of Neurological Disorders and Stroke (Bethesda) grant (R01NS102161). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author summary Chronic pain is a highly prevalent and debilitating condition, which is more common in women than in men. Sex differences in this condition may be a result of several factors, including differences between the sexes in genetic variation related to chronic pain and gene expression differences related to sex. To explore sex differences in chronic pain from a genetic perspective, we looked for genetic variants associated with chronic pain in men and women separately in a large general-population cohort, and compared the variants we identified between the sexes. We assessed the degree of overlap between genetic vari- ants associated with chronic pain in each sex and those associated with a wide range of other traits, including major depression, body-mass index and suicidality. We also investi- gated gene expression patterns across a range of tissues for genes associated with chronic pain in each sex, in particular examining expression in neural and non-neural human and mouse tissues and assessing the degree of Dorsal Root Ganglion (DRG) enrichment, an important peripheral nervous system component involved in chronic pain. This work contributes to understanding of chronic pain as a trait and of sex differences in chronic pain at the levels of genetics and gene expression. Competing interests: The authors have declared that no competing interests exist. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 Introduction Chronic pain is widely defined as pain persisting beyond 3 months [1,2], and can be a primary disorder [3] or secondarily associated with injury, surgery or a range of medical conditions. Chronic pain is highly prevalent worldwide [4–9] and imparts a significant socioeconomic and public health burden [10]. Factors influencing susceptibility to chronic pain, and the mechanisms underlying its development and maintenance, are not fully understood. Several aspects of chronic pain including Chronic Pain Grade [11], severe chronic pain and low back pain have been studied from a genetic perspective and found to be complex traits. Heritability estimates vary from ~30–46% in twin, pedigree and factor analysis studies [12– 15], while single nucleotide polymorphism (SNP) heritability has been estimated from genome-wide association studies (GWAS) to be ~7–10% [16,17]. It is increasingly recognised that sex differences in many complex human traits are biologi- cally important, with genetic architecture for many traits being to some extent sex-specific [18], and a ‘sex-aware’ approach to genetic analysis has been widely advocated [19]. Sex as a biological variable has wide-ranging effects on the functioning of the genome and on resultant phenotypes. These effects can be mediated via sex-differential gene expression [20,21], sex differences in methylation [22–26] and expression quantitative trait locus (eQTL) effects [27,28], or differing levels and actions of hormones [29,30]. Sex can also influence traits through environmental fac- tors strongly correlated with sex [23,24] and sex-specific pleiotropy [25,31]. Chronic pain exhibits sex-related prevalence differences, and is more common in women than in men [32–34]. There are also potential sex differences in the impact of pain on functioning in daily life, and in the suc- cess of specific coping strategies [35]. In addition to differences in prevalence between the sexes, sex differences in underlying pain mechanisms and their modulation by immune cells have been recently reported [36–38], and immune responses in general can differ by sex [39]. Multisite Chronic Pain (MCP) is a derived chronic pain phenotype, defined as the sum of the number of sites of chronic pain on the body, here expressed on a scale from 0–7 [17]. GWAS of MCP in males and females separately To detect sex-differential genetic influences on multisite chronic pain, GWASs were run sepa- rately for males and females in UK Biobank. In men, a total of 123 SNPs at five independent loci were associated with MCP at a genome-wide significance threshold of p < 5 x 10−8 (Table 1, Fig 1). In women, a total of 286 genome-wide significant SNPs at ten independent loci were discovered (Table 1, Fig 1). All 15 of these loci were differentially associated with sex —none of the genome-wide significant SNPs at these loci had p < 5 x 10−8 in the GWAS con- ducted in the opposite sex. However, a total of 257 SNPs were found to have suggestive levels of association with MCP (p < 5 x 10−5) in both men and women. Two SNPs had suggestive evidence in men and were genome-wide significant in women, and eight SNPs had suggestive evidence in women and were genome-wide significant in men. In addition, the genome-wide significant loci on chromosome 6 in each sex were separated by less than 1 Mbp and may potentially exert their influence via differential effects on the same gene. lead SNPs associated with MCP (p < 5 x 10−8) in male and female sex-stratified GWAS. CHR, chromosome; BP, here A1 is the effect allele; BETA/SE, coefficient and its standard error for the effect allele; Gene = ANNOVAR annota- i i ANNOVAR) Table 1. Genome-wide significant independent lead SNPs associated with MCP (p < 5 x 10−8) in male and female sex-stratified GWAS. CHR, chromosome; BP, chromosome coordinate; A1/A2, alleles 1 and 2, where A1 is the effect allele; BETA/SE, coefficient and its standard error for the effect allele; Gene = ANNOVAR annota- tion gene symbol for variant (N/A, no gene annotation in ANNOVAR). Introduction We have previously shown in UK Biobank [17] that genetic predisposition to MCP (as captured by a polygenic risk score; PRS) was associated with Chronic Widespread Pain (CWP), a separate PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 2 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS but related chronic pain phenotype, in women but not men [17]. Additional unpublished find- ings using the Generation Scotland study [40] demonstrated that the PRS-MCP was associated with both chronic pain grade and an MCP-like phenotype in both men and women, but that the magnitude of effect was roughly twice as great in women as it was in men. There was also a significant PRS-by-sex interaction. These findings suggest that sets of variants contributing to chronic pain in males and females may act differently, or have different genetic effect sizes, in the two sexes. Here we report on an exploration of this preliminary evidence for the existence of sex-specific loci associated with MCP using a sex-stratified GWAS analysis approach in UK Biobank, and identify several sex-specific MCP loci. A meta-analysis of the female- and male- specific GWASs also revealed novel MCP loci not identified in the original MCP GWAS. We have also investigated possible functional effects associated with sex-specific MCP-associated genes as revealed by gene expression data in multiple relevant tissues, including dorsal root ganglion (DRG) samples, in both human and mouse. GWAS of MCP in males and females separately SNP rsID CHR BP A1 A2 BETA SE P Sex Gene rs35072907 1 51189556 G C 0.020 0.004 2.40E-08 Female FAF1 rs59898460 1 150493004 T C 0.025 0.004 4.90E-12 Female LINC00568; RP11-54A4.2 rs147903676 2 5835352 C CT -0.031 0.006 2.00E-08 Female SOX11 rs13135092 4 103198082 A G -0.038 0.006 2.30E-09 Female SLC39A8 rs3080367 5 57576558 TACAC T 0.024 0.004 2.90E-08 Female PGAM1P1; PLK2 rs62381120 5 120176330 T C -0.021 0.004 3.50E-08 Female CTD-2334D19.1; AC008565.1 rs74274428 5 170842428 CA C 0.020 0.004 2.80E-08 Female NPM1; FGF18 rs151060048 6 34633069 CA C -0.035 0.006 5.40E-09 Female C6orf106 rs34003284 13 53902876 C A -0.024 0.004 3.20E-10 Female RN7SL618P; AL450423.1 rs11079993 17 50301552 G T -0.021 0.004 4.50E-09 Female CA10; snoZ178 rs10660361 6 33741371 C CG 0.020 0.004 1.80E-08 Male LEMD2 9:140251458_G_A 9 140251458 G A -0.030 0.005 3.00E-09 Male EXD3 rs16909443 11 6192462 T C -0.040 0.007 4.40E-08 Male RP11-290F24.3 18:50442591_TTTC_T 18 50442591 TTTC T -0.020 0.004 1.60E-08 Male N/A 20:19709268_AAAAT_A 20 19709268 AAAAT A 0.030 0.005 1.20E-08 Male SLC24A3; AL121761.1 htt //d i /10 1371/j l 1009428 t001 Table 1. Genome-wide significant independent lead SNPs associated with MCP (p < 5 x 10−8) in male and female sex-stratified GWAS. CHR, chromosome; BP, chromosome coordinate; A1/A2, alleles 1 and 2, where A1 is the effect allele; BETA/SE, coefficient and its standard error for the effect allele; Gene = ANNOVAR annota- tion gene symbol for variant (N/A, no gene annotation in ANNOVAR). PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 3 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS Fig 1. Manhattan plots for the stratified GWAS analyses. Upper panel: female, lower panel: male. Red dotted line indicates genome-wide significant p-value threshold. https://doi.org/10.1371/journal.pgen.1009428.g001 Fig 1. Manhattan plots for the stratified GWAS analyses. Upper panel: female, lower panel: male. Red dotted line indicates genome-wide significant p-value threshold. https://doi.org/10.1371/journal.pgen.1009428.g001 https://doi.org/10.1371/journal.pgen.1009428.g001 LDSR analysis demonstrated that inflation of test statistics in each GWAS was due to poly- genicity (Table 2; LDSR intercept). SNP heritability was moderate, estimated as 0.125 and 0.106 in females and males, respectively (Table 2). The genetic correlation between male and female MCP was high (rg = 0.92, SE 0.03; p = 3.32 x 10−213), but significantly less than 100% (based on confidence intervals calculated as +/- 2 x SE). Meta-Analysis of Sex-Stratified MCP GWAS Outputs 87 independent SNPs were found to be associated with MCP at genome-wide significance in total, 11 of which were novel (not found in the unstratified or in each sex-stratified GWAS analysis). Each of the 87 independent significant SNPs showed consistent direction of effect between males and females (Table 3), but seven showed significant heterogeneity in effect size (I2 p < 0.05). In total, 49 lead SNPs across 46 genomic risk loci were found to be associated with MCP in meta-analysis of sex-stratified outputs. Genetic correlations between sex-stratified MCP and other disorders and i A range of complex trait phenotypes were selected for LDSR analysis with male and female MCP based on previous evidence for phenotypic correlation [17], with the addition of newly available trait data such as GWAS outputs on suicide and self-harm [41] and mood instability [42]. Suicidality and self-harm are important comorbidities of chronic pain, an issue Table 2. Trait genetic attributes from the male and female MCP GWASs. SNP-heritability, BOLT-LMM pseudo- heritability estimate; λGC1000, λGC value adjusted for sample size; LDSR_intercept (SE), LD-score regression intercept value and its standard error. Attribute female male SNP-heritability 0.125 0.106 λGC1000 1.002 1.001 LDSR_intercept (SE) 1.03 (0.006) 1.025 (0.005) https://doi.org/10.1371/journal.pgen.1009428.t002 4 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS Table 3. Independent, genome-wide significant (GWS) SNPs from meta-analysis of sex-stratified MCP GWASs. Genomic Locus = genomic risk locus context of SNP with multiple independent GWS SNPs present at some of the 46 loci, rsID = SNP rsID identifier, Position = genomic position (chromosome: base-pair start position), Meta p = p value for association from the GWAS meta-analysis, Direction = direction of effect in female MCP and male MCP GWASs, respectively (+ if association beta value for effect allele > 0,—if < 0), I2 = effect size heterogeneity estimate, I2 p = p value for heterogeneity estimate, Symbol = ANNOVAR gene annotation for variant (N/ A, no gene annotation in ANNOVAR). Significant (unadjusted) heterogeneity I2 p values (I2 p < 0.05) are marked with . Table 3. Independent, genome-wide significant (GWS) SNPs from meta-analysis of sex-stratified MCP GWASs. Genomic Locus = genomic risk locus context of SNP with multiple independent GWS SNPs present at some of the 46 loci, rsID = SNP rsID identifier, Position = genomic position (chromosome: base-pair start position), Meta p = p value for association from the GWAS meta-analysis, Direction = direction of effect in female MCP and male MCP GWASs, respectively (+ if association beta value for effect allele > 0,—if < 0), I2 = effect size heterogeneity estimate, I2 p = p value for heterogeneity estimate, Symbol = ANNOVAR gene annotation for variant (N/ A, no gene annotation in ANNOVAR). Significant (unadjusted) heterogeneity I2 p values (I2 p < 0.05) are marked with . Genetic correlations between sex-stratified MCP and other disorders and i value for effect allele > 0,—if < 0), I2 = effect size heterogeneity estimate, I2 p = p value for heterogeneity estimate, Symbol = ANNOVAR gene annotation for variant (N/ A, no gene annotation in ANNOVAR). Significant (unadjusted) heterogeneity I2 p values (I2 p < 0.05) are marked with . compounded by the common co-occurrence of mental health traits, such as major depressive disorder (MDD), with chronic pain, and by the fact that use of certain medication in chronic pain contributes to increased risk for self-harm, suicidal ideation and suicide attempt [43–48]. Genetic correlations between sex-stratified MCP and other disorders and i rsID Genomic Locus Position Meta p Direction I2 I2 p Symbol rs909001 1 1:32196647 2.80E-08 – 7.6 0.298 BAI2 1:51042504_CT_C 2 1:51042504 2.35E-08 – 59.1 0.118 FAF1 rs197441 3 1:112283655 4.33E-10 – 0 0.644 FAM212B-AS1 rs12033257 3 1:112318484 2.79E-08 ++ 0 0.824 KCND3 rs509345 4 1:150276022 1.42E-10 – 71.3 0.062 MRPS21 rs367563576 4 1:150495378 2.24E-11 ++ 89.6 0.002 LINC00568; RP11-54A4.2 rs9700909 5 1:243255124 4.07E-09 – 30 0.232 RP11-261C10.3 1:243461350_CT_C 5 1:243461350 9.48E-09 – 53 0.145 SDCCAG8 rs6721975 6 2:5832667 2.86E-08 – 67 0.082 SOX11 rs4852567 7 2:80703379 3.51E-08 ++ 25 0.248 CTNNA2 rs5832889 8 2:100503396 2.06E-08 ++ 74.9 0.046 AFF3 rs112908707 9 3:49865628 4.35E-10 – 0 0.866 TRAIP rs62260755 9 3:49898318 5.36E-09 – 0 0.619 CAMKV 3:50098024_CAA_C 9 3:50098024 4.79E-09 ++ 0 0.75 RBM6 rs13067082 9 3:50221715 3.66E-08 ++ 0 0.534 SEMA3F rs144433312 10 3:84591507 1.07E-08 ++ 26.7 0.243 AC107025.1; LINC00971 rs62263345 11 3:107252190 5.01E-10 – 0 0.848 BBX rs28750366 12 3:136361055 3.55E-08 – 0 0.498 STAG1 rs56203712 13 4:25342606 1.37E-11 ++ 0 0.783 ZCCHC4 rs201081507 14 4:102681041 8.93E-09 – 51.9 0.149 BANK1 rs13109404 14 4:102896591 1.17E-09 – 0 0.768 BANK1 rs13135092 14 4:103198082 2.61E-14 – 3.4 0.309 SLC39A8 rs6869446 15 5:65570607 3.23E-08 – 3.4 0.309 snoU13; RP11-305P14.1 rs10076888 16 5:103786487 1.06E-08 ++ 0 0.872 RP11-6N13.1 rs147831713 16 5:103787168 9.96E-09 – 0 0.66 RP11-6N13.1 rs325485 16 5:103995368 3.15E-08 ++ 0 0.576 RP11-6N13.1 rs1976423 16 5:104042643 1.48E-08 – 58.3 0.121 RP11-6N13.1 rs137863733 17 5:160890323 8.13E-10 ++ 23.3 0.254 GABRB2 rs6915136 18 6:33651322 1.09E-08 – 37.2 0.207 ITPR3 rs482786 18 6:33707599 1.51E-09 – 29.8 0.233 IP6K3 6:33709752_CA_C 18 6:33709752 1.08E-08 ++ 0 0.5 IP6K3 rs28651968 18 6:33717424 2.16E-08 ++ 0 0.945 IP6K3; LEMD2 rs17529077 18 6:33793332 1.27E-09 ++ 0 0.997 MLN; LINC01016 rs17600945 18 6:33802263 1.21E-09 ++ 0 0.365 MLN; LINC01016 rs6907508 19 6:34592090 2.81E-08 – 50.3 0.156 C6orf106 rs151060048 19 6:34633069 6.92E-09 – 84.4 0.011 C6orf106 rs142415291 19 6:34755312 9.43E-09 – 81.6 0.020 SNRPC; UHRF1BP1 rs6926377 20 6:145105354 7.17E-09 – 49.6 0.159 UTRN rs148148187 21 7:3602520 1.29E-08 ++ 0 0.839 SDK1 rs7798894 22 7:21552995 3.44E-08 ++ 17 0.273 SP4 rs6966540 23 7:95727967 1.09E-08 – 0 0.67 DYNC1I1 rs10156143 23 7:95844896 6.43E-09 – 0 0.349 SLC25A13 rs1450833 24 7:113865735 1.16E-08 – 0 0.715 FOXP2 7:113945981_CCACTTATAAATACTGTCCCTTGGGCA_C 24 7:113945981 1.24E-08 ++ 0 0.657 FOXP2 ( d) PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 5 / 27 https://doi.org/10.1371/journal.pgen.1009428.t003 PLOS GENETICS PLOS GENETICS Sex-stratified multisite chronic pain GWAS Table 3. (Continued) rsID Genomic Locus Position Meta p Direction I2 I2 p Symbol rs1527146 24 7:113987281 4.56E-08 ++ 27.4 0.241 FOXP2 7:114058731_CA_C 24 7:114058731 5.08E-10 ++ 23.2 0.254 AC073626.2 rs55671932 25 7:150556803 4.07E-08 ++ 68.2 0.076 AOC1 rs6997840 26 8:141658361 2.97E-08 – 0 0.37 AGO2; PTK2 9:96168164_CT_C 27 9:96168164 1.83E-08 ++ 0 0.87 RNU6-829P; Y_RNA rs7869969 27 9:96217447 9.87E-10 ++ 0 0.797 FAM120A rs6478241 28 9:119252629 6.61E-09 ++ 5.6 0.303 ASTN2 9:140247497_A_C 29 9:140247497 5.65E-12 – 0 0.567 EXD3 9:140249861_A_C 29 9:140249861 6.70E-09 ++ 23.9 0.252 EXD3 9:140260266_T_G 29 9:140260266 3.38E-14 – 0 0.571 EXD3 rs2183271 30 10:21957229 4.47E-08 – 4.3 0.307 MLLT10 10:99784552_CCA_C 31 10:99784552 1.05E-08 – 40.4 0.195 CRTAC1 rs11599236 32 10:106454672 3.33E-08 ++ 0 0.351 SORCS3 rs17553733 33 11:16362089 1.70E-10 – 0 0.392 SOX6 rs2118362 33 11:16373083 1.32E-08 – 31.9 0.226 SOX6 rs55670730 34 11:43620008 4.18E-08 – 0 0.925 N/A rs7303462 35 12:23974911 4.61E-08 – 32.5 0.224 SOX5 rs184483429 36 12:107620106 4.87E-08 ++ 0 0.58 RP11-797M17.1; SETP7 rs2759694 37 13:53695378 3.17E-08 ++ 76.8 0.038 OLFM4; LINC01065 rs67128127 37 13:53889000 7.48E-09 ++ 3.4 0.309 RN7SL618P; AL450423.1 rs1443914 37 13:53917230 4.73E-10 ++ 76.7 0.038 RN7SL618P; AL450423.1 rs7335163 37 13:53989975 7.23E-09 ++ 8.9 0.295 AL450423.1; LINC00558 rs17574479 37 13:54049489 2.97E-09 – 88.3 0.003 AL450423.1; LINC00558 rs34521521 38 14:73832318 4.99E-08 – 0 0.512 NUMB rs4886649 39 15:75328595 2.25E-08 – 0 0.892 PPCDC 15:75348905_CAACA_C 39 15:75348905 1.62E-08 – 0 0.924 PPCDC rs2386584 40 15:91539572 6.68E-12 – 0 0.578 PRC1 rs285027 41 16:77100932 9.39E-09 – 0 0.748 CTD-2336H13.2; MON1B rs11871043 42 17:43172849 1.49E-09 ++ 0 0.945 NMT1 rs967823 43 17:50317276 1.98E-11 – 58.8 0.119 snoZ178; RP11-429O1.1 rs35518690 44 18:42136963 2.15E-08 ++ 41.5 0.191 CTC-782O7.1; RP11-456K23.1 rs2043187 45 18:50394405 3.58E-10 – 0 0.668 DCC rs72922230 45 18:50394407 4.12E-10 – 29.7 0.233 DCC rs767443167 45 18:50622162 1.43E-08 – 0 0.469 DCC rs8089828 45 18:50669725 9.63E-10 ++ 0 0.783 DCC rs8099145 45 18:50743672 2.15E-12 – 0 0.871 DCC rs12968428 45 18:50750225 1.77E-09 ++ 0 0.413 DCC rs17410557 45 18:50776391 1.03E-11 – 0 0.561 DCC rs773737322 45 18:50846440 9.82E-11 ++ 0 0.529 DCC rs1367635 45 18:50861409 5.25E-09 – 0 0.659 DCC rs766498304 45 18:50871256 7.87E-09 – 35.6 0.213 DCC rs10164055 45 18:50919600 2.53E-08 – 0 0.659 DCC rs16980973 46 20:19648493 1.51E-10 ++ 0 0.322 SLC24A3 https://doi.org/10.1371/journal.pgen.1009428.t003 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 6 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS Fig 2. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 PLOS GENETICS https://doi.org/10.1371/journal.pgen.1009428.g004 Fig 4. MCP-associated gene expression tissue enrichment analysis. Significant (-log10 p > 3) results in female analyses and their corresponding tissue results in male analyses are shown for brevity. No significant enrichment for expression in any tissue was found for MCP-associated genes in males. Dashed line = significance threshold. ACC_BA24 = anterior cingulate cortex BA24, Caudate_BG = caudate basal ganglia, NAc_BG = Basal ganglia— Nucleus accumbens region, Putamen_BG = Basal ganglia—putamen region. Full results for all 53 tissues can be found in S1 and S2 Figs. htt //d i /10 1371/j l 1009428 004 Fig 4. MCP-associated gene expression tissue enrichment analysis. Significant (-log10 p > 3) results in female analyses and their corresponding tissue results in male analyses are shown for brevity. No significant enrichment for expression in any tissue was found for MCP-associated genes in males. Dashed line = significance threshold. ACC_BA24 = anterior cingulate cortex BA24, Caudate_BG = caudate basal ganglia, NAc_BG = Basal ganglia— Nucleus accumbens region, Putamen_BG = Basal ganglia—putamen region. Full results for all 53 tissues can be found in S1 and S2 Figs. https://doi.org/10.1371/journal.pgen.1009428.g004 https://doi.org/10.1371/journal.pgen.1009428.g004 PLOS GENETICS Genetic correlations (rg) between male (M) and female (F) MCP and multiple traits (rg values with 95% CI error bars). Phenotypes differentially correlated with MCP between the sexes are plotted in orange. https://doi.org/10.1371/journal.pgen.1009428.g002 Fig 2. Genetic correlations (rg) between male (M) and female (F) MCP and multiple traits (rg values with 95% CI error bars). Phenotypes differentially correlated with MCP between the sexes are plotted in orange. https://doi.org/10.1371/journal.pgen.1009428.g002 MCP in both men and women was found to be significantly genetically correlated with a range of traits and disorders, including psychiatric and mood phenotypes such as anhedonia, mood instability, depressive symptoms, MDD, anxiety, suicidality and subjective wellbeing (Fig 2, S1 Table). In addition, as expected, genetic correlations between both male and female MCP and unstratified MCP from our previous analysis [17]were essentially perfect (rg = 1.00, p < 1 x 10−120 for both sexes). PTSD, schizophrenia, autism spectrum disorder, anorexia ner- vosa, PGC cross-disorder phenotype and primary biliary cirrhosis were found to be signifi- cantly correlated with MCP in one sex and not the other. Several phenotypes were found not to be genetically correlated with MCP in either sex (pfdr > 0.05), including inflammatory bowel diseases, Parkinson’s disease, bipolar disorder, rheumatoid arthritis, and low relative amplitude (a circadian rhythmicity-related phenotype). Fig 3. Venn diagram of number of genes found significantly associated with MCP in MAGMA gene-based test analyses. Sectors: Original = non-sex-stratified MCP GWAS, Meta = meta-analysed female and male GWAS output, Female = female MCP GWAS, Male = male MCP GWAS. https://doi.org/10.1371/journal.pgen.1009428.g003 Fig 3. Venn diagram of number of genes found significantly associated with MCP in MAGMA gene-based test analyses. Sectors: Original = non-sex-stratified MCP GWAS, Meta = meta-analysed female and male GWAS output, Female = female MCP GWAS, Male = male MCP GWAS. https://doi.org/10.1371/journal.pgen.1009428.g003 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 7 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS Fig 4. MCP-associated gene expression tissue enrichment analysis. Significant (-log10 p > 3) results in female analyses and their corresponding tissue results in male analyses are shown for brevity. No significant enrichment for expression in any tissue was found for MCP-associated genes in males. Dashed line = significance threshold. ACC_BA24 = anterior cingulate cortex BA24, Caudate_BG = caudate basal ganglia, NAc_BG = Basal ganglia— Nucleus accumbens region, Putamen_BG = Basal ganglia—putamen region. Full results for all 53 tissues can be found in S1 and S2 Figs. Gene-level analysis Genes enriched for variants associated with MCP were identified using a gene-level association analysis (gene-based test) [49] approach implemented by MAGMA as part of the FUMA suite that tests 19,012 separate genes. The results are summarised in Fig 3 and S2 Table. In females and males, 31 and 37 genes, respectively, were found to be significantly (Bonferroni-adjusted significance criterion: p < 2.63 x 10−6) associated with MCP. The only gene found to be signifi- cantly associated with both male and female MCP was DCC. 24 out of the 31 genes signifi- cantly associated with MCP in females in the sex-stratified analyses, and 31 out of the 37 genes significantly associated in males, were also significantly associated in our previous non-strati- fied analysis [17]. Six genes were significantly associated with MCP only in females (NCAN, SPATS2L, TBC1D9, CAMK1D, SOX11, GON4L), while 4 genes were associated only in males (CENPW, MTCH2, NICN1, DNAJA4). Twenty-four genes were identified as significantly asso- ciated with MCP only in the meta-analysis of the sex-stratified GWAS outputs (Fig 3 and S3 Table). Nineteen genes found in previous sex-combined GWAS analyses [17] were not found to be associated with MCP in either sex-stratified GWAS, or in GWAS meta-analysis (Fig 3 and S2 Table). None of the six and four genes associated with MCP in females and males only respectively had a mouse orthologue listed in the Pain Genes Database [50], an interactive web browser listing mouse genes associated with pain-related phenotypes when knocked out. None of the six and four genes associated with MCP in females and males only respectively had a mouse ortholog listed in the Pain Genes Database [50], an interactive web browser of pain-related transgenic knockout studies. Of the twenty-four novel genes found to be associated with MCP in GWAS meta-analyses and associated downstream analyses, one had an ortholog listed in the Pain Genes Database (Gnaq)[51]. Gene expression analysis Tissue-enrichment of MCP-associated gene expression was analysed using FUMA, which implements a MAGMA gene-property analysis [52] using GTEx [53] gene expression datasets to determine association between trait-associated genes and expression in a range of bodily 8 / 27 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 PLOS GENETICS Sex-stratified multisite chronic pain GWAS Fi M ll ifi i f diff i d MCP i d Th b Sex stratified multisite chronic pain G Fig 5. Mouse cell-type-specific expression patterns for sex-differentiated MCP-associated genes. The mousebrain. org database was used to identify cell types of expression in the mouse nervous system (brain neurons and glia have been collapsed to two lines at the base of the plot for clarity) for orthologues of genes identified in this study. Trinarization score (posterior probability of detection in a particular cell type) was used, rather than expression levels. Fig 5. Mouse cell-type-specific expression patterns for sex-differentiated MCP-associated genes. The mousebrain. org database was used to identify cell types of expression in the mouse nervous system (brain neurons and glia have been collapsed to two lines at the base of the plot for clarity) for orthologues of genes identified in this study. Trinarization score (posterior probability of detection in a particular cell type) was used, rather than expression levels. The analysis was carried out for 25 genes selected from the male-specific (n = 14) and female-specific (n = 11; note that Dcc appears in both lists) MCP-associated sets as being enriched for neural tissue expression (neural proportion score > 0.5, S4 and S5 Tables). While most of these show pan-neuronal expression, a few are expressed in mouse glial subpopulations (Cdhr4), primarily in CNS neurons (Gabrb2), primarily in DRG neurons (Amigo3), or in limited subsets of neurons (Dcc, Slc4a10, Camkv). https://doi.org/10.1371/journal.pgen.1009428.g005 https://doi.org/10.1371/journal.pgen.1009428.g005 https://doi.org/10.1371/journal.pgen.1009428.g005 tissues. MCP-associated genes in females were found to be enriched for expression in the brain, particularly the cerebellum, and frontal cortex (Fig 4). There was no significant tissue enrichment for expression of MCP genes found in males (Fig 4). tissues. MCP-associated genes in females were found to be enriched for expression in the brain, particularly the cerebellum, and frontal cortex (Fig 4). There was no significant tissue enrichment for expression of MCP genes found in males (Fig 4). Gene expression analysis PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 9 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS For genes identified (using MAGMA, as reported above) as significantly associated with MCP in either the male-only or female-only GWAS, we carried out further analyses of gene expression at tissue and cell-type level by querying existing transcriptomic data- sets, focusing on neural tissues and tissues specific to each sex. For most tissues GTEx data were used, but because GTEx does not contain data for dorsal root ganglion (DRG) neu- rons, which are key for the generation of the nociceptive signals that initiate pain in chronic pain patients, we assessed expression in this tissue using other comparable data- sets [54], which additionally contained measures of gene expression enrichment in human neural tissues (neural proportion score) and in the DRG (DRG enrichment score) (details in Methods). We also used single cell RNA-seq datasets to estimate whether genes of inter- est are likely to be expressed in neurons in the peripheral or central nervous systems (CNS). Full results of this analysis are given in S4 and S5 Tables, while those for a subset of genes (those enriched for neural tissue expression) are shown in Fig 5. Most of the 37 genes identified from the male-only GWAS were observed to be expressed in the nervous system. Two of the 37 genes, IP6K3 and FAM129A, had low neural proportion scores, sug- gesting that they are non-neuronal and non-glial. All 37 genes, however, showed DRG expression, although expression level was very low for DCC and IP6K3, which were more highly expressed in the CNS. One gene, AMIGO3, was found to be enriched solely in the DRG; its orthologue has also been found to be primarily expressed only in mouse DRG neurons in the www.mousebrain.org dataset (Fig 5). Of the 30 female-specific MCP-asso- ciated genes, all showed high neural expression except CPS1, which was not expressed in neural tissue at all. Again, we noted that all 30 genes except GABRB2 (whose mouse ortho- logue is primarily expressed in CNS neurons, Fig 5) did show expression in DRG (S5 Table), though none of them showed enriched expression in the DRG compared to the CNS. Several genes in either the male-only and female-only lists (e.g. Gene expression analysis CPS1, SEMA3F, MST1, MST1R, SDK1, ECM1; S4 and S5 Tables) that were expressed in neural tissues but with low (< 0.5) neural proportion scores were found to be involved in immune function. Among the genes with high (> 0.5) neural proportion scores, mouse orthologues of many are pan-neuro- nal in expression based on the mousebrain.org dataset (Fig 5), with a few genes having ubiqui- tous but neural-enriched expression (Mrps21, Ip6k1), solely glial expression (Cdhr4), or being expressed in limited neuronal subpopulations including CNS, sensory and enteric neuronal subtypes (Dcc, Camkv, Slc4a10 respectively). Several of the neuronally expressed genes are known to be involved in axon pathfinding and neurite outgrowth. Many of these genes have elevated expression levels in sex-specific tissues like testis and ovary, and / or are somewhat differentially expressed (10% or more difference in median TPMs across sexes) between male and female cohorts in brain sub-regions in the GTEx database (S4 and S5 Tables), suggesting they may be androgen- or estrogen-regulated. Comparing male and female multisite chronic pain Prevalence, coping strategies, and, potentially, mechanisms of development and maintenance of chronic pain vary between the sexes. To explore underlying genetic differences that may contribute to these sex differences in chronic pain, we carried out a large-scale sex-stratified GWAS of a quantitative chronic pain phenotype, MCP. We found both male and female MCP to be moderately heritable. Although the estimated female SNP heritability was higher than that in males (12.5% versus 10.6% respectively), this difference was not significant. Pathway analyses No significant results were found for male MCP-associated genes using FUMA GENE2FUNC [55]. There was no overrepresentation of male MCP-associated genes within any of the Molec- ular Signatures Database (MSigDB) gene sets (h, c1-c7) [56–58]. Two of the male MCP-associ- ated genes were associated with disease-related entries in OMIM (Online Mendelian Inheritance in Man) [59], neither of which mention a pain-related phenotype, and none of the genes were listed as previously identified drug targets in DrugBank [60] (S6 Table). Amongst the female MCP-associated genes, overrepresentation was only found for two of the positional PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 10 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS gene sets listed in MsigDB (S3 Fig). Nineteen female MCP genes were associated with an OMIM entry, and five were listed as drug targets in DrugBank (S7 Table). Polygenic risk score analysis Polygenic risk scores were used to assess whether MCP and chronic widespread pain (CWP), a related but distinct chronic pain phenotype, are likely to be related biologically. For both men and women, the sex-specific PRS-MCP was significantly associated with CWP (p < 2 x 10−17; O.R. = 1.0034 (female) and 1.0026 (male); S8 and S9 Tables), indicating that increased genetic risk for MCP is significantly associated with having chronic widespread pain. MCP-associated SNP loci Twice as many genomic risk loci were identified in GWAS analyses in females as in males (10 versus 5 respectively), with no risk loci shared between the sexes. This may be due to lower sample size in the male MCP GWAS, both in comparison to previous non-stratified analyses and to sample size in the female MCP GWAS analysis (roughly 30,000 more participants are included in the female MCP GWAS than in the male GWAS). Loci were found across the genome (Fig 1 and Table 3) in both males and females, with genomic location varying by sex. We note also, however, that significant loci were found in both men and women on chromo- some 6, only 0.89 Mbp apart (Table 3). Additional trait-associated SNPs were discovered when the sex-stratified GWAS outputs were meta-analysed, likely due to increased power. Twenty-four loci that had not reached genome-wide significance in our previous sex-combined analysis were found to be associated with MCP after meta-analysis. The fact that these loci were not identified in the non-stratified GWAS could be due to effect heterogeneity, in terms either of direction or magnitude, between the sexes reducing the overall signal at these loci. Previous studies have highlighted sex-specific or sex-differentiated loci in a range of disor- ders and traits, such as ASD, anthropometric traits and asthma [31,61–63]. Four of the loci found to be associated with MCP in our meta-analysis of sex-stratified GWAS outputs, includ- ing 7 SNPs in total, showed signs of heterogeneity of effect size between the sexes (I2 p < 0.05), though the evidence was not significant after FDR-adjustment. Nevertheless, if these results are replicated in future studies, it may be that these loci are found to contribute to sex differ- ences in chronic pain. Genes of interest Genes associated with MCP in males. The sex-stratified gene-level analysis discovered a number of genes with significant evidence for a genetic contribution to MCP. Significant genes in males included CENPW, MTCH2, NICN1, AMIGO3, DNAJA4, CTBP2 and NOP14, with the latter two also being significant in the meta-analysis gene-level testing (S2 and S3 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 11 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS Tables). CENPW encodes centromere protein W, involved in kinetochore assembly and func- tion, and associated with diseases such as type 1 diabetes [64–66]. MTCH2 (mitochondrial car- rier 2) encodes a member of the SLC25 family, a family of transporters localised to the inner membrane of mitochondria and involved in a wide range of cell metabolism functions [67]. SNPs in this locus have previously been associated with obesity [68–70], and this gene may be involved in regulation of development of adipocytes. NICN1 (nicolin 1) encodes a nuclear pro- tein of unknown function expressed in a variety of tissues [71]. AMIGO3 (adhesion molecule with Ig-like domain 3), the only male-specific MCP-associated gene whose expression was enriched in DRG, is a member of a small family of cell-surface immunoglobulin domain- and leucine-rich repeat-containing adhesion molecules. Its function is not well understood, but it is expressed in a range of DRG neuronal subtypes and may play a specialized role in nocicep- tion or other sensory modalities. Interestingly, AMIGO3 is located almost next to and within 40 kbp of IP6K1, which was also found to be associated in the male gene-level analysis (S2 Table). It may be that there is coordinate regulation of these two genes in tissues relevant to MCP, or that a number of separate functional variants are distributed across this genomic locus but in fact only influence expression of one of these two genes. DNAJA4 (DnaJ Heat Shock Protein Family (Hsp40) Member A4) encodes a heat shock protein [72] previously shown to be involved in melanoma metastasis and angiogenesis regulation, but is generally poorly characterised [73]. Genes associated with MCP in females. Genes found to be associated with MCP in females included NCAN, SPATS2L, TBC1D9, CAMK1D, SOX11, GON4L, and DAGLB, the last of which was also significantly associated in the meta-analysis. NCAN (neurocan) encodes a chondroitin sulfate proteoglycan [74] potentially involved in the modulation of cell adhesion and migration, and previously linked to bipolar disorder in GWAS and mouse model studies [75,76]. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 Genes of interest SPATS2L (spermatogenesis associated serine rich 2 like) encodes a protein that may be involved in ribosome biogenesis and translational control as a response to oxidative cellular stress [77]. TBC1D9 (TBC1 domain family member 9) encodes a potential GTPase and was found to be overexpressed in mantle cell lymphoma [78]. TBC1D9 was also recently found to be involved in a Ca2+-dependent cellular response to infection [79]. CAMK1D (calcium/cal- modulin dependent protein kinase ID) encodes a member of the calcium/calmodulin-depen- dent protein kinase 1 family involved in granulocyte regulation, activating CREB-dependent gene transcription, the activation and differentiation of neutrophils, promotion of basal den- dritic growth of hippocampal neurons, and apoptosis in erythroleukemia cells [80]. SOX11 (SRY-box transcription factor 11) encodes a member of the SOX (SRY-related HMG-box) family of transcription factors, with potential roles both in nervous system development and in neurogenesis during adulthood [81–85]. De novo mutations in this gene have also been associated with Coffin-Siris syndrome [86]. GON4L (Gon-4 like) encodes a protein involved in transcriptional repression [87,88]. DAGLB (diacylglycerol lipase beta) encodes an enzyme that participates in the endocannabinoid synthesis pathway and is required for axonal growth during development and for retrograde synaptic signalling in mature synapses [89]. Overall, it was notable that genes found to be significantly associated with male and female MCP were largely different. Only one gene, DCC (DCC netrin 1 receptor; a.k.a. deleted in colorectal carcinoma), was associated with both male and female MCP. DCC encodes a recep- tor for the guidance cue netrin 1, and is important for development of the nervous system, par- ticularly the dopaminergic system [90]. Mutations in the DCC gene have been found in those with congenital mirror movement disorder (MRMV-1; [91] and it has also been previously associated with a range of complex brain-related traits, including suicidality, mood instability, intelligence and putamen volume [41,42,92–94]. DCC has also been highlighted as a risk gene PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 12 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS for major depression, and may be involved in the pathology of depression through effects on axon guidance in the developing and adult CNS [95,96]. Gene expression differences in male and female MCP. Genes of interest Gene expression analyses in GTEx (which does not have DRG samples) carried out using FUMA indicated that expression of the female-specific MCP-associated genes was enriched primarily in brain tissue, and this pattern was also seen when meta-analysed sex-stratified GWAS outputs were analysed simi- larly (not shown). Almost all of these genes were also expressed in the human DRG—it is inter- esting to speculate as to whether the role of these genes in initiation or maintenance of chronic pain phenotypes is mediated through roles in the brain or via effects on cells located within the DRG. No significant enrichment for specific tissues was seen in analyses of male MCP-associ- ated genes using GTEx and the human DRG expression profiles. The lack of tissue-enrichment findings for the male-specific genes may, as with the lower number of MCP-associated loci, be due to reduced power resulting from lower sample size in the male GWAS. However, these patterns of tissue-level gene expression in GTEx may also indicate differing gene expression between the sexes, with more ubiquitous expression across all tissues for genes associated with MCP in males, while genes conferring risk in females may tend to have more tissue-specific expression patterns. These expression patterns may also be associated with the fact that the GTEx resource is enriched for male tissue samples (V8 release; 67.1% male)–sex-differential enrichment of certain genes may be conflated with tissue-differential gene enrichment. It is still notable that almost all the male or female MCP-associated genes were found to be expressed in human CNS tissues and in DRG. A subset of these are enriched in human neural tissues and additionally are expressed in mouse neuronal subpopulations when examining sin- gle cell sequencing databases (Fig 4). All of these lines of evidence, together, suggest putative central and peripheral neuronal roles for some of these genes, many of which have not been historically well studied in the field of chronic pain. Genetic correlations. For both males and females, MCP was genetically correlated with non-stratified MCP [17], at rg = 1. In contrast, the genetic correlation between male MCP and female MCP was found to be high but significantly less than 1 (rg = 0.92), with around 8% of common SNP-tagged trait variation therefore not shared between the two traits. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 Genes of interest However, it could be argued that although this figure suggests that a small subset of genetic variation linked to MCP is unique to each sex, this difference is not large enough to consider MCP in the two sexes as biologically distinct to a considerable extent—traits correlated at lower rg are routinely used as proxies for one another in GWAS settings e.g. educational attainment as proxy for intelligence (rg ~ 70%) [92], or current age as a proxy for life span (rg ~40–70%) [97]. g g g y g Genetic correlations with a range of psychiatric disorders, psychological traits and somatic traits and disorders were explored and notable sex-related commonalities and differences were observed. Significant genetic correlations of similar magnitude for both sexes were found between MCP and a range of psychiatric, autoimmune and anthropometric traits. Some phe- notypes were significantly genetically correlated only with female MCP (schizophrenia, rg = 0.13; PGC cross-disorder phenotype, rg = 0.14; PTSD, rg = 0.44; anorexia nervosa, rg = -0.08), while others were significantly genetically correlated only with male MCP (autism spectrum disorder, rg = -0.16; primary biliary cholangitis, rg = 0.14). In most cases, where one sex was significant and the other was not, the rg values for both sexes were not very different, suggest- ing that the underlying biology is only subtly, quantitatively different. The sex differences that were observed may reflect sex-differential patterns of pleiotropy, with some genetic factors contributing differentially to multiple phenotypes in males and females. It would be of interest to investigate the relative effect size of loci contributing to MCP and to schizophrenia and PTSD in the two sexes and the biological mechanisms underlying this difference. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 13 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS Observed genetic correlation differences may also, however, be due to differences in sample size between male and female MCP GWASs (with lower sample size for men compared to women). Differences may also be a result of either men or women being over-represented in the comparison GWAS. For example, in the GWAS meta-analysis of autism spectrum disorder [98] contributing cohorts had M:F ratios from 1.2:1 to as high as 8.6:1, PBC GWASs contribut- ing to the discovery set in the PBC GWAS meta-analysis [99] contained > 90% female cases, and the anorexia nervosa GWAS [100] contained only female cases. Genes of interest Analysis of the cohorts used in the schizophrenia and PTSD GWAS meta-analyses [101–106], suggests that the sex dif- ference in genetic correlation with MCP for these phenotypes is not primarily driven by an overrepresentation of one sex. Comparing the relationship between chronic widespread pain and MCP in males and females Each sex-specific PRS was significantly associated with CWP in the corresponding sex, but the magnitude of association was much lower in comparison to the sex-combined PRS analysis reported previously [17], which may reflect the smaller sample sizes in the sex-stratified analy- ses. As previously found in sex combined analyses, results indicated a moderate degree of shared genetic basis for MCP and chronic widespread pain in both sexes, with degree of shar- ing potentially slightly stronger in females than in males. It is possible that this difference is driven at least in part by the overrepresentation of females in CWP cases (M:F ratio 1:1.74). Clinical perspective on findings and potential impact on treatment Overall, our findings suggest that MCP shows genetic (and therefore biological) differences between men and women. If women with MCP are more likely to be at risk of PTSD and schizophrenia (and vice versa) specific screening for these potential comorbidities could be appropriate, with a view to instigating additional appropriate management or referrals. Except for PTSD, however, these sex-differential genetic correlations were relatively small in compari- son with those that affected both sexes, particularly a range of mood phenotypes. Nevertheless, the shared biology evident in the significant genetic correlations should stimulate investigation of whether screening for these potential clinically important co-morbidities can be used to improve management of chronic pain patients. Enhanced attention to the sex differences in manifestation and underlying biology of chronic pain is also merited, and may lead to improvements in clinical assessment, awareness of risks and choice of medical treatment. For example, if MCP in women is more strongly associated with immune function (based on evidence from MCP-associated genes), efficacy and side-effect profiles for drugs targeting immune system function may be different in women and men. Inappropriate treatments, such as chronic opioid prescribing, might also have sex-differential consequences—opioids are known to adversely affect immune function [107,108]. These sex differences in MCP biology may also inform the search for new or re-pur- posed drugs that can be prescribed in a sex specific manner. It is already known that specific proteins play sex-specific roles in pain processing and that certain drugs have been found to have sex-specific analgesic effects (reviewed by [109]). Limitations These analyses were carried out using UK Biobank, which was used in our previous sex-com- bined MCP GWAS [17]. In comparison to our previous analysis, sample size in each individ- ual sex-stratified GWAS was lower, which leads to somewhat reduced power. However, sample sizes are still larger than many sex-combined GWAS analyses of chronic pain PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 14 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS phenotypes, and meta-analysis of the sex-stratified GWAS outputs resulted in an increase in power to find MCP-associated SNPs overall. True replication is difficult due to heterogeneity in chronic pain phenotyping and available sample sizes of potential independent cohorts, but in an independent subset of the UK Biobank, a PRS constructed from each sex-stratified GWAS output was found to be significantly associated with CWP, a related but distinct chronic pain phenotype of interest. Although this work was focused on sex differences in the genetics of MCP we examined only autosomal variation. An important extension of this work would be to assess genetic asso- ciations with X chromosome loci, which is likely to provide an additional heritability contribu- tion and give a fuller picture of sex differences in MCP at the genetic level. Inclusion of the X chromosome in GWAS analyses is associated with specific methodological and statistical issues including lower quality genotyping array coverage of the X chromosome compared to autosomes, differences in how imputation needs to be implemented, differences in X chromo- some dosage between the sexes leading to differences in population genetics/demographic his- tory of the X chromosome relative to the autosomes, and changes to quality control required (and differing QC protocols between sexes) [110,111]. We aim to address this in future work as we adapt our BOLT-LMM pipeline and downstream analysis pathway. These GWAS analyses were carried out on a white British subset of UK Biobank, and there- fore may not generalise to admixed or non-white populations. GTEx donors are also primarily white (v8 release: 84.6% white, 12.9% African American, 0.2% American Indian, 1.3% Asian, 1.1% Unknown, [https://www.gtexportal.org/home/tissueSummaryPage]), as are donors who provided DRG tissue (white females [54]), again potentially limiting generalisability of our findings to non-white populations. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 Chronic pain phenotyping UK Biobank participants were asked via a touchscreen questionnaire about “pain type(s) expe- rienced in the last month” (field ID 6159), with possible answers: ‘None of the above’; ‘Prefer not to answer’; pain at seven different body sites (head, face, neck/shoulder, back, stomach/ abdomen, hip, knee); or ‘all over the body’. The seven individual body-site pain options were not mutually exclusive, but those who chose ‘all over the body’ could not also select from the seven individual body sites. Where patients reported recent pain at one or more body sites, or all over the body, they were additionally asked (category ID 100048) whether this pain had lasted for 3 months or longer. Chronic Widespread Pain (CWP) was defined as reported [112], and the ‘case’ group included only those participants who answered that they had pain ‘all over the body’ that was longer than 3 months in duration in the touchscreen questionnaire. These individuals were excluded from analyses of other chronic pain phenotypes as there is some evidence that this phenotype can be substantially different from more localised chronic pain [113]. Multisite Chronic Pain (MCP) was a quasi-quantitative variable defined as previously reported [17]; briefly, this variable captures the number of body sites at which chronic pain (at least 3 months duration) was recorded (excluding those with CWP): phenotypic values therefore ranged from 0 to 7. 10,000 randomly selected individuals reporting no chronic pain were excluded from the GWAS to use as controls in subsequent polygenic risk score (PRS) analyses. Conclusions We also observed that several MCP-associated loci in both sexes have been linked to immune function. Overall, our findings indicate the existence of potential sex differences in chronic pain at multiple levels, from SNP-level to transcript abundance and the results support theories of strong nervous system and immune involvement in chronic pain in both sexes. These findings may inform development of novel treatment approaches in future, as well as adding to our understanding of the physiology of chronic pain. Conclusions Sex differences in chronic pain likely have, at least in part, a genetic basis and the study of com- plex traits such as chronic pain is likely to benefit from “sex-aware” analytical approaches. This study comprises one of the largest sex-stratified genetic analyses of a chronic pain phenotype, and highlights sex-differential MCP-associated loci, genes, genetic correlations, and patterns of tissue expression. We also examined transcriptome abundance of key sex-differential MCP- associated genes in a range of neural and non-neural tissues, including DRG, an important nervous system component in chronic pain which is not part of the GTEx resource and so may be understudied in GWASs with follow-up conducted solely using FUMA. Sex-stratified GWASs can provide an increase in power if heterogeneity in effects of trait- associated variants is seen between the sexes. Here, 24 novel genes and 11 novel independent lead SNPs were associated with MCP, in addition to the findings from previous non-sex-strati- fied work, further contributing to understanding of genetic variation predisposing to chronic pain. Genetic correlation results indicated possible sex-differential pleiotropy, including differing genetic correlations between certain psychiatric disorders and traits and chronic pain in women compared to men. However, it is of note that genetic correlations between the sexes are largely similar, particularly with respect to psychiatric disorders and traits including MDD, anhedonia and depressive symptoms. This is the first study to use novel GWAS outputs from studies of suicidality and of RDoC mental health traits such as mood instability and anhedonia in genetic correlation analyses with chronic pain and it provides an important insight into shared genetic factors between these comorbidities of chronic pain and MCP. The patterns of gene expression enrichment associated with the identified predisposing genes supports and enhances our previous conclusion that MCP derives more strongly from PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 15 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS brain and/or CNS-based mechanisms than from other organ systems and functions. We did observe, however, that almost all the sex-specific MCP-associated genes identified are expressed in the DRG as well as in the brain, with one sex differentiated MCP gene, AMIGO3, being DRG-specific amongst the tissues assessed, raising the possibility that cells in the DRG that play a specialised role in nociception or other sensory function might be involved in chronic pain mechanisms. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 BOLT-LMM GWAS and gene-level analysis in FUMA Sex-stratified GWASs of MCP, modelled as a quantitative trait, were carried out using BOLT-LMM [115], adjusting for age and chip (genotyping array), under the infinitesimal model of genetic risk, as previously described for our unstratified MCP GWAS [17]. BOLT-LMM uses a genetic relatedness matrix in the model to adjust for population stratifica- tion and familial relationships. We therefore did not include genetic principal components (GPCs) in the model. The SNP-level GWAS summary statistics were then analysed using FUMA [55] to obtain genome-wide plots and carry out MAGMA [49] gene-set and gene- based test analyses and gene expression analysis using GTEx [53] for male-enriched and female-enriched MCP genes. Significant independent lead SNPs were determined according to FUMA. Briefly, FUMA defines lead SNPs as the subset of independent significant SNPs (SNPs associated with the trait at p < 5 x 10−8 and having LD r2 < 0.6 with any other signifi- cant SNP) that are not in LD (r2 > 0.1) with any other lead SNP [55]. In addition, when these LD blocks of independent significant SNPs are in close proximity (< 250kbp apart), separate loci based on LD thresholds are merged into a single genomic locus, and thus each genomic risk locus can contain multiple lead and independent significant SNPs [55]. Meta-analysis of male and female GWAS Summary statistics. Meta-analysis of the two sex-specific GWAS summary statistics datasets was carried out using METAL [116], deploying a fixed-effects model and weighted by standard error (‘SCHEME STDERR’) with default options aside from selecting ‘heterogeneity’ in order to analyse heterogeneity (‘ANALYZE HETEROGENEITY’). A meta-analysis p-value of < 5 x 10−8 was selected as the significance threshold for association. Gene-level analysis (MAGMA) was also carried out using meta-anal- ysis output. Genetic quality control For the GWAS analyses, SNPs with an imputation quality score of less than 0.3, minor allele frequency (MAF) < 0.01 and/or Hardy-Weinberg equilibrium (HWE) test p < 10−6 were excluded. Participants whose self-reported sex did not match their genetically-determined sex, those who had putative sex-chromosome aneuploidy, those considered outliers in UK Biobank QC in terms of missingness or heterozygosity [114], and those who were not of self-reported white British ancestry were excluded from analyses. A list of “poor quality” samples (due to missingness, putative genetic and reported sex mismatch and/or unexpectedly high heterozy- gosity) was derived by Bycroft et al [114] and is available to all researchers using UK Biobank, and was used here as part of genetic quality control. Briefly, putative sex chromosome aneu- ploidy was defined by visual inspection of scatterplots of mean log2 ratio (L2R) on X and Y chromosomes, and 652 UKB participants meet these criteria for putative sex-chromosome aneuploidy (Supplemental Information S 3.6 [114]). Samples with a heterozygosity value, adjusted for both ancestry and genetic principal components (GPCs), above the mean PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 16 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS Table 4. Number of participants per MCP phenotype level group included in each GWAS (male or female sex- stratified analysis). MCP N female % female N male % male 0 113148 54.11 105474 59.07 1 49984 23.91 42734 23.93 2 26000 12.43 18612 10.42 3 12376 5.92 7771 4.35 4 5319 2.54 2970 1.66 5 1723 0.82 780 0.44 6 471 0.23 181 0.10 7 72 0.03 34 0.02 total n in each GWAS 209093 NA 178556 NA https://doi.org/10.1371/journal.pgen.1009428.t004 Table 4. Number of participants per MCP phenotype level group included in each GWAS (male or female sex- stratified analysis). ber of participants per MCP phenotype level group included in each GWAS (male or female sex- ysis) heterozygosity value (0.1903) and missing rate greater than 0.05 as computed using PLINK ‘— miss’ command were also flagged as potentially poor quality ([114]; 968 such samples are listed in this paper’s Supplemental Information S 3.5.3). heterozygosity value (0.1903) and missing rate greater than 0.05 as computed using PLINK ‘— miss’ command were also flagged as potentially poor quality ([114]; 968 such samples are listed in this paper’s Supplemental Information S 3.5.3). A summary of participant MCP phenotypic information for those included in each GWAS is shown in Table 4. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 Transcriptome analysis of sex-specific association gene lists We further analysed the tissue and cell type of expression of male-specific and female-specific genes identified from the MAGMA gene-based analysis of the sex-stratified GWAS results. Specifically, we characterized gene expression in mammalian nervous system tissues and cell PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 17 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS types as a potential starting point for identifying the functions of these genes with respect to pain. We also characterised expression for the one gene found to be associated with both male and female MCP in the gene-based analyses (DCC). types as a potential starting point for identifying the functions of these genes with respect to pain. We also characterised expression for the one gene found to be associated with both male and female MCP in the gene-based analyses (DCC). RNA-seq-derived gene expression values have been previously reported [54] as relative abundances in TPMs (standardised Transcripts per Million mapped reads) for 12 adult human tissues (6 neural and 6 non-neural). Additionally, the study notes 3 metrics on a scale of 0 to 1 for each gene based on expression in these 12 tissues: normalized Shannon’s entropy as a mea- sure of tissue specificity (0 for highly tissue-specific and 1 for tissue-agnostic gene expression in the quantified tissues), neural proportion score as a measure of enriched expression (possi- bly neuronal and/or glial) in the nervous system (0 for genes not expressed in the nervous sys- tem and 1 for genes expressed solely in neural tissues with respect to these 12 tissues), and DRG enrichment score for identifying specificity of gene expression in the DRG with respect to the other 11 profiled tissues (0 for no expression in the DRG or for tissue-agnostic gene expression, and 1 for DRG-specific gene expression in the context of the set of profiled tissues). The mathematical formulations for these scores are provided in detail in Ray et al. 2018 [54]. The corresponding tables are presented in S4 Table (male) and S5 Table (female). Genes with neural proportion scores > 0.5 (with overall more neural than non-neural tissue expression), were further characterized by the putative cell type(s) of gene expression in the mammalian nervous system (Fig 4). While human single cell resolution RNA-seq datasets are not publicly available for the peripheral nervous system, a comprehensive database of gene expression in the mouse nervous system exists: the www.mousebrain.org repository [117]. Transcriptome analysis of sex-specific association gene lists While it is true that nervous system expression patterns may be different between human and mouse between nervous system subpopulations due to regulatory evolution or due to differ- ences in nervous system cell types [118], it is unlikely that expression would change categories across the 4 broad nervous system cell type categories: neurons, glia, immune and vascular cells) between human and mouse, given overall conservation of tissue gene expression profiles across humans and mice [54]. Trinarization scores, defined as the posterior probability (using a Bayesian framework) of detecting reads from a particular gene in a cell type subpopulation (details are given in ref. [117]), were used to characterise gene expression by cell-type. Fig 4 visualizes trinarization scores for a range of peripheral nervous system cell types (sensory neu- rons and glia, enteric neurons and glia, and sympathetic neurons). While peripheral nervous system vascular and immune cells have not been profiled so far in www.mousebrain.org, we depict CNS vascular immune cells and vascular cells as surrogate cell types for their PNS coun- terparts. Both CNS neurons and glia play a critical role in chronic pain, but due to high diver- sity of cell types were not ideal for summarizing expression patterns in each subpopulation of these categories succinctly in a single figure. Instead, summary rows for CNS neurons and glia expression for relevant genes are provided at the bottom of the figure. For further details of expression in these subtypes, www.mousebrain.org can be queried for mouse gene expression profiles. Finally, for genes with neural proportion scores > 0.5, the GTEx database was queried for sex differential gene expression in profiled CNS regions, and for high expression in sex-specific tissues, such as testis and ovary, noted in the comments section of S4 and S5 Tables. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 Polygenic risk score analysis Previous analyses showed that a polygenic risk score (PRS) for MCP was associated with the phenotype of chronic widespread pain (CWP) in women but not in men, and that this PRS was associated more strongly with chronic pain phenotypes in women than in men in an inde- pendent cohort (Johnston K.J.A. et al., unpublished). To further explore the relationship between MCP and CWP, we assessed how separate male and female PRSs for MCP were asso- ciated with CWP. Separate sex-specific PRSs for MCP, based on the sex-specific GWAS results reported here, were calculated for a ‘case’ group consisting of participants who reported pain all over the body that lasted for three months or longer (a proxy phenotype for Chronic Wide- spread Pain; CWP; N = 6, 813)), and for a ‘control’ group consisting of 10,000 randomly selected UKB controls, both of which had been excluded from the GWAS analyses (demo- graphic data for this subsample are given in Table 5). SNPs associated with MCP at p < 0.01 in the original sex-specific GWAS were selected and LD-pruned (at a threshold of r2 < 0.1 within a 250kbp window using the PLINK ‘—clump’ command). Sex-specific PRS-MCPs were calculated for each individual in the analysis as the sum of risk alleles at each SNP, weighted by effect size (beta value) in the GWAS [122]. PRS values were standardised and z-scores were used in the analysis. Association between stan- dardised sex-specific PRS-MCP and CWP status was investigated separately in males and in females in the target case-control subsample using logistic regression, adjusted for chip (geno- typing array), age and the first eight genetic principal components. Genetic correlations Genetic correlations with a range of neuropsychiatric disorders and traits were assessed by LD-score regression (LDSR) for the male and female MCP GWAS outputs separately. Sum- mary statistics datasets employed were publicly available or available via LD Hub [119,120], or were results from published and unpublished in-house GWASs. Pre-computed LD scores PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 18 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS Table 5. Summary of sample sizes of participants used in each of the two PRS analyses (male or female). Female Male Combined Totals N mean age (years) N mean age N mean age Control 5135 56.68 4865 56.96 10000 56.81 CWP 4328 57.00 2485 57.27 6813 57.10 Total 9463 56.83 7350 57.07 16813 56.93 https://doi.org/10.1371/journal.pgen.1009428.t005 Summary of sample sizes of participants used in each of the two PRS analyses (male or female). were used, along with HapMap3 SNPs as a reference. By default the munging part of LDSR fil- ters out SNPs with an info score of < 0.9, a MAF of < 0.01, a GWAS p value outside the range 0–1, strand-ambiguous SNPs, non-SNP (e.g. indel) variants, and SNPs with low sample size. The default option of no constraint on the LDSR intercept was used. LDSR p-values for genetic correlation were FDR-corrected within each sex. LDSR was also used to assess trait polygeni- city and to calculate a SNP-heritability estimate. Note that although some of the GWAS results used in this analysis came from studies that included UK Biobank data, genetic correlations estimated using LDSR are not subject to bias caused by sample overlap [121]. were used, along with HapMap3 SNPs as a reference. By default the munging part of LDSR fil- ters out SNPs with an info score of < 0.9, a MAF of < 0.01, a GWAS p value outside the range 0–1, strand-ambiguous SNPs, non-SNP (e.g. indel) variants, and SNPs with low sample size. The default option of no constraint on the LDSR intercept was used. LDSR p-values for genetic correlation were FDR-corrected within each sex. LDSR was also used to assess trait polygeni- city and to calculate a SNP-heritability estimate. Note that although some of the GWAS results used in this analysis came from studies that included UK Biobank data, genetic correlations estimated using LDSR are not subject to bias caused by sample overlap [121]. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 S6 Table. OMIM and DrugBank derived information (male MCP). (PDF) S6 Table. OMIM and DrugBank derived information (male MCP). (PDF) S7 Table. OMIM and DrugBank derived information (female MCP). (PDF) PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 Supporting information S1 Table. Genetic correlations between MCP and other disorders and traits. Results of LDSR analysis using summary statistics from the sex-stratified GWASs of MCP versus a range of potentially related disorders and traits. Genetic correlations are given as rg values (and FDR-corrected p-values) sorted in order of numerically decreasing rg for female MCP vs other traits. f_rg and m_rg = genetic correlation value for female and male MCP versus trait, respec- tively, f_p_fdr and m_p_fdr = FDR-corrected p value for genetic correlation, source = source of trait GWAS data, PMID = PubMed ID of associated publication for GWAS of trait. Signifi- cant genetic correlations (FDR-corrected p value < 0.05) within each sex are highlighted PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 19 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS orange, non-significant in blue. (PDF) S2 Table. Table accompanying Venn diagram of genes associated with multisite chronic pain in MAGMA gene-level analyses. ‘Found in’ refers to the GWAS and corresponding MAGMA gene-level analyses where genes were found to be significantly associated with MCP: Female = sex-stratified GWAS (female), Male = sex-stratified GWAS (male), Meta = GWAS meta-analysis of male and female sex-stratified GWAS outputs, Original = sex-combined GWAS analysis described previously [17]. Total = total number of genes in category. Elements = gene names. (PDF) S3 Table. Genes associated with MCP in MAGMA gene-level analyses of GWAS meta-anal- ysis output. (PDF) S4 Table Expression of genes associated with male MCP in MAGMA analyses across neural and non-neural tissues TPM = transcripts per million, DRG = dorsal root ganglion h_DRG_enrich = DRG enrichment score h_entropy = normalized Shannon’s entropy h_neural_propn = neural proportion score. (PDF) S5 Table. Expression of genes associated with female MCP in MAGMA analyses across neural and non-neural tissues. TPM = transcripts per million, DRG = dorsal root ganglion. h_DRG_enrich = DRG enrichment score. h_entropy = normalized Shannon’s entropy. h_neural_propn = neural proportion score. (PDF) S6 Table. OMIM and DrugBank derived information (male MCP). (PDF) S7 Table. OMIM and DrugBank derived information (female MCP). (PDF) S8 Table. Association between male MCP PRS and CWP in men. Full results (chip, PCs) not shown for brevity. SE = standard error, Z = Z value, P = p value, OR = odds ratio, PRS = z- standardised PRS value. (PDF) S9 Table. Association between female MCP PRS and CWP in women. Full results (chip, PCs) not shown for brevity. Supporting information SE = standard error, Z = Z value, P = p value, OR = odds ratio, PRS = z-standardised PRS value. (PDF) S1 Fig. Tissue Expression of MCP-associated Genes (male) (53 tissues). (PDF) S2 Fig. Tissue Expression of MCP-associated Genes (female) (53 tissues). (PDF) S3 Fig. FUMA gene set analysis results for female MCP genes from the gene-level analysis. (TIFF) ( ) S2 Table. Table accompanying Venn diagram of genes associated with multisite chronic pain in MAGMA gene-level analyses. ‘Found in’ refers to the GWAS and corresponding MAGMA gene-level analyses where genes were found to be significantly associated with MCP: Female = sex-stratified GWAS (female), Male = sex-stratified GWAS (male), Meta = GWAS meta-analysis of male and female sex-stratified GWAS outputs, Original = sex-combined GWAS analysis described previously [17]. Total = total number of genes in category. Elements = gene names. (PDF) S3 Table. Genes associated with MCP in MAGMA gene-level analyses of GWAS meta-anal- ysis output. (PDF) S3 Table. Genes associated with MCP in MAGMA gene-level analyses of GWAS meta-anal- ysis output. (PDF) S4 Table Expression of genes associated with male MCP in MAGMA analyses across neural and non-neural tissues TPM = transcripts per million, DRG = dorsal root ganglion h_DRG_enrich = DRG enrichment score h_entropy = normalized Shannon’s entropy h_neural_propn = neural proportion score. (PDF) S4 Table Expression of genes associated with male MCP in MAGMA analyses across neural and non-neural tissues TPM = transcripts per million, DRG = dorsal root ganglion h_DRG_enrich = DRG enrichment score h_entropy = normalized Shannon’s entropy h_neural_propn = neural proportion score. (PDF) S5 Table. Expression of genes associated with female MCP in MAGMA analyses across neural and non-neural tissues. TPM = transcripts per million, DRG = dorsal root ganglion. h_DRG_enrich = DRG enrichment score. h_entropy = normalized Shannon’s entropy. h_neural_propn = neural proportion score. (PDF) S7 Table. OMIM and DrugBank derived information (female MCP). (PDF) S8 Table. Association between male MCP PRS and CWP in men. Full results (chip, PCs) not shown for brevity. SE = standard error, Z = Z value, P = p value, OR = odds ratio, PRS = z- standardised PRS value. (PDF) S9 Table. Association between female MCP PRS and CWP in women. Full results (chip, PCs) not shown for brevity. SE = standard error, Z = Z value, P = p value, OR = odds ratio, PRS = z-standardised PRS value. (PDF) S1 Fig. Tissue Expression of MCP-associated Genes (male) (53 tissues). (PDF) S2 Fig. Tissue Expression of MCP-associated Genes (female) (53 tissues). (PDF) S3 Fig. FUMA gene set analysis results for female MCP genes from the gene-level analysis. (TIFF) PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 20 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS Author Contributions Conceptualization: Keira J. A. Johnston, Daniel J. Smith, Mark E. S. Bailey. Conceptualization: Keira J. A. Johnston, Daniel J. Smith, Mark E. S. Bailey. Data curation: Joey Ward, Pradipta R. Ray. Data curation: Joey Ward, Pradipta R. Ray. Formal analysis: Keira J. A. Johnston, Joey Ward, Pradipta R. Ray, Theodore J. Price. Supervision: Mark J. Adams, Andrew M. McIntosh, Daniel J. Smith, Barbara I. Nicholl, Mark E. S. Bailey. Writing – original draft: Keira J. A. Johnston, Pradipta R. Ray, Blair H. Smith, Theodore J. Price. Writing – review & editing: Keira J. A. Johnston, Joey Ward, Pradipta R. Ray, Mark J. Adams, Andrew M. McIntosh, Blair H. Smith, Rona J. Strawbridge, Theodore J. Price, Daniel J. Smith, Barbara I. Nicholl, Mark E. S. Bailey. Acknowledgments We thank participants in the UK Biobank study. UK Biobank was established by the Wellcome Trust, Medical Research Council, Department of Health, Scottish Government and Northwest Regional Development Agency. This work was carried out under approved UK Biobank appli- cations 6553 and 7155. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 References Eur J Pain (United Kingdom). 2012; 16(7):1053–63. https://doi.org/10.1002/j.1532- 2149.2011.00095.x PMID: 22337623 13. Junqueira DRG, Ferreira ML, Refshauge K, Maher CG, Hopper JL, Hancock M, et al. Heritability and lifestyle factors in chronic low back pain: Results of the Australian Twin Low Back Pain Study (The AUTBACK study). Eur J Pain (United Kingdom). 2014; 18(10):1410–8. https://doi.org/10.1002/ejp.506 PMID: 24733726 14. Williams FMK, Spector TD, MacGregor AJ. Pain reporting at different body sites is explained by a sin- gle underlying genetic factor. Rheumatology. 2010; 49(9):1753–5. https://doi.org/10.1093/ rheumatology/keq170 PMID: 20525736 15. McIntosh AM, Hall LS, Zeng Y, Adams MJ, Gibson J, Wigmore E, et al. Genetic and Environmental Risk for Chronic Pain and the Contribution of Risk Variants for Major Depressive Disorder: A Family- Based Mixed-Model Analysis. PLoS Med. 2016; 13(8):1–17. https://doi.org/10.1371/journal.pmed. 1002090 PMID: 27529168 16. Suri P, Palmer MR, Tsepilov YA, Freidin MB, Boer CG, Yau MS, et al. Genome-wide meta-analysis of 158,000 individuals of European ancestry identifies three loci associated with chronic back pain. 2018; 48(7):1–23. 17. Johnston KJA, Adams MJ, Nicholl BI, Ward J, Strawbridge RJ, Ferguson A, et al. Genome-wide asso- ciation study of multisite chronic pain in UK Biobank. PLoS Genet. 2019; 15(6):1–22. https://doi.org/ 10.1371/journal.pgen.1008164 PMID: 31194737 18. Rawlik K, Canela-Xandri O, Tenesa A. Evidence for sex-specific genetic architectures across a spec- trum of human complex traits. Genome Biol [Internet]. 2016; 17(1):1–8. Available from: http://dx.doi. org/10.1186/s13059-016-1025-x PMID: 27473438 19. Khramtsova EA, Davis LK, Stranger BE. The role of sex in the genomics of human complex traits. Nat Rev Genet [Internet]. 2019; 20(3):173–90. Available from: https://doi.org/10.1038/s41576-018-0083-1 PMID: 30581192 20. Xu X, Coats JK, Yang CF, Wang A, Ahmed OM, Alvarado M, et al. Modular genetic control of sexually dimorphic behaviors. Cell [Internet]. 2012; 148(3):596–607. Available from: https://doi.org/10.1016/j. cell.2011.12.018 PMID: 22304924 21. Quinn MA, Cidlowski JA. Endogenous hepatic glucocorticoid receptor signaling coordinates sex- biased inflammatory gene expression. FASEB J. 2016; 30(2):971–82. https://doi.org/10.1096/fj.15- 278309 PMID: 26581598 22. McCormick H, Young PE, Hur SSJ, Booher K, Chung H, Cropley JE, et al. Isogenic mice exhibit sexu- ally-dimorphic DNA methylation patterns across multiple tissues. BMC Genomics. 2017; 18(1):1–9. https://doi.org/10.1186/s12864-016-3406-7 PMID: 28049423 23. Gilks WP, Abbott JK, Morrow EH. Sex differences in disease genetics: Evidence, evolution, and detec- tion. Trends Genet [Internet]. 2014; 30(10):453–63. Available from: https://doi.org/10.1016/j.tig.2014. 08.006 PMID: 25239223 24. Ge T, Chen CY, Neale BM, Sabuncu MR, Smoller JW. Phenome-wide heritability analysis of the UK Biobank. PLoS Genet. 2017; 13(4):1–21. References 1. Merskey H, Bogduk N. Classification of Chronic Pain. IASP Pain Terminology. 1994. 240 p. 2. IASP. International Association for the Study of Pain [Internet]. [cited 2020 Apr 14]. Available from: https://www.iasp-pain.org/ 3. Nicholasa M, Vlaeyenb JWS, Riefe W, Barkee A, Azizf Q, Benolielg R, et al. The IASP classification of chronic pain for ICD-11: Chronic primary pain. Pain. 2019; 160(1):53–9. https://doi.org/10.1097/j.pain. 0000000000001365 PMID: 30586071 4. Gureje O, Von Korff M, Kola L, Demyttenaere K, He Y, Posada-Villa J, et al. The relation between mul- tiple pains and mental disorders: Results from the World Mental Health Surveys. Pain. 2008; 135(1– 2):82–91. https://doi.org/10.1016/j.pain.2007.05.005 PMID: 17570586 5. Von Korff M, Crane P, Lane M, Miglioretti DL, Simon G, Saunders K, et al. Chronic spinal pain and physical-mental comorbidity in the United States: Results from the national comorbidity survey replica- tion. Pain. 2005; 113(3):331–9. https://doi.org/10.1016/j.pain.2004.11.010 PMID: 15661441 6. Santos-Eggimann B, Wietlisbach V, Rickenbach M, Paccaud F, Gutzwiller F. One-year prevalence of low back pain in two Swiss regions. Estimates from the population participating in the 1992–1993 MONICA project. Spine (Phila Pa 1976). 2000; 25(19):2473–9. https://doi.org/10.1097/00007632- 200010010-00009 PMID: 11013499 7. Palmer KT, Walsh K, Bendall H, Cooper C, Coggon D. Back pain in Britain: Comparison of two preva- lence surveys at an interval of 10 years. Br Med J. 2000; 320(7249):1577–8. https://doi.org/10.1136/ bmj.320.7249.1577 PMID: 10845966 8. Goldberg DS, McGee SJ. Pain as a global public health priority. BMC Public Health [Internet]. 2011; 11(1):770. Available from: http://www.biomedcentral.com/1471-2458/11/770 https://doi.org/10.1186/ 1471-2458-11-770 PMID: 21978149 9. Breivik H, Collett B, Ventafridda V, Cohen R, Gallacher D. Survey of chronic pain in Europe: Preva- lence, impact on daily life, and treatment. Eur J Pain. 2006; 10(4):287–333. https://doi.org/10.1016/j. ejpain.2005.06.009 PMID: 16095934 10. James SL, Abate D, Abate KH, Abay SM, Abbafati C, Abbasi N, et al. Global, regional, and national incidence, prevalence, and years lived with disability for 354 Diseases and Injuries for 195 countries and territories, 1990–2017: A systematic analysis for the Global Burden of Disease Study 2017. Lan- cet. 2018;1789–858. https://doi.org/10.1016/S0140-6736(18)32279-7 PMID: 30496104 11. Von Korff M, Ormel J, Keefe FJ, Dworkin SF. Grading the severity of chronic pain. Pain [Internet]. 1992; 50(1092):133–49. Available from: http://www.ncbi.nlm.nih.gov/pubmed/1408309 https://doi.org/ 10.1016/0304-3959(92)90154-4 PMID: 1408309 21 / 27 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 PLOS GENETICS Sex-stratified multisite chronic pain GWAS 12. Hocking LJ, Morris AD, Dominiczak AF, Porteous DJ, Smith BH. Heritability of chronic pain in 2195 extended families. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 References https://doi.org/10.1371/journal.pgen.1006711 PMID: 28388634 25. Rahmioglu N, MacGregor S, Drong AW, Hedman ÅK, Harris HR, Randall JC, et al. Genome-wide enrichment analysis between endometriosis and obesity-related traits reveals novel susceptibility loci. Hum Mol Genet. 2015; 24(4):1185–99. https://doi.org/10.1093/hmg/ddu516 PMID: 25296917 26. Hall E, Volkov P, Dayeh T, Esguerra JL o. S, Salo¨ S, Eliasson L, et al. Sex differences in the genome- wide DNA methylation pattern and impact on gene expression, microRNA levels and insulin secretion in human pancreatic islets. Genome Biol. 2014; 15(12):522. https://doi.org/10.1186/s13059-014- 0522-z PMID: 25517766 27. Kukurba KR, Parsana P, Balliu B, Smith KS, Zappala Z, Knowles DA, et al. Impact of the X chromo- some and sex on regulatory variation. Genome Res. 2016; 26(6):768–77. https://doi.org/10.1101/gr. 197897.115 PMID: 27197214 28. Yao C, Joehanes R, Johnson AD, Huan T, Esko T, Ying S, et al. Sex- and age-interacting eQTLs in human complex diseases. Hum Mol Genet [Internet]. 2013 Nov 15; 23(7):1947–56. Available from: https://doi.org/10.1093/hmg/ddt582 PMID: 24242183 29. Ko´sa JP, Balla B, Speer G, Kiss J, Borsy A, Podani J, et al. Effect of menopause on gene expression pattern in bone tissue of nonosteoporotic women. Menopause. 2009; 16(2):367–77. https://doi.org/10. 1097/gme.0b013e318188b260 PMID: 19512969 30. Gomez-Santos C, Hernandez-Morante JJ, Margareto J, Larrarte E, Formiguera X, Martı´nez CM, et al. Profile of adipose tissue gene expression in premenopausal and postmenopausal women: Site- 22 / 27 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 PLOS GENETICS Sex-stratified multisite chronic pain GWAS specific differences. Menopause. 2011; 18(6):675–84. https://doi.org/10.1097/gme. 0b013e31820641da PMID: 21358552 specific differences. Menopause. 2011; 18(6):675–84. https://doi.org/10.1097/gme. 0b013e31820641da PMID: 21358552 31. Mitra I, Tsang K, Ladd-Acosta C, Croen LA, Aldinger KA, Hendren RL, et al. Pleiotropic Mechanisms Indicated for Sex Differences in Autism. PLoS Genet. 2016; 12(11):1–27. https://doi.org/10.1371/ journal.pgen.1006425 PMID: 27846226 32. Bartley EJ, Fillingim RB. Sex differences in pain: A brief review of clinical and experimental findings. Br J Anaesth. 2013; 111(1):52–8. https://doi.org/10.1093/bja/aet127 PMID: 23794645 33. Fillingim RB, King CD, Ribeiro-Dasilva MC, Rahim-Williams B, Riley JL. Sex, Gender, and Pain: A Review of Recent Clinical and Experimental Findings. J Pain [Internet]. 2009; 10(5):447–85. Available from: https://doi.org/10.1016/j.jpain.2008.12.001 PMID: 19411059 34. Fillingim RB. Biopsychosocial contributions to sex differences in pain. BJOG An Int J Obstet Gynaecol. 2015; 122(6):769. https://doi.org/10.1111/1471-0528.13337 PMID: 25752330 35. El-Shormilisy N, Strong J, Meredith PJ. Associations among gender, coping patterns and functioning for individuals with chronic pain: A systematic review. Pain Res Manag. 2015; 20(1):48–55. https://doi. org/10.1155/2015/490610 PMID: 24927488 36. References Sorge RE, Mapplebeck JCS, Rosen S, Beggs S, Taves S, Alexander JK, et al. Different immune cells mediate mechanical pain hypersensitivity in male and female mice. Nat Neurosci. 2015; 18(8):1081–3. https://doi.org/10.1038/nn.4053 PMID: 26120961 37. Sorge RE, Totsch SK. Review Sex Differences in Pain. 2017; 1281(February 2016):1271–81. 7. Sorge RE, Totsch SK. Review Sex Differences in Pai 38. Agalave NM, Rudjito R, Farinotti AB, Khoonsari PE, Sandor K, Nomura Y, et al. Sex-dependent role of microglia in disulfide HMGB1-mediated mechanical hypersensitivity. Pain. 2020;Articles i. 39. Klein SL, Flanagan KL. Sex differences in immune responses. Nat Rev Immunol. 2016; 16(10):626– 38. https://doi.org/10.1038/nri.2016.90 PMID: 27546235 40. Smith BH, Campbell A, Linksted P, Fitzpatrick B, Jackson C, Kerr SM, et al. Cohort profile: Generation scotland: Scottish family health study (GS: SFHS). The study, its participants and their potential for genetic research on health and illness. Int J Epidemiol. 2013; 41. Strawbridge RJ, Ward J, Ferguson A, Graham N, Shaw RJ, Cullen B, et al. Identification of novel genome-wide associations for suicidality in UK Biobank, genetic correlation with psychiatric disorders and polygenic association with completed suicide. EBioMedicine [Internet]. 2019; 41:517–25. Avail- able from: https://doi.org/10.1016/j.ebiom.2019.02.005 PMID: 30745170 42. Ward J, Tunbridge EM, Sandor C, Lyall LM, Ferguson A, Strawbridge RJ, et al. The genomic basis of mood instability: identification of 46 loci in 363,705 UK Biobank participants, genetic correlation with psychiatric disorders, and association with gene expression and function. Mol Psychiatry [Internet]. 2019; Available from: http://dx.doi.org/10.1038/s41380-019-0439-8 43. Okifuji A, Benham B. Suicidal and Self-Harm Behaviors in Chronic. J Appl Biobehav Res. 2011; 16 (2):57–77. 44. Nock MK, Borges G, Bromet EJ, Cha CB, Kessler RC, Lee S. Suicide and Suicidal Behavior Matthew. Epidemiol Rev. 2008; 30(1):133–54. 45. Cheatle MD. Depression, chronic pain, and suicide by overdose: On the edge. Pain Med. 2011; 12 (SUPPL. 2):43–8. 46. Tang NKY, Crane C. Suicidality in chronic pain: A review of the prevalence, risk factors and psycholog- ical links. Psychol Med. 2006; 36(5):575–86. https://doi.org/10.1017/S0033291705006859 PMID: 16420727 47. Triñanes Y, Gonza´lez-Villar A, Go´mez-Perretta C, Carrillo-de-la-Peña MT. Suicidality in Chronic Pain: Predictors of Suicidal Ideation in Fibromyalgia. Pain Pract. 2015; 15(4):323–32. https://doi.org/10. 1111/papr.12186 PMID: 24690160 48. Smith MT, Edwards RR, Robinson RC, Dworkin RH. Suicidal ideation, plans, and attempts in chronic pain patients: Factors associated with increased risk. Pain. 2004; 111(1–2):201–8. https://doi.org/10. 1016/j.pain.2004.06.016 PMID: 15327824 49. de Leeuw CA, Mooij JM, Heskes T, Posthuma D. MAGMA: Generalized Gene-Set Analysis of GWAS Data. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 References PLoS Comput Biol. 2015; 11(4). https://doi.org/10.1371/journal.pcbi.1004219 PMID: 25885710 50. LaCroix-Fralish ML, Ledoux JB, Mogil JS. The Pain Genes Database: An interactive web browser of pain-related transgenic knockout studies. Pain. 2007; 131(1–2):3.e1–3.e4. https://doi.org/10.1016/j. pain.2007.04.041 PMID: 17574758 51. Tappe-Theodor A, Constantin CE, Tegeder I, Lechner SG, Langeslag M, Lepcynzsky P, et al. Gα q/11 signaling tonically modulates nociceptor function and contributes to activity-dependent sensitization. Pain [Internet]. 2012; 153(1):184–96. Available from: https://doi.org/10.1016/j.pain.2011.10.014 PMID: 22071319 23 / 27 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 PLOS GENETICS Sex-stratified multisite chronic pain GWAS 52. Watanabe K, Taskesen E, Van Bochoven A, Posthuma D. Functional mapping and annotation of genetic associations with FUMA. Nat Commun. 2017; 8(1):1–10. https://doi.org/10.1038/s41467-016- 0009-6 PMID: 28232747 53. Aguet F, Brown AA, Castel SE, Davis JR, He Y, Jo B, et al. Genetic effects on gene expression across human tissues. Nature. 2017; 550(7675):204–13. https://doi.org/10.1038/nature24277 PMID: 29022597 54. Ray P, Torck A, Quigley L, Wangzhou A, Neiman M, Rao C, et al. Comparative transcriptome profiling of the human and mouse dorsal root ganglia. Pain. 2018; 159(7):1325–45. https://doi.org/10.1097/j. pain.0000000000001217 PMID: 29561359 55. Watanabe K, Taskesen E, Van Bochoven A, Posthuma D. Functional mapping and annotation of genetic associations with FUMA. Nat Commun [Internet]. 2017; 8(1):1–10. Available from: https://doi. org/10.1038/s41467-016-0009-6 PMID: 28232747 56. Liberzon A, Birger C, Ghandi M, Jill P, Tamayo P, Jolla L, et al. The Molecular Signatures Database (MSigDB) hallmark gene set collection. Cell Syst. 2015; 1(6):417–25. https://doi.org/10.1016/j.cels. 2015.12.004 PMID: 26771021 57. Liberzon A, Subramanian A, Pinchback R, Thorvaldsdo´ttir H, Tamayo P, Mesirov JP. Molecular signa- tures database (MSigDB) 3.0. Bioinformatics. 2011; 27(12):1739–40. https://doi.org/10.1093/ bioinformatics/btr260 PMID: 21546393 58. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrich- ment analysis: A knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005; 102(43):15545–50. https://doi.org/10.1073/pnas.0506580102 PMID: 16199517 59. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore M. Online Men- delian Inheritance in Man, OMIM [Internet]. [cited 2020 Oct 7]. Available from: omim.org 60. Wishart DS, Knox C, Guo AC, Shrivastava S, Hassanali M, Stothard P, et al. DrugBank: a comprehen- sive resource for in silico drug discovery and exploration. Nucleic Acids Res. 2006; 34(Database issue):668–72. https://doi.org/10.1093/nar/gkj067 PMID: 16381955 61. Winkler TW, Justice AE, Graff M, Barata L, Feitosa MF, Chu S, et al. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 References Cloning and characterization of the mam- malian-specific nicolin 1 gene (NICN1) encoding a nuclear 24 kDA protein. Eur J Biochem. 2002; 269 (21):5240–5. https://doi.org/10.1046/j.1432-1033.2002.03232.x PMID: 12392556 72. Alderson TRR, Kim JHH, Markley JLL. Dynamical Structures of Hsp70 and Hsp70-Hsp40 Complexes. Structure [Internet]. 2016; 24(7):1014–30. Available from: https://doi.org/10.1016/j.str.2016.05.011 PMID: 27345933 73. Pencheva N, Tran H, Buss C, Huh D, Drobnjak M, Busam K, et al. Convergent multi-mRNA Targeting of ApoE Drives LRP1/LRP8-Dependent Melanoma Metastasis and Angiogenesis. Cell. 2012; 151 (5):1068–82. https://doi.org/10.1016/j.cell.2012.10.028 PMID: 23142051 74. Rauch U, Karthikeyan L, Maurel P, Margolis RU, Margolis RK. Cloning and primary structure of neuro- can, a developmentally regulated, aggregating chondroitin sulfate proteoglycan of brain. J Biol Chem. 1992; 267(27):19536–47. PMID: 1326557 75. Cichon S, Mu¨hleisen TW, Degenhardt FA, Mattheisen M, Miro´ X, Strohmaier J, et al. Genome-wide association study identifies genetic variation in neurocan as a susceptibility factor for bipolar disorder. Am J Hum Genet. 2011; 88(3):372–81. https://doi.org/10.1016/j.ajhg.2011.01.017 PMID: 21353194 76. Miro´ X, Meier S, Dreisow ML, Frank J, Strohmaier J, Breuer R, et al. Studies in humans and mice impli- cate neurocan in the etiology of mania. Am J Psychiatry. 2012; 169(9):982–90. https://doi.org/10. 1176/appi.ajp.2012.11101585 PMID: 22952076 77. Zhu CH, Kim J, Shay JW, Wright WE. SGNP: An essential stress granule/nucleolar protein potentially involved in 5.8s rRNA processing/transport. PLoS One. 2008; 3(11). https://doi.org/10.1371/journal. pone.0003716 PMID: 19005571 78. Islam TC, Asplund AC, Lindvall JM, Nygren L, Liden J, Kimby E, et al. High level cannabinoid receptor 1, resistance of regulator G protein signaling 13 and differential expression of Cyclin D1 in mantle cell lymphoma. Leukemia. 2003; 17(9):1880–90. https://doi.org/10.1038/sj.leu.2403057 PMID: 12970790 79. Nozawa T, Sano S, Minowa-Nozawa A, Toh H, Nakajima S, Murase K, et al. TBC1D9 regulates TBK1 activation through Ca2+ signaling in selective autophagy. Nat Commun [Internet]. 2020; 11(1):1–16. Available from: https://doi.org/10.1038/s41467-019-13993-7 PMID: 31911652 80. Verploegen S, Lammers JWJ, Koenderman L, Coffer PJ. Identification and characterization of CKLiK, a novel granulocyte Ca++/calmodulin-dependent kinase. Blood. 2000; 96(9):3215–23. PMID: 11050006 81. Bhattaram P, Penzo-Me´ndez A, Sock E, Colmenares C, Kaneko KJ, Vassilev A, et al. Organogenesis relies on SoxC transcription factors for the survival of neural and mesenchymal progenitors. Nat Com- mun. 2010; 1(1):1–12. https://doi.org/10.1038/ncomms1008 PMID: 20596238 82. Jay P, Goze´ C, Marsollier C, Taviaux S, Hardelin JP, Koopman P, et al. The human SOX11 Gene: Cloning, chromosomal assignment and tissue expression. Genomics. 1995; 29(2):541–5. https://doi. org/10.1006/geno.1995.9970 PMID: 8666406 83. References The Influence of Age and Sex on Genetic Associations with Adult Body Size and Shape: A Large-Scale Genome-Wide Interaction Study. Vol. 11, PLoS Genetics. 2015. 1–42 p. https://doi.org/10.1371/journal.pgen.1005378 PMID: 26426971 62. Randall JC, Winkler TW, Kutalik Z, Berndt SI, Jackson AU, Monda KL, et al. Sex-stratified Genome- wide Association Studies Including 270,000 Individuals Show Sexual Dimorphism in Genetic Loci for Anthropometric Traits. PLoS Genet. 2013; 9(6). https://doi.org/10.1371/journal.pgen.1003500 PMID: 23754948 63. Myers RA, Scott NM, Gauderman WJ, Qiu W, Mathias RA, Romieu I, et al. Genome-wide interaction studies reveal sex-specific asthma risk alleles. Hum Mol Genet. 2014; 23(19):5251–9. https://doi.org/ 10.1093/hmg/ddu222 PMID: 24824216 64. Prendergast L, van Vuuren C, Kaczmarczyk A, Doering V, Hellwig D, Quinn N, et al. Premitotic Assembly of Human CENPs -T and -W switches centromeric Chromatin to a mitotic state. PLoS Biol. 2011; 9(6):1–12. https://doi.org/10.1371/journal.pbio.1001082 PMID: 21695110 65. Lee S, Gang J, Jeon SB, Choo SH, Lee B, Kim YG, et al. Molecular cloning and functional analysis of a novel oncogene, cancer-upregulated gene 2 (CUG2). Biochem Biophys Res Commun. 2007; 360 (3):633–9. https://doi.org/10.1016/j.bbrc.2007.06.102 PMID: 17610844 66. Chun Y, Park B, Koh W, Lee S, Cheon Y, Kim R, et al. New centromeric component CENP-W Is an RNA-associated nuclear matrix protein that interacts with nucleophosmin/B23 protein. J Biol Chem. 2011; 286(49):42758–69. https://doi.org/10.1074/jbc.M111.228411 PMID: 22002061 67. Schwarz M, Andrade-Navarro MA, Gross A. Mitochondrial carriers and pores: Key regulators of the mitochondrial apoptotic program? Apoptosis. 2007; 12(5):869–76. https://doi.org/10.1007/s10495- 007-0748-2 PMID: 17453157 68. Willer CJ, Speliotes EK, Loos RJF, Li S, Lindgren CM, Heid IM, et al. Six new loci associated with body mass index highlight a neuronal influence on body weight regulation. Nat Genet. 2009; 41(1):25–34. https://doi.org/10.1038/ng.287 PMID: 19079261 69. Yu K, Ganesan K, Tan LK, Laban M, Wu J, Xiao DZ, et al. A precisely regulated gene expression cas- sette potently modulates metastasis and survival in multiple solid cancers. PLoS Genet. 2008; 4(7). https://doi.org/10.1371/journal.pgen.1000129 PMID: 18636107 70. Renstro¨m F, Payne F, Nordstro¨m A, Brito EC, Rolandsson O, Hallmans G, et al. Replication and extension of genome-wide association study results for obesity in 4923 adults from northern Sweden. Hum Mol Genet. 2009; 18(8):1489–96. https://doi.org/10.1093/hmg/ddp041 PMID: 19164386 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 24 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS 71. Backofen B, Jacob R, Serth K, Gossler A, Naim HY, Leeb T. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 References Haslinger A, Schwarz TJ, Covic M, Chichung Lie D. Expression of Sox11 in adult neurogenic niches suggests a stage-specific role in adult neurogenesis. Eur J Neurosci. 2009; 29(11):2103–14. https:// doi.org/10.1111/j.1460-9568.2009.06768.x PMID: 19490090 84. Bergsland M, Werme M, Malewicz M, Perlmann T, Muhr J. The establishment of neuronal properties is controlled by Sox4 and Sox11. Genes Dev. 2006; 20(24):3475–86. https://doi.org/10.1101/gad. 403406 PMID: 17182872 85. Larson BL, Ylostalo J, Lee RH, Gregory C, Prockop DJ. Sox11 is expressed in early progenitor human multipotent stromal cells and decreases with extensive expansion of the cells. Tissue Eng—Part A. 2010; 16(11):3385–94. https://doi.org/10.1089/ten.tea.2010.0085 PMID: 20626275 86. Tsurusaki Y, Koshimizu E, Ohashi H, Phadke S, Kou I, Shiina M, et al. De novo SOX11 mutations cause Coffin-Siris syndrome. Nat Commun. 2014; 5:1–7. https://doi.org/10.1038/ncomms5011 PMID: 24886874 87. Kuryshev VY, Vorobyov E, Zink D, Schmitz J, Rozhdestvensky TS, Mu¨nstermann E, et al. An anthro- poid-specific segmental duplication on human chromosome 1q22. Genomics. 2006; 88(2):143–51. https://doi.org/10.1016/j.ygeno.2006.02.002 PMID: 16545939 88. Kikuno R, Nagase T, Ishikawa K, Hirosawa M, Miyajima N, TANAKA A, et al. Prediction of the Coding Sequences of Unidentified Human Genes. XIV. The Complete Sequences of 100 New cDNA Clones from Brain Which Code for Large Proteins in vitro. DNA Res. 1999; 205:197–205. https://doi.org/10. 1093/dnares/6.3.197 PMID: 10470851 89. Bisogno T, Howell F, Williams G, Minassi A, Cascio MG, Ligresti A, et al. Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain. J Cell Biol. 2003; 163(3):463–8. https://doi.org/10.1083/jcb.200305129 PMID: 14610053 25 / 27 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 PLOS GENETICS Sex-stratified multisite chronic pain GWAS 90. Manitt C, Mimee A, Eng C, Pokinko M, Stroh T, Cooper HM, et al. The Netrin Receptor DCC Is Required in the Pubertal Organization of Mesocortical Dopamine Circuitry. J Neurosci. 2011; 31 (23):8381–94. https://doi.org/10.1523/JNEUROSCI.0606-11.2011 PMID: 21653843 91. Srour M, Rivière JB, Pham JMT, Dube´ MP, Girard S, Morin S, et al. Mutations in DCC cause congeni- tal mirror movements. Science (80-). 2010; 328(5978):592. https://doi.org/10.1126/science.1186463 PMID: 20431009 92. Savage JE, Jansen PR, Stringer S, Watanabe K, Bryois J, De Leeuw CA, et al. Genome-wide associa- tion meta-analysis in 269,867 individuals identifies new genetic and functional links to intelligence. Nat Genet. 2018; 50(7):912–9. https://doi.org/10.1038/s41588-018-0152-6 PMID: 29942086 93. Lee JJ, Wedow R, Okbay A, Kong E, Maghzian O, Zacher M, et al. References Gene discovery and polygenic pre- diction from a genome-wide association study of educational attainment in 1.1 million individuals. Nat Genet. 2018; 50(8):1112–21. https://doi.org/10.1038/s41588-018-0147-3 PMID: 30038396 94. Satizabal CL, Adams HHH, Hibar DP, White CC, Knol MJ, Stein JL, et al. Genetic architecture of sub- cortical brain structures in 38,851 individuals. Nat Genet. 2019; 51(11):1624–36. https://doi.org/10. 1038/s41588-019-0511-y PMID: 31636452 95. Li HJ, Qu N, Hui L, Cai X, Zhang CY, Zhong BL, et al. Further confirmation of netrin 1 receptor (DCC) as a depression risk gene via integrations of multi-omics data. Transl Psychiatry. 2020; 10(1). https:// doi.org/10.1038/s41398-020-0777-y PMID: 32184385 96. Torres-Berrı´o A, Hernandez G, Nestler EJ, Flores C. The Netrin-1/DCC Guidance Cue Pathway as a Molecular Target in Depression: Translational Evidence. Biol Psychiatry [Internet]. 2020; Available from: https://doi.org/10.1016/j.biopsych.2020.04.025 PMID: 32593422 97. Wright KM, Rand KA, Kermany A, Noto K, Curtis D, Garrigan D, et al. A prospective analysis of genetic variants associated with human lifespan. G3 Genes, Genomes, Genet. 2019; 9(9):2863–78. https:// doi.org/10.1534/g3.119.400448 PMID: 31484785 98. Consortium TASDWG of TPG. Meta-analysis of GWAS of over 16,000 individuals with autism spec- trum disorder highlights a novel locus at 10q24.32 and a significant overlap with schizophrenia. Mol Autism. 2017; 8(21). 99. Cordell HJ, Han Y, Mells GF, Li Y, Hirschfield GM, Greene CS, et al. International genome-wide meta- analysis identifies new primary biliary cirrhosis risk loci and targetable pathogenic pathways. Nat Com- mun [Internet]. 2015; 6:1–11. Available from: https://doi.org/10.1038/ncomms9019 PMID: 26394269 100. Boraska V, Franklin CS, Floyd JAB, Thornton LM, Huckins LM, Southam L, et al. A genome-wide association study of anorexia nervosa. Mol Psychiatry. 2014; 19(10):1085–94. https://doi.org/10.1038/ mp.2013.187 PMID: 24514567 101. Jung SJ, Winning A, Roberts AL, Nishimi K, Chen Q, Gilsanz P, et al. Posttraumatic stress disorder symptoms and television viewing patterns in the Nurses’ Health Study II: A longitudinal analysis. PLoS One. 2019; 14(3):1–13. https://doi.org/10.1371/journal.pone.0213441 PMID: 30897111 102. Liberzon I, King AP, Ressler KJ, Almli LM, Zhang P, Ma ST, et al. Interaction of the ADRB2 gene poly- morphism with childhood trauma in predicting adult symptoms of posttraumatic stress disorder. JAMA Psychiatry. 2014; 71(10):1174–82. https://doi.org/10.1001/jamapsychiatry.2014.999 PMID: 25162199 103. Bierut LJ, Strickland JR, Thompson JR, Afful SE, Cottler LB. Drug use and dependence in cocaine dependent subjects, community-based individuals, and their siblings. Drug Alcohol Depend. 2008; 95 (1–2):14–22. https://doi.org/10.1016/j.drugalcdep.2007.11.023 PMID: 18243582 104. Baker DG, Nash WP, Litz BT, Geyer MA, Risbrough VB, Nievergelt CM, et al. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 References Predictors of Risk and Resilience for Posttraumatic Stress Disorder Among Ground Combat Marines: Methods of the Marine Resiliency Study. Prev Chronic Dis. 2012; 9(5):1–11. https://doi.org/10.5888/pcd9.110134 PMID: 22575082 105. Kimbrel NA, Hauser MA, Garrett M, Ashley-Koch A, Liu Y, Dennis MF, et al. Effect of the APOE ε4 allele and combat exposure on PTSD among Iraq/Afghanistan-era veterans. Depress Anxiety. 2015; 32(5):307–15. https://doi.org/10.1002/da.22348 PMID: 25709077 106. Nievergelt CM, Maihofer AX, Klengel T, Atkinson EG, Chen C-Y,. . ., et al. Largest genome-wide asso- ciation study for PTSD identifies genetic risk loci in European and African ancestries and implicates novel biological pathways. bioRxiv [Internet]. 2018;(Nov). Available from: http://dx.doi.org/10.1101/ 458562 107. Liang X, Liu R, Chen C, Ji F, Li T. Opioid System Modulates the Immune Function: A Review Xuan. Transl Perioper Pain Med. 2016; 1(1):5–13. PMID: 26985446 108. Diasso PDK, Birke H, Nielsen SD, Main KM, Højsted J, Sjøgren P, et al. The effects of long-term opioid treatment on the immune system in chronic non-cancer pain patients: A systematic review. Eur J Pain (United Kingdom). 2020; 24(3):481–96. https://doi.org/10.1002/ejp.1506 PMID: 31705699 26 / 27 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 PLOS GENETICS Sex-stratified multisite chronic pain GWAS 109. Mogil JS. Qualitative sex differences in pain processing: emerging evidence of a biased literature. Nat Rev Neurosci 2020 [Internet]. 2020;1–13. Available from: http://www.nature.com/articles/s41583-020- 0310-6%0Ahttps://www.nature.com/articles/s41583-020-0310-6 https://doi.org/10.1038/s41583-020- 0310-6 PMID: 32440016 110. Ko¨nig IR, Loley C, Erdmann J, Ziegler A. How to Include Chromosome X in Your Genome-Wide Asso- ciation Study. Genet Epidemiol. 2014; https://doi.org/10.1002/gepi.21782 PMID: 24408308 111. Accounting for sex in the genome. Nat Med. 2017; 23(11):1243. https://doi.org/10.1038/nm.4445 PMID: 29117171 112. Macfarlane GJ, Barnish MS, Jones GT. Persons with chronic widespread pain experience excess mortality: longitudinal results from UK Biobank and meta-analysis. Ann Rheum Dis [Internet]. 2017; 76 (11):1815–22. Available from: http://ard.bmj.com/lookup/doi/10.1136/annrheumdis-2017-211476 PMID: 28733474 113. Kamaleri Y, Natvig B, Ihlebaek CM, Benth JS, Bruusgaard D. Number of pain sites is associated with demographic, lifestyle, and health-related factors in the general population. Eur J Pain. 2008; 12 (6):742–8. https://doi.org/10.1016/j.ejpain.2007.11.005 PMID: 18160318 114. Bycroft C, Freeman C, Petkova D, Band G, Elliott LT, Sharp K, et al. The UK Biobank resource with deep phenotyping and genomic data. Nature. 2018; 562(7726):203–9. https://doi.org/10.1038/ s41586-018-0579-z PMID: 30305743 115. Loh PR, Tucker G, Bulik-Sullivan BK, Vilhja´lmsson BJ, Finucane HK, Salem RM, et al. Efficient Bayes- ian mixed-model analysis increases association power in large cohorts. Nat Genet [Internet]. 2015; 47 (3):284–90. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 References Available from: https://doi.org/10.1038/ng.3190 PMID: 25642633 116. Willer CJ, Li Y, Abecasis GR, Overall P. METAL: fast and efficient meta-analysis of genomewide asso- ciation scans. Bioinformatics. 2010; 26(17):2190–1. https://doi.org/10.1093/bioinformatics/btq340 PMID: 20616382 117. Zeisel A, Hochgerner H, Lo¨nnerberg P, Johnsson A, Memic F, van der Zwan J, et al. Molecular Archi- tecture of the Mouse Nervous System. Cell. 2018; 174(4):999–1014.e22. https://doi.org/10.1016/j.cell. 2018.06.021 PMID: 30096314 118. Won H, Huang J, Opland CK, Hartl CL, Geschwind DH. Human evolved regulatory elements modulate genes involved in cortical expansion and neurodevelopmental disease susceptibility. Nat Commun [Internet]. 2019; 10(1):1–11. Available from: https://doi.org/10.1038/s41467-018-07882-8 PMID: 30602773 119. Zheng J, Erzurumluoglu AM, Elsworth BL, Kemp JP, Howe L, Haycock PC, et al. LD Hub: A central- ized database and web interface to perform LD score regression that maximizes the potential of sum- mary level GWAS data for SNP heritability and genetic correlation analysis. Bioinformatics. 2017; 33 (2):272–9. https://doi.org/10.1093/bioinformatics/btw613 PMID: 27663502 120. Bulik-Sullivan B, Loh PR, Finucane HK, Ripke S, Yang J, Patterson N, et al. LD score regression dis- tinguishes confounding from polygenicity in genome-wide association studies. Nat Genet [Internet]. 2015; 47(3):291–5. Available from: https://doi.org/10.1038/ng.3211 PMID: 25642630 121. Bulik-sullivan B, Finucane HK, Anttila V, Gusev A, Day FR, Case T, et al. An atlas of genetic correla- tions across human diseases and traits. Nat Publ Gr [Internet]. 2015; 47(11):1236–41. Available from: https://doi.org/10.1038/ng.3406 PMID: 26414676 122. Dudbridge F. Power and Predictive Accuracy of Polygenic Risk Scores. PLoS Genet. 2013; 9(3). https://doi.org/10.1371/journal.pgen.1003348 PMID: 23555274 27 / 27
https://openalex.org/W2982426240	https://europepmc.org/articles/pmc6982640?pdf=render	English	null	Accuracy of the neurosurgeons estimation of extent of resection in glioblastoma	Acta neurochirurgica	2,019	cc-by	3,781	Acta Neurochirurgica https://doi.org/10.1007/s00701-019-04089-8 (2020) 162:373–378 Acta Neurochirurgica https://doi.org/10.1007/s00701-019-04089-8 (2020) 162:373–378 ORIGINAL ARTICLE - TUMOR - GLIOMA * Mark ter Laan Mark.terLaan@radboudumc.nl Abstract Background The surgeons’ estimate of the extent of resection (EOR) shows little accuracy in previous literature. Considering the developments in surgical techniques of glioblastoma (GBM) treatment, we hypothesize an improvement in this estimation. This study aims to compare the EOR estimated by the neurosurgeon with the EOR determined using volumetric analysis on the post- operative MR scan. Methods Pre- and post-operative tumor volumes were calculated through semi-automatic volumetric assessment by three ob- servers. Interobserver agreement was measured using intraclass correlation coefficient (ICC). A univariate general linear model was used to study the factors influencing the accuracy of estimation of resection percentage. Results ICC was high for all three measurements: pre-operative tumor volume was 0.980 (0.969–0.987), post-operative tumor volume 0.974 (0.961–0.984), and EOR 0.947 (0.917–0.967). Estimation of EOR by the surgeon showed moderate accuracy and agreement. Multivariable analysis showed a statistically significant effect of operating neurosurgeon (p = 0.01), use of fluores- cence (p < 0.001), and resection percentage (p < 0.001) on the accuracy of the EOR estimation. Conclusion All measurements through semi-automatic volumetric analysis show a high interobserver agreement, suggesting this to be a reliable assessment of EOR. We found a moderate reliability of the surgeons’ estimate of EOR. Therefore, (early) post- operative MRI scanning for evaluation of EOR remains paramount. Keywords Extent of resection . Glioblastoma multiforme . Interobserver agreement . Tumor volume measurement Sümeyye Sezer1 & Martin J. van Amerongen2 & Hans H. K. Delye1 & Mark ter Laan1 Sümeyye Sezer1 & Martin J. van Amerongen2 & Hans H. K. Delye1 & Mark ter Laan1 Received: 16 April 2019 /Accepted: 24 September 2019 # The Author(s) 2019 /Published online: 28 ctober 2019 O 2 Department of Radiology, Radboud University Medical Center, Geert Grooteplein Zuid 10, 6525 GA Nijmegen, The Netherlands 1 Department of Neurosurgery, Radboud University Medical Center, Geert Grooteplein Zuid 10, 6525 GA Nijmegen, The Netherlands Introduction Increasing the extent of resection (EOR) of GBM is associated with prolonged survival [2]. Also, adjuvant radiochemothera- py showed higher survival rates in patients with complete resection (EOR ≥90%), compared with partial resection (EOR < 90%) [3]. Glioblastoma (GBM) represents 47.1% of malignant primary brain tumors, making it the most common type of malignant primary brain tumor [1]. Even though survival rates have im- proved, prognosis of GBM remains poor. Surgery aimed at as much resection as safely possible is the main treatment option. Using volumetric analysis, it is possible to quantify the EOR using MR imaging. Prior studies using semi-automated methods for this purpose found a high interobserver agree- ment [4, 5]. However, when manual segmentation is used, a low interobserver agreement in the assessment of tumor resec- tion rates on magnetic resonance imaging (MRI) is described. This applies particularly for post-operative tumor volume and residual tumor volume [6]. This article is part of the Topical Collection on Tumor - Glioma Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00701-019-04089-8) contains supplementary material, which is available to authorized users. Before the general use of post-operative scanning, intra- operative estimation by the neurosurgeon was used to deter- mine partial, subtotal, or total tumor resection. The only study that compared this estimation with the presence of residual tumor mass on a MR image, dates back to 1994 [7]. It showed that neurosurgeons often underestimate the presence of resid- ual tumor. Residual tumor mass was three times more often * Mark ter Laan Mark.terLaan@radboudumc.nl Acta Neurochir (2020) 162:373–378 374 including the possibly present necrotic area. All three ob- servers were instructed to include any tissue with a high post-contrast signal in their estimation. The minimal detect- able residual would be one voxel, which corresponds with 0.954 mm3 using our settings. To eliminate hemorrhage inclu- sion in post-operative tumor volume (Post-opTV) determina- tion, any parts around the resection area with high signal on T1-weighed image without contrast were subtracted from the hyperintense mass on the contrast-enhanced T1-weighed im- age. Extent of resection (EOR) was calculated as: seen on early post-operative MRI than estimated by the neu- rosurgeon [7]. With the advancement of microsurgical techniques and the introduction of adjuncts such as fluorescence, we hypothesize a high accuracy of the surgeons’ estimation of the EOR. Possibly, the post-op MRI scan can be omitted after resection using fluorescence. Power calculation Previous literature comparing the presence of residual tumor on MR images with the estimation made by the neurosurgeon is scarce. We predicted to find an accuracy of 80%, meaning that the estimation about the presence of residual tumor mass should agree with the findings on MRI in 80% of the cases. Sample size calculation with a 95% confidence probability, resulted in a sample size of 62. Test methods All patients were treated according to protocol; neuronavigation was used in all patients and 5-ALA was used for intra-operative fluorescence in 22 patients. The EOR by the neurosurgeon was estimated by the operating surgeon post-operatively (EORsurgeon), before the post-operative MR scan was made. This estimation was based on intra-operative microscopic visu- alization and neuronavigation. Post-operative MR imaging was performed within 72 h after surgery. Introduction This study aims to compare the EOR es- timated by the neurosurgeon with the EOR determined using volumetric analysis on post-operative MR images. Participants 100% 100% Because the study implied no burden for the patients, ethical approval was waived by the ethical committee. In this retro- spective study, patient characteristic, operation details, and tumor characteristics were prospectively collected. Therefore, no informed consent was required. Adult patients (≥18 years old) who underwent a resection for GBM were included. Exclusion criteria were as follows: (1.) patients without a pre-operative contrast-enhanced MR image (i.e., less than 48 h before surgery) or without an early post- operative MRI (i.e., within 72 h after surgery) and (2.) patients that had prior treatment for the target tumor. This EOR calculation was done for the measurements of the surgeon, the radiologist, and the student, respectively, resulting in an EORobs1, EORobs2, and EORobs3. The mean of these three measurements is defined as the EORMRI. Methods EOR in% ð Þ ¼ Pre−opTV−Post−opTV ð Þ=Pre−opTV ð Þ Analysis All analyses were performed on SPSS software version 25 (IBM Inc., Armonk, New York) for Windows (Microsoft Inc., Redmond, Washington). We have used an altered Bland-Altmann plot to assess agreement between more than two observers [8]. Pre- and post-operative T1-weighed MR images (slice thickness 1.0 mm) with and without gadolinium contrast were used for semi-automated segmentation of the tumor. Tumor volumes were calculated through semi-automated volumetric analysis using Brainlab software (Brainlab, iPlanNet version 2.3.1.215.1, Munich, Germany) by three observers (a neuro- surgeon, a radiologist, and a trained medical student). Previous literature stated the use of this method to be reliable for tumor measurements [4, 5]. All observers are skilled in interpreting MR images for brain tumors. Each observer was blinded for the data measured by the other observers and for the surgeons’ estimate. The neurosurgeon did not measure the tumors of his own patients. For this reason, there was a second neurosurgeon measuring these patients’ MR images. Interobserver agreement for pre-opTV, post-opTV, and EOR was measured using intraclass correlation coefficient (ICC) based on a two-way random model for absolute agree- ment. Using semi-automated volumetric analysis methods, even the smallest remnants are detected. Therefore, a cut-off value of 99% was used as a surrogate for residual tumor only for accuracy calculation. A univariate multivariable general linear model was used to study the factors influencing the accuracy of EORsurgeon. Here, we used Δ = EORsurgeon −EORMRI as the dependent variable. The following variables were included in analysis: Pre-opTV (mean Pre-opTV of the three observers), blood loss during surgery, use of fluorescence, operating neurosurgeon, and EORMRI. Significance for all analysis was set at a p value < 0.05. Our definition of tumor volume was in accordance with previous descriptions [6]: the contrast enhancing mass, Acta Neurochir (2020) 162:373–378 375 Table 1 Patient characteristics (n = 62) Variable No. Participants Between November 2012 and May 2018, 62 patients were included in the study. A flowchart of the selection process can be found in Fig. 1. The characteristics of the study group are summarized in Table 1. The group consisted of 38 male (61%) and 24 female (39%) patients. The median age at diag- nosis was 63.5 year (range 27–78 years). Agreement between EORobs1, EORobs2, and EORobs3 The altered Bland-Altmann plot for pre-opTVand post-opTV showed good agreement between observers (see Supplemental Digital Content). An ICC of 0.980 (95% CI 0.969–0.987) for pre-opTV and 0.974 (95% CI 0.961–0.984) for post-opTV showed an excellent reliability between ob- servers for these measurements [9]. The Bland-Altmann plot for EOR can be found in Fig. 2. The estimated limits of agreement were – 12.39 to 12.39. The plot shows no systematic over or underestimation of EOR by any observer. There was an excellent reliability for EOR mea- surement, with an ICC of 0.947 (0.917–0.967) (Table 2). Analysis of patients (%) Sex Male 38 (61) Female 24 (39) Age at diagnosis Median 63.5 Range 27–78 Side of tumor Left 32 (52) Right 30 (48) Tumor location Frontal lobe 18 (29) Temporal lobe 28 (45) Parietal lobe 13 (21) Occipital 2 (3) Basal ganglia 1 (2) Necrosis No 10 (16) Yes 52 (84) Fluorescence guided resection (use of 5-ALA) No 40 (65) Yes 22 (35) Use of neuronavigation No 0 (0) Yes 62 (100) Blood loss (mL) Median 200 Range 20–1300 Pre-operative tumor volume* (cm3) Median 33.7 Range 1.0–169.8 Numbers are absolute values (percentages) The mean pre-operative volume of neurosurgeon, radiologist, and med- ical student EORsurgeon, EORMRI There is a higher agreement between EORsurgeon and EORMRI with increasing resection percentage and when fluorescence is used (Fig. 3). The cases are equally distributed above and below the identity line, so there was no systematic EOR over or underestimation by the surgeon. Presence of residual tumor (defined as EOR < 99%) was estimated by the neurosurgeon almost as often as it was seen Numbers are absolute values (percentages) Biopsy (n = 58) Adult patients diagnosed with primary GBM n = 160 Patients included n = 102 Selected patients n = 62 No early post-op MRI (n = 22) Missing data EORsurgeon (n = 18) Fig. 1 Flowchart describing the selection process of the participants between November 2012 and May 2018. GBM, glioblastoma. Neurosurgeon, extent of resection estimated by the operating surgeon The mean pre-operative volume of neurosurgeon, radiologist, and med- ical student Adult patients diagnosed with primary GBM n = 160 on the MRI: surgeons expected residual tumor presence in 76% of the patients, whilst residual was seen in 79% of the MR images (Table 3). The accuracy of predicting presence of residual tumor (defined as EOR < 99%) was 77%, meaning that 77% of the estimations about presence of residual tumor was correct. Biopsy (n = 58) Patients included n = 102 The ICC for EORsurgeon and EORMRI was 0.641 (95% CI 0.404–0.784). The latter indicates a moderate reliability of the EORsurgeon [9]. No early post-op MRI (n = 22) Missing data EORsurgeon (n = 18) Selected patients n = 62 Multivariable analysis showed a statistically significant ef- fect of operating neurosurgeon (p = 0.01), use of fluorescence (p < 0.001), and EORMRI (p < 0.001) on Δsurgeon - MRI. Blood loss and pre-operative tumor volume were not statically sig- nificant factors. Fig. 1 Flowchart describing the selection process of the participants between November 2012 and May 2018. GBM, glioblastoma. Neurosurgeon, extent of resection estimated by the operating surgeon 376 Acta Neurochir (2020) 162:373–378 Fig. 2 Bland-Altman plot for three observers [8]. EORmri (x-axis) is plotted against EORmri minus the EOR measured by the individual observer (y-axis). This graph displays per patient the agreement between the three observers. High agreement corresponds with the three colors being close together. The lines represent the limits of agreement. EORsurgeon, EORMRI EOR, extent of resection; EORmri, mean EOR of three observers; EORobs1, EOR measured by neurosurgeon; EORobs2, EOR measured by radiologist; EORobs3, EOR measured by student colors being close together. The lines represent the limits of agreement. EOR, extent of resection; EORmri, mean EOR of three observers; EORobs1, EOR measured by neurosurgeon; EORobs2, EOR measured by radiologist; EORobs3, EOR measured by student Fig. 2 Bland-Altman plot for three observers [8]. EORmri (x-axis) is plotted against EORmri minus the EOR measured by the individual observer (y-axis). This graph displays per patient the agreement between the three observers. High agreement corresponds with the three Following these findings, an estimated marginal means was calculated for use of fluorescence. Use of fluorescence showed an estimated marginal mean of 12.48 (95% CI 7.26; 17.71), meaning with the use of fluorescence, there is a sig- nificant overestimation by the surgeon. result and this way of reporting data is similar to that of the previous study on this subject [7]. However, with the in- creased precision of statistical analysis in this era, we added a more statistical approach to display our findings. So, we found an accuracy of 77% and a moderate reliability [9]. Higher resection percentage contributed to a higher agree- ment between EORsurgeon and EORMRI. The use of fluores- cence also contributed to a higher agreement, with a tendency of overestimation of the EOR by the surgeon (marginal means 12.48 95% CI 7.26; 17.71). The reliability of the surgeons’ estimate was surgeon dependant. Table 2 Measurements of observers and corresponding intraclass correlation coefficients Discussion The identity line (y = x) is shown for reference clinically more relevant than percentages, we have plotted sur- geons estimating complete resection against residual volume. There was no estimation of complete resection when residual was above 6 mL. With minimal residual volume (< 2 mL, 2– 4 mL, and 4–6 mL), 17 to 36% of surgeons estimated a complete resection (Supplemental Digital Content, Fig. 3). on MRI than estimated by the surgeon. Our data shows the findings of residual tumor on MRI to be equal to the estima- tion of the neurosurgeon. At first hand, this seems as an im- provement in reliability of surgeons’ estimate as hypothe- sized, but further analysis shows only moderate reliability. The strength of our study is the use of semi-automatic volu- metric assessment of EOR and showing this method of determin- ing EOR is highly reliable. Another strength is the statistical assessment of reliability of EORsurgeon. We found an equal dis- tribution of over and underestimation of EOR by the surgeon, so we did not confirm the systematic overestimation of the surgeon as suggested by others [7]. We did find that accuracy of the surgeons’ estimate is surgeon dependant. So, some surgeons es- timate the resection percentage better than others. Even though the use of fluorescence resulted in a slight overestimation, it did increase the accuracy of surgeons’ estimate. Conclusion Semi-automatic volumetric analysis showed a high interob- server agreement and should therefore be considered the gold standard for assessment of EOR. The introduction of fluores- cence has resulted in better resections [10]. We found it to increase the accuracy of the surgeons’ estimate of fluores- cence, whilst resulting in a tendency towards overestimation. Even though surgeons’ estimate of extent of resection has clearly improved since the report of Albert et al., the reliability of their estimation is statistically moderate. Therefore, early post-operative MRI scanning for evaluation of EOR remains paramount. Semi-automatic volumetric analysis showed a high interob- server agreement and should therefore be considered the gold standard for assessment of EOR. The introduction of fluores- cence has resulted in better resections [10]. We found it to increase the accuracy of the surgeons’ estimate of fluores- cence, whilst resulting in a tendency towards overestimation. There were a few limitations in our study. Our study was powered to detect accuracy of 80%, but the low number of sub- jects limits the factors that can be included in our multivariate analysis. We have calculated accuracy in an indirect way, using a dichotomy with 99% EOR as the cut-off value. Nevertheless, the different statistical approaches in this study all show a compara- ble result: moderate agreement for surgeons’ estimate and volu- metric assessment by MRI. Finally, since residual volume is Discussion Our main purpose in this study was to compare the EOR estimated by the neurosurgeon post-operatively (EORsurgeon) with the EOR determined using volumetric analysis on the post-operative MR scan (EORMRI). Using manual segmentation, others have reported high in- terobserver agreement only for pre-operative tumor volume, but not regarding post-operative volume and EOR [6]. Prior studies using semi-automated methods found a high interob- server agreement for calculation of resection percentage [4, 5]. Our results agree, showing that semi-automatic volume as- sessment in GBM is a reliable method for detecting post- operative residual and determining EOR. In order to achieve this, we first studied the reliability of semi-automated volumetric analysis for determining EOR. There was a very high interobserver agreement between the three observers for measurements of pre-operative tumor vol- ume, post-operative tumor volume, and extent of resection. Therefore, semi-automatic volumetric measurement of extent resection of glioblastoma is reliable. Only one prior study has reported on the reliability of sur- geons’ estimate of EOR [7]. In this study, data collection was nominal: presence of residual tumor was answered with “yes,” “no,” or “?.” Residual tumor was detected 3 times more often Our data shows that residual tumor (defined as EOR < 99%) is seen on MR images as often as suggested by the neurosurgeon. This simple comparison shows an evident Table 2 Measurements of observers and corresponding intraclass correlation coefficients Neurosurgeon Radiologist Student ICC (95% CI) Mean pre-opTV (SD) 38.15 (33.47) 37.72 (29.43) 39.92 (33.08) 0.980 (0.969–0.987) Mean post-opTV (SD) 6.01 (12.01) 5.69 (8.25) 5.68 (10.71) 0.974 (0.961–0.984) Mean EOR (SD) 86.41 (17.66) 83.21 (16.06) 86.96 (17.96) 0.947 (0.917–0.967) The ICC was calculated using a two-way random model for absolute agreement. Pre-opTV, post-opTV, and EOR all showed a high ICC, indicating an excellent reliability between observers. ICC, intraclass correlation coeffi- cient; Pre-opTV, pre-operative tumor volume; Post-opTV, post-operative tumor volume; EOR, extent of resection The ICC was calculated using a two-way random model for absolute agreement. Pre-opTV, post-opTV, and EOR all showed a high ICC, indicating an excellent reliability between observers. ICC, intraclass correlation coeffi- cient; Pre-opTV, pre-operative tumor volume; Post-opTV, post-operative tumor volume; EOR, extent of resection 377 Acta Neurochir (2020) 162:373–378 Fig. 3 Scatterplot for extent of resection (EOR) based on semi- automatic calculation on MR (EORmri) versus EOR estimated by the operating surgeon (EORsurgeon). Triangles repre- sent resections where fluores- cence was used, circles represent resections without fluorescence. Informed consent For this type of study, formal consent is not required. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. Table 3 Assessment of completeness of tumor resection: comparison between the neurosurgeon’s estimation and the findings on MRI Residual tumor Neurosurgeon (%) MRI (%) Yes (EOR < 99%) 76 79 No (EOR ≥99%) 24 21 Table 3 Assessment of completeness of tumor resection: comparison between the neurosurgeon’s estimation and the findings on MRI Ethical approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the institu- tional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent For this type of study, formal consent is not required. Acta Neurochir (2020) 162:373–378 378 5. Kanaly CW, Mehta AI, Ding D, Hoang JK, Kranz PG, Herndon JE 2nd, Coan A, Crocker I, Waller AF, Friedman AH, Reardon DA, Sampson JH (2014) A novel, reproducible, and objective method for volumetric magnetic resonance imaging assessment of enhanc- ing glioblastoma. J Neurosurg 121:536–542. https://doi.org/10. 3171/2014.4.Jns121952 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. 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. 6. Kubben PL, Postma AA, Kessels AG, van Overbeeke JJ, van Santbrink H (2010) Intraobserver and interobserver agreement in volumetric assessment of glioblastoma multiforme resection. Neurosurgery 67:1329–1334. https://doi.org/10.1227/NEU. 0b013e3181efbb08 References 7. Albert FK, Forsting M, Sartor K, Adams HP, Kunze S (1994) Early postoperative magnetic resonance imaging after resection of malig- nant glioma: objective evaluation of residual tumor and its influence on regrowth and prognosis. Neurosurgery 34:45–60 discussion 60- 41 1. Ostrom QT, Gittleman H, Liao P, Vecchione-Koval T, Wolinsky Y, Kruchko C, Barnholtz-Sloan JS (2017) CBTRUS statistical report: primary brain and other central nervous system tumors diagnosed in the United States in 2010-2014. Neuro-Oncology 19:v1–v88. https://doi.org/10.1093/neuonc/nox158 1. Ostrom QT, Gittleman H, Liao P, Vecchione-Koval T, Wolinsky Y, Kruchko C, Barnholtz-Sloan JS (2017) CBTRUS statistical report: primary brain and other central nervous system tumors diagnosed in the United States in 2010-2014. Neuro-Oncology 19:v1–v88. https://doi.org/10.1093/neuonc/nox158 8. Jones M, Dobson A, O'Brian S (2011) A graphical method for assessing agreement with the mean between multiple observers using continuous measures. Int J Epidemiol 40:1308–1313. https://doi.org/10.1093/ije/dyr109 2. Sanai N, Polley MY, McDermott MW, Parsa AT, Berger MS (2011) An extent of resection threshold for newly diagnosed glioblasto- mas. J Neurosurg 115:3–8. https://doi.org/10.3171/2011.2. jns1099810.3171/2011.7.jns10238 2. Sanai N, Polley MY, McDermott MW, Parsa AT, Berger MS (2011) An extent of resection threshold for newly diagnosed glioblasto- mas. J Neurosurg 115:3–8. https://doi.org/10.3171/2011.2. jns1099810.3171/2011.7.jns10238 9. Koo TK, Li MY (2016) A guideline of selecting and reporting intraclass correlation coefficients for reliability research. J Chiropr Med 15:155–163. https://doi.org/10.1016/j.jcm.2016.02.012 3. Stummer W, van den Bent MJ, Westphal M (2011) Cytoreductive surgery of glioblastoma as the key to successful adjuvant therapies: new arguments in an old discussion. Acta Neurochir 153:1211– 1218. https://doi.org/10.1007/s00701-011-1001-x 10. Eljamel S (2015) 5-ALA fluorescence image guided resection of glioblastoma multiforme: a meta-analysis of the literature. Int J Mol Sci 16:10443–10456. https://doi.org/10.3390/ijms160510443 4. Chow DS, Qi J, Guo X, Miloushev VZ, Iwamoto FM, Bruce JN, Lassman AB, Schwartz LH, Lignelli A, Zhao B, Filippi CG (2014) Semiautomated volumetric measurement on postcontrast MR im- aging for analysis of recurrent and residual disease in glioblastoma multiforme. AJNR Am J Neuroradiol 35:498–503. https://doi.org/ 10.3174/ajnr.A3724 Publisher’s note Springer Nature remains neutral with regard to jurisdic- tional claims in published maps and institutional affiliations. Publisher’s note Springer Nature remains neutral with regard to jurisdic- tional claims in published maps and institutional affiliations.
https://openalex.org/W2399563817	https://europepmc.org/articles/pmc4876404?pdf=render	English	null	Emission energy, exciton dynamics and lasing properties of buckled CdS nanoribbons	Scientific reports	2,016	cc-by	6,761	Emission energy, exciton dynamics and lasing properties of buckled CdS nanoribbons Qi Wang1,2, Liaoxin Sun1, Jian Lu3, Ming-Liang Ren4, Tianning Zhang1, Yan Huang1, Xiaohao Zhou1, Yan Sun1, Bo Zhang1, Changqing Chen2, Xuechu Shen1, Ritesh Agarwal4 & Wei Lu1 received: 04 March 2016 accepted: 05 May 2016 Published: 23 May 2016 We report the modulation of emission energy, exciton dynamics and lasing properties in a single buckled CdS nanoribbon (NR) by strain-engineering. Inspired by ordered structure fabrication on elastomeric polymer, we develop a new method to fabricate uniform buckled NRs supported on polydimethylsiloxane (PDMS). Wavy structure, of which compressive and tensile strain periodically varied along the CdS NR, leads to a position-dependent emission energy shift as large as 14 nm in photoluminescence (PL) mapping. Both micro-PL and micro-reflectance reveal the spectral characteristics of broad emission of buckled NR, which can be understood by the discrepancy of strain-induced energy shift of A- and B-exciton of CdS. Furthermore, the dynamics of excitons under tensile strain are also investigated; we find that the B-exciton have much shorter lifetime than that of redshifted A-exciton. In addition, we also present the lasing of buckled CdS NRs, in which the strain- dominated mode selection in multi-mode laser and negligible mode shifts in single-mode laser are clearly observed. Our results show that the strained NRs may serve as new functional optical elements for flexible light emitter or on-chip all-optical devices. Strain-engineering has been demonstrated to be a very efficient way to tune the optical and electronic properties of materials, which may lead to new functional devices with great flexibility1–9. Especially in nanomaterials, high crystal quality and much larger elastic limit because of large surface-volume ratio, facilitate the strain-related new functionalities. Many remarkable reports on tuning electronic and optical properties of diverse nanostructures by strain-engineering have been published8–17. For example, strain effect on electron-transport of carbon nano- tubes1,8; silicon nanomembranes and nanowires10–14; piezoelectricity or optical-mechanical coupling of ZnO, CdS nanowires15–24; strain-related bandgap-engineering and electronic properties in 2D nanomaterials25–28. And very recently, hundreds of meV exciton energy shift modulated by strain were observed in both GaAs and CdS nano- wires29,30. Undoubtedly, Strain engineering provided a new method to tailor the bandgap of materials instead of traditional ways such as component ratio control, quantum confinement effect. if Nanoribbons (NRs) with an out-of-plane buckled geometry, which have great bendability and mechanical stretchability, are functional nanostructures with great application potential in both electronic and optical device. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| 6:26607 \| DOI: 10.1038/srep26607 Emission energy, exciton dynamics and lasing properties of buckled CdS nanoribbons In last decade, both mechanical and electronic properties of these flexible NR devices have been intensively inves- tigated31–37. For example, stretchable “wavy” silicon was functioned as flexible field-effect transistors31–34, the buckled GaAs NRs with very high stretchability and compressibility36, 70% enhancement of piezoelectric effect in wavy PZT ribbons37, etc. Some NRs (e.g. CdS NR, ZnO NR) can also serve as laser sources, waveguides and even semiconductor optical cryocooler, which are building blocks for all-optical on-chip devices38–43. It is highly desirable to realize flexible NR-based optical devices, but in above reports, NRs usually have a length of millimeter and perfect alignment along the prestrain direction of elastomeric substrate, which allows for a periodic buck- ling just via adhesive Van der Waals forces between NRs and elastomeric substrate. Neither of them is fulfilled (maximal hundred micrometers and not good alignment) for CdS or ZnO NRs, thus it is hard to get a periodic 1National Lab for Infrared Physics, Shanghai Institute of Technical Physics, Chinese Academy of Science, Shanghai, 200083, China. 2Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China. 3Shanghai Advanced Research Institute, Chinese Academy of Science, Shanghai 201210, China. 4Department of Materials Science and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania, 19104, United States. Correspondence and requests for materials should be addressed to L.S. (email: sunlx@mail. sitp.ac.cn) or W.L. (email: luwei@mail.sitp.ac.cn) Scientific Reports \| 6:26607 \| DOI: 10.1038/srep26607 1 www.nature.com/scientificreports/ Figure 1. The fabrication process and the characteristics of the samples. (a–c) Schematic diagrams of the fabrication process for buckled CdS NRs. (d) Optical image of a typical buckled CdS NR. (e) Atomic Force Microscopy image (top panel) of the buckled CdS NR which is shown in (d), and the surface height (bottom panel) of CdS as a function of position along wavy CdS NR on PDMS, the red solid curve represent sinusoidal fits to the data. Figure 1. The fabrication process and the characteristics of the samples. (a–c) Schematic diagrams of the fabrication process for buckled CdS NRs. (d) Optical image of a typical buckled CdS NR. (e) Atomic Force Microscopy image (top panel) of the buckled CdS NR which is shown in (d), and the surface height (bottom panel) of CdS as a function of position along wavy CdS NR on PDMS, the red solid curve represent sinusoidal fits to the data. buckling with a stable geometry. Experiments and measurements CdS NRs were grown via the vapor-liquid-solid method at the temperature of 850 °C, which have the wurtzite crystal structure with the [0001] direction (c-axis) perpendicular to the nanoribbon long axis (a-axis)42,52. To measure the optical properties of buckled NRs, we utilized our home-made micro-PL and micro-reflectance setup based on a microscope (as shown in Supplementary Materials, Figure S1). A semiconductor laser (457 nm continuous laser used for PL and 355 nm pulse laser used for lasing) was focused to a spot of ~1 μm by a 50X (NA, 0.5) objective. The emission was collected by the same objective and directed to spectrometer. Spatial scanning measurements were performed in cryostat mounted on a translation stage with the spatial resolution of 1 μm. All the spectral measurements were performed at a temperature of 77 K to ensure that A- and B-exciton of CdS are clearly visible in the spectra. The time-correlated single-photon-counting system (TCSPC) was adopted to study the exciton dynamics. Emission energy, exciton dynamics and lasing properties of buckled CdS nanoribbons Moreover, flexibility of NR-based optical devices inevitably induces deformation and thus strain effect on materials which is expected to modulate the optical properties of NR, just like buckled nanowires29. But as far as we know, the optical tuning (e.g. PL, exciton’s dynamics and lasing properties) of NRs by strain-engineering has rarely been investigated before44,45. In this work, CdS, of which nanostructures have been widely investigated in their optical properties46–51, is chosen as the test bed because of its high optical quality and very large fracture strength limit30. We present a new method to fabricate wavy structured CdS NRs and studied their optical response to strain. CdS NRs were wrinkled to a nearly uniform buckled structure, which was well characterized by atomic force microscope (AFM) and PL mapping (a periodic emission shift with 14 nm). Moreover, the tuning of emission energy, lifetime of excitons and lasing properties under strain were investigated by using micro-PL, micro-reflectance, and time-resolved PL techniques, respectively. This uniform buckled struc- tures and related optical tuning may greatly expand their potential applications in large-scale integrated flexible light-emitting devices. Results and Discussions (Here, to avoid the resonant modes of cavity, formed between the top and bottom surface of thick NR, in the reflectance spectra, a thin buckled NR (thickness, 200 nm) with small tensile strain was selected to perform micro-reflectance measurements). Features corresponding to the A- and B-exciton are clearly observed from both the reflectance and PL spectra. Figure 2. PL and Polarization-resolved reflectance spectra measured on periodic buckled CdS NRs. (a) Spatially resolved PL mapping of the buckled NR, exciton emission peak shows a periodic energy shift. (b) Spectra measured at flat part, valley part and crest part are presented, respectively. Polarization-resolved reflectance (c) and PL (d) spectra measured at the crest part of thin buckled CdS NR. (Here, to avoid the resonant modes of cavity, formed between the top and bottom surface of thick NR, in the reflectance spectra, a thin buckled NR (thickness, 200 nm) with small tensile strain was selected to perform micro-reflectance measurements). Features corresponding to the A- and B-exciton are clearly observed from both the reflectance and PL spectra. without a layer of Al2O3 and a corresponding discussion are shown in Supplementary Materials (Figure S2). After hat, we released the strain of PDMS and obtained CdS NRs with wavy geometry (Fig. 1(a–c)).h The optical image of a typical buckled NR is presented in Fig. 1(d). One can see clearly that the periodic out-of-plane buckled structure of a single CdS NR is formed. To understand its geometrical characteristics, AFM with tapping mode was adopted to scan along the buckled surface of NR and its atomic force micrograph map- ping is shown in Fig. 1e (top panel). For clarity, the profile of buckled ribbon (black dots) is also shown at bottom panel; a sinusoidal profile of buckled CdS NR, which is the same as that of buckled silicon ribbons36, is clearly visible. The red curve is the sinusoidal fit to experimental data, from which the amplitude of 2.13 μm and a period of 30.5 ± 1.4 μm are obtained. μ Strain-induced exciton energy shifts have been demonstrated in bent ZnO, CdS and GaAs nanowires recently19,29,30. Out-of-plane CdS buckled NRs actually have a similar buckled geometry as bent nanowires, which the tensile strain localized at the outer surface and the compressive strain at the inner surface, respectively. Thus it is expected that the exciton energies of CdS should be modulated by the periodic strain variation of the buckled NR. Results and Discussions To fabricate out-of-plane buckled CdS NRs, shown in Fig. 1(d), a prestrained PDMS substrate was prepared by a custom-built strain stage. We first dry transferred as-grown CdS NRs from the growth substrate to a polyimide film, and then carefully contacted the NRs on polyimide film onto the prestrained PDMS surface and mechani- cally slid it along the PDMS prestrain direction. In this manner, most NRs are transferred to PDMS with the long axis roughly following the prestrain direction. But unlike the buckled GaAs or PZT ribbons36,37, here, adhesive Van der Waals forces between NRs and PDMS cannot ensure a stable periodic buckled CdS NRs formed. To over- come this limit, we deposited a 5 nm thick Al2O3 film on the NRs by Atom Layer Deposition to tightly fixed NRs on PDMS surface without any influence on PL measurement. A comparison of PL spectra of nanoribbon with and Scientific Reports \| 6:26607 \| DOI: 10.1038/srep26607 2 www.nature.com/scientificreports/ Figure 2. PL and Polarization-resolved reflectance spectra measured on periodic buckled CdS NRs. (a) Spatially resolved PL mapping of the buckled NR, exciton emission peak shows a periodic energy shift. (b) Spectra measured at flat part, valley part and crest part are presented, respectively. Polarization-resolved reflectance (c) and PL (d) spectra measured at the crest part of thin buckled CdS NR. (Here, to avoid the resonant modes of cavity, formed between the top and bottom surface of thick NR, in the reflectance spectra, a thin buckled NR (thickness, 200 nm) with small tensile strain was selected to perform micro-reflectance measurements). Features corresponding to the A- and B-exciton are clearly observed from both the reflectance and PL spectra. Figure 2. PL and Polarization-resolved reflectance spectra measured on periodic buckled CdS NRs. ( ) ll l d f h b kl d k h d h f Figure 2. PL and Polarization-resolved reflectance spectra measured on periodic buckled CdS NRs. (a) Spatially resolved PL mapping of the buckled NR, exciton emission peak shows a periodic energy shift. (b) Spectra measured at flat part, valley part and crest part are presented, respectively. Polarization-resolved reflectance (c) and PL (d) spectra measured at the crest part of thin buckled CdS NR. Scientific Reports \| 6:26607 \| DOI: 10.1038/srep26607 Results and Discussions The redshifted emission peak shows broadening compared to the black spectrum, this can be attributed to the strain gradient distribution both along the thickness direction and along the a-axis of the NR. In addition, other possibility is that bending deformation-induced broadening of the gaps between different sub- bands in the conduction band of CdS, which was predicted by theoretical calculation29. At last, one should note that the strain-induced piezoelectric field in CdS NRs may affect the PL properties. In regard to this issue, some previous literatures have demonstrated that the influence of piezoelectric field on the PL is strongly dependent on the size of nanostructures and the binding energy of excitons53–55. For CdS NRs, it is believed that the PL redshift is dominated by the tensile strain-induced bandgap shift, and the piezoelectric field may have slight contribution for thin buckled CdS NRs (~200 nm)54. ( ) Tensile strain induces redshift and broadening of emission peak, but beside that, the other emission peak at the wavelength of 489 nm is always accompanied the redshifted emission peak. This phenomenon is quite similar to the double peaks observed on bent CdS nanowires, in which the PL peak at high energy side was shown to be originating from the emission of B-exciton at inner part (compressive strain part) of a bent nanowire29. But in a buckled NR, we notice that laser mainly excite the tensile part because of penetration depth of laser is no more than 100 nm56,57, Even though 30% of the incident light may penetrate more into the CdS NRs and excite the compressive part, considering that the emission coming from the compressive part may be re-absorbed by the tensile part because of tensile strain-induced redshifted energy band, it is hard to generate a strong emission peak at around 489 nm, as shown in Fig. 2(b). Thus, this phenomenon is totally different from the in-plane bent nanowire where both tensile and compressive parts are covered by the laser spot, and the emission peak at 489 nm should not originate from the compressive part. To explore its fundamental properties and the physical mech- anism, the micro-PL and micro-reflectance spectral techniques with different polarization configurations were used to study the excitons’ characteristics. Results and Discussions Figure 2(a) shows the micro-PL spectral mapping, including around 60 spectra measured on the top surface of buckled nanoribbon with a scanning step of ~2.5 μm along the long axis. One can see that the emission peak shifts periodically, which can be attributed to the tensile and compressive strain alternation along the buckled NR. The waterfall plot shown in Supplementary Materials, Figure S3, shows that the maximum redshifted emis- sion peaks (~503.5 nm) and the emission peaks (~489.5 nm) correspond exactly to the crest and the valley of the buckled NR, respectively. Due to the nearly perfect sinusoidal geometry, the redshifted emission peaks measured at six crests of the buckled NR are around 503.5 nm, which show uniform and periodic tuning of emission energy. As mentioned above, the redshift of the exciton emission is attributed to the tensile strain (εtop) localized at the top surface of the crest part, which can be roughly estimated by the local radius of curvature, r, and thickness, d, of NR (εtop = d/2r)19. As characterized by AFM, the NR has a thickness of ~0.35 μm, and the inscribed circle radius of the crest part is ~2.75 μm. With these values, the tensile strain εtop can be calculated to be around 6.36%, resulting in the redshift of exciton emission of ~14 nm (~68 meV). This redshift amount may be further improved by increasing the pre-tensile strength of PDMS to obtain a larger curvature. For comparison, we also measured the PL spectrum from the flat part of NR on PDMS (as shown in Fig. 2(b), black curve). The red curve (meas- ured at the crest part) is clearly redshifted and the blue curve (obtained from the valley region) shows a little bit Scientific Reports \| 6:26607 \| DOI: 10.1038/srep26607 3 www.nature.com/scientificreports/ Figure 3. Time-resolved PL. The time-resolved PL of A- and B-exciton measured at crest part of buckled CdS NR where the tensile strain dominated (top panel) and at flat part of buckled CdS NR where strain effect can be ignored (bottom panel). Figure 3. Time-resolved PL. The time-resolved PL of A- and B-exciton measured at crest part of buckled CdS NR where the tensile strain dominated (top panel) and at flat part of buckled CdS NR where strain effect can be ignored (bottom panel). blueshift. This is consistent with the previous results of exciton energy under tensile or compressive strain in CdS nanowires29. Results and Discussions To avoid the impacts from resonant modes of cavity, usually formed between the top and bottom surface of thick NR, in the reflectance spectra, a thin NR (thickness, 200 nm) was selected to perform micro-reflectance measurements. Figure 2(c,d) shows the micro-reflectance (c) and micro-PL (d) spectra with different polarization configurations, which are measured from the crest. One can see that the polarization measurements in reflectance show different spectral characteristics for the two emission peaks in PL spectra. As known, CdS has a wurtzite crystal structure, the conduction band is s-like state, whereas the valence band is p-like state, which is split into three bands (labeled as A, B, and C) due to the crystal-field effect and spin-orbit interaction58. According to the selection rules, all three excitonic transitions are allowed for transverse electric (TE) polarization (E-field component of light perpendicular to the c-axis of NR), while in transverse magnetic (TM) polarization (E-field component of light parallel to c-axis), the A-exciton is entirely forbidden, while B and C excitons are allowed. The redshifted emission peak (~2.505 eV) is originally TE polarized in both polarized reflectance and PL spectra, which allows us to assign it to A-exciton. Whereas for the emission peak at the high energy side (~2.551 eV), the polarized reflectance spectra show that both TE and TM polarizations are allowed, and thus it is reasonable to attribute this emission peak to B-exciton. Compared to the energy difference (~20 meV) between A- (2.540 eV) and B-exciton (2.560 eV) of unstrained CdS29, the tensile strain-induced the A- (2.505 eV) and B-exciton (2.551 eV) energy difference becomes larger (~46 meV) in the buckled CdS NR. This can be understood by the different energy shift trend of A- and B-exciton under uniaxial strain, which has also been experimentally shown in refs [29 and 30]. Experimental study of CdS nanowires show that the A-exciton shifts more than that of B-exciton under the same strain, which explains why we observed a larger energy splitting between A- and B-exciton of buckled NR. Strain modulates not just the exciton energies but also the dynamics of exciton emission22. Figure 3 (bottom panel) shows the time resolved spectra of A- and B-exciton obtained from the flat part of NR, where the tensile strain effect can be ignored. Fitted by single exponential function, the lifetime of A- and B-exciton was obtained Scientific Reports \| 6:26607 \| DOI: 10.1038/srep26607 4 www.nature.com/scientificreports/ Figure 4. Results and Discussions Lasing properties of buckled CdS NRs. (a) The lasing spectra measured at flat part (w/o strain), valley part (compressive strain) and crest part (tensile strain) of CdS NRs under the same laser excitation power. Two lasing modes with very little energy shift under different strains are observed. (b) Only one lasing mode is observable at gain regime measured on a buckled CdS NR with narrow width (shorter cavity length). (c) The intensity of lasing modes (I and II) as a function of excitation power, which shows an almost the same lasing threshold. Figure 4. Lasing properties of buckled CdS NRs. (a) The lasing spectra measured at flat part (w/o strain), valley part (compressive strain) and crest part (tensile strain) of CdS NRs under the same laser excitation power. Two lasing modes with very little energy shift under different strains are observed. (b) Only one lasing mode is observable at gain regime measured on a buckled CdS NR with narrow width (shorter cavity length). (c) The intensity of lasing modes (I and II) as a function of excitation power, which shows an almost the same lasing threshold. as 1.469 ns and 1.459 ns, respectively. The discrepancy between them is within the time resolution of TCSPS (~50 ps). However, the same measurements at the crest part of buckled NR, where a large tensile strain is applied, show a large lifetime variation for A- and B-exciton (as shown in the top panel). The lifetime of B-exciton becomes much shorter (~1.079 ns), while for A-exciton, it becomes a little longer (~1.573 ns). The difference between them is around 500 ps, which is clearly visible in the time-resolved spectra. This difference can be explained by the enhanced coupling of exciton with LO phonon due to larger energy gap between A- and B-exction. In unstrained CdS NR, the energy gap between A- and B-exciton is around 20 meV, which is much smaller than the LO pho- non (~38 meV), this suppressed the decay channel of B-exciton to lower energy via transferring energy to LO phonon59. But the case is totally changed when the tensile strain induced a relatively large energy gap (>50 meV) between A and B excitons, as shown in Fig. 2(c). In this case, the B excitons can decay to the lower energy states efficiently via coupling of exciton and LO phonon. Scientific Reports \| 6:26607 \| DOI: 10.1038/srep26607 Results and Discussions And furthermore, this decay channel may be enhanced by the energy broadening of A- and B- excitons under gradient tensile strain which lead to that the transition rule of exciton decay via LO phonon can be easily satisfied. Carrier’s dynamics plays a key role in light efficiency or response rate of optoelectronic devices, and these results show that the strain-induced carrier’s dynamics tuning may be an efficient way to improve the performance of optical devices. yfi y p p p NRs served as nanolaser sources have been widely reported in previous works39,41,42, however, the lasing prop- erties of NR under strain have not been investigated. In our samples, the waveguide cavity formed between the two side edges and the optical gains under pulse laser excitation allow us to realize stimulated emission in single CdS NRs. As shown in Fig. 4 (inset), the emission pattern (lasing) from the NR exhibits interference fringes which result from interference between two laser emitting spots from edges42. The typical laser spectrum measured from the flat part of NR is shown in Fig. 4(a) (black curve), and two lasing modes (labeled as I and II) are visible in the optical gain region near the bandedge. But when we measure the lasing both at the valley part and the crest part, two obvious features are observed in the spectra (blue and red curves). One is that the competition between two lasing mode is modulated by applying different strain. The compressive strain (valley part) supports the lasing mode I, while the tensile strain (crest part) is favorable for the lasing mode II. This behavior can be well explained by the strain-induced gain shift. Initially, lasing mode I is stronger than lasing mode II (black spectrum), which implies that the highest optical gain located at mode I rather than mode II. At valley part, a compressive strain caused the bandedge blueshifted, and thus the optical gain regime blueshifted accordingly. This suppressed lasing Scientific Reports \| 6:26607 \| DOI: 10.1038/srep26607 5 www.nature.com/scientificreports/ mode II further and made it invisible in blue spectrum. But at crest part, a tensile strain oppositely induced a redshifted bandedge and the optical gain at lasing mode II would be enhanced, leading to stronger lasing mode II than lasing mode I. The other feature is that the energies of lasing modes are quite stable and change very little with strain. Results and Discussions As known, the lasing modes are mainly determined by the cavity length and the refractive index of the material. In the planar waveguide cavity formed along the short axis of NR, the width and the thickness of NR is hardly affected because strain is mainly applied along the long-axis of NR. Since CdS has a large dispersion near the bandedge owing to strong coupling between light and excitons, the energy redshift of A-exciton under ten- sile strain could modulate the refractive index of CdS and thus the cavity modes’ energies. However, under high excitation power, free electron-hole carriers with a large density are generated and all the empty states around the bandedge are completely occupied, in which case, the refractive index is determined by the carrier density (the quasi-Fermi surface in the conduction and valence band) rather than by the energy band60. So under the same excitation power, the carrier density in the buckled NR is almost the same regardless of strain, and thus the influ- ence of strain on the refractive index can be ignored. This is why the energies of lasing modes change very little even though a large tensile or compressive strain is applied. In addition, from a point of view of practical appli- cations, the single-mode nanolaser is highly desirable in the future optical digitized communication and signal processing61,62. Thus we measured a buckled CdS NR with very narrow width (short cavity length, ~1.5 μm) where only one lasing mode is observable in the gain range. Similar to the above multi-mode laser measurements, the lasing spectra measured at the flat part, crest part and valley part of buckled NR are shown in Fig. 4(b). Just as we expected, the lasing mode indeed shifts very little under different strain, which is almost negligible. Furthermore, the emission intensity as a function of excitation power measured at three different strain parts are also presented in Fig. 4(c). Similar to the energies of lasing modes, the threshold of lasing with average power of around 4.7 μW is almost independent of strain. Our results demonstrates that the nanoribbon-based laser sources can retain their optical properties even though a large strain is generated by deformation of devices, this could be very helpful for flexible nanodevices in which the functionalities are not affected by the deformation at all. Summary and Conclusions y By using a new but relatively easy method, we have fabricated uniform sinusoidal out-of-plane buckled CdS NRs. This led to a periodic modulation of emission energy with wavelength variation of 14 nm. The contribution of A- and B-exciton to the broadening spectrum under tensile strain were studied experimentally by polarized micro-PL and micro-reflectance, the underlying mechanism is believed to be the fact that A-exciton redshift more than B-exciton under the same tensile strain. Besides that, the dynamics of excitons and the lasing properties under strain were also investigated. Buckled CdS NRs may serve as one of optical elements in flexible optoelec- tronic devices. References 1. Tombler, T. W. et al. Reversible electromechanical characteristics of carbon nanotubes under local-probe manipulation. Nature 405 769–772 (2000). 2. Ieong, M. et al. Silicon device scaling to the sub-10-nm regime. Science 306, 2057–2060 (2004). et al. Elastically relaxed free-standing strained-silicon nanomembra 3. Roberts, M. M. et al. Elastically relaxed free-standing strained-silicon nanomembranes. Nat. Mater. 5, 388–393 (2006). 3. Roberts, M. M. et al. Elastically relaxed free-standing strained-silicon nanomembranes. Nat. Mater. 5, 388–393 (2006). 4 Sun, X C et al Direct gap photoluminescence of n-type tensile-strained Ge-on-Si Appl Phys Lett 95, 011911 (2009) g p p yp pp y 5. Huo, Y. J. et al. Strong enhancement of direct transition photoluminescence with highly tensile-strained Ge grown by molecular beam epitaxy. Appl. Phys. Lett. 98, 011111 (2011). p y pp y 6. Sanchez-Perez, J. R. et al. Direct-bandgap light-emitting germanium in tensilely strained nanomembranes. Proc. Natl. Acad. Sci USA 108, 18893–18898 (2011). 7. Smith, A. M. et al. Tuning the optical and electronic properties of colloidal nanocrystals by lattice strain. Nat. Nanotechnol. 4, 56–63 (2009). 8. Huang, M. Y. et al. Direct measurement of strain-induced changes in the band structure of carbon nanotubes. Phys. Rev. Lett. 100 106803 (2008).lifi 9. Someya, T. et al. A large-area, flexible pressure sensor matrix with organic field-effect transistors for artificial skin applications. Proc Natl. Acad. Sci. USA 101, 9966–9970 (2004). 9. Someya, T. et al. A large-area, flexible pressure sensor matrix with organic field-effect transistors for artificial s Natl. Acad. Sci. USA 101, 9966–9970 (2004). h l ll h bl “ ” l b ( ) ( ) 10. Choi, W. M. et al. Biaxially stretchable “Wavy” silicon nanomembranes. Nano Lett. 7, 1655–1663 (2007). 10. Choi, W. M. et al. Biaxially stretchable “Wavy” silicon nanomem 10. Choi, W. M. et al. Biaxially stretchable “Wavy” silicon nanomembranes. Nano Lett. 7, 1655–1663 (2007). y y & Rogers, J. A. Bend, Buckle, and Fold: Mechanical Engineering with Nanomembranes. ACS Nano 3, 498–501 (2009). 11. Kim, D. H. & Rogers, J. A. Bend, Buckle, and Fold: Mechanical Engineering with Nanomembranes. ACS Nano 3, g g g 12. Wu, Z. G. et al. Charge Separation via Strain in Silicon Nanowires. Nano Lett. 9, 2418–2422 (2009).f g p 13. Lugstein, A. et al. Anomalous Piezoresistance Effect in Ultrastrained Silicon Nanowires. Nano Lett. 10, 3204–3208 t al. Anomalous Piezoresistance Effect in Ultrastrained Silicon Nan gf 14. Niquet, Y. M. et al. g gyt 30. Signorello, G. et al. Tuning the Light Emission from GaAs Nanowires over 290 meV with Uniaxial Strain. Nano Lett. 13, 917 (2013). References Effects of Strain on the Carrier Mobility in Silicon Nanowires. Nano Lett. 12, 3545–3550 (2012). f 15. Wang, Z. L. & Song, J. H. Piezoelectric nanogenerators based on zinc oxide nanowire arrays. Science 312, 242–246 (2 16. Qin, Y. et al. Microfibre-nanowire hybrid structure for energy scavenging. Nature 451, 809–813 (2008). i 17. Wang, Z. L. ZnO nanowire and nanobelt platform for nanotechnology. Mater. Sci. Eng. R-Rep. 64, 33–71 (2009) Progress in Piezotronics and Piezo-Phototronics. Adv. Mater. 24, 4 19. Han, X. B. et al. Electronic and Mechanical Coupling in Bent ZnO Nanowires. Adv. Mater. 21, 4937–4941 (2009).it 20. Liao, Z. M. et al. Strain induced exciton fine-structure splitting and shift in bent ZnO microwires. Sci. Rep. 2, 452 (2012). i 21. Wei, B. et al. Size-Dependent Bandgap Modulation of ZnO Nan 22. Fu, X. W. et al. Exciton Drift in Semiconductors under Uniform Strain Gradients: Application to Bent ZnO Microwires. A 8, 3412–3420 (2014). Author Contributions Q.W., L.S. and W.L. conceived the idea, designed the experiments and wrote the manuscript; M.-L.R., T.Z. and Y.S. prepared samples used in experiments; Q.W., L.S. and J.L. contributed to time-resolved measurement and interpretation; Y.H., X.Z. and B.Z. each participated in the experiment and contributed to the data analysis; C.C., X.S. and R.A. discussed the results and reviewed the manuscript. , J R p 134, 12394–12397 (2012). l ff h l f Z ( ) 3. Han, X. B. et al. Strain-Gradient Effect on Energy Bands in Bent ZnO Microwires. Adv. Mater. 24, 4707–4711 (2012). 4. Xu, S. G. et al. Piezotronic Effects on the Optical Properties of ZnO Nanowires. Nano Lett. 12, 5802–5807 (2012). l d d d d f l f 54. Xu, S. G. et al. Piezotronic Effects on the Optical Properties of ZnO Nanowires. Nano Lett. 12, 5802–5807 (2012). f 55. Fu, X. W. et al. Strain Loading Mode Dependent Bandgap Deformation Potential in ZnO Micro/Nanowires. ACS Na 11960–11967 (2015). ( ) 56. Ninomiya, S. & Adachi, S. Optical-properties of Wurtzite CdS. J. Appl. Phys. 78, 1183–1190 (1995). 57. Li, D. H. et al. Electric-Field-Dependent Photoconductivity in CdS Nanowires and Nanobelts: Exciton Ionization, Franz Kel and Stark Effects. Nano Lett. 12, 2993–2999 (2012).hii f . Thomas, D. G. & Hopfield, J. J. Exciton spectrum of Cadmium Su f 58. Thomas, D. G. & Hopfield, J. J. Exciton spectrum of Cadmium Sulfide. Phys. Rev. 116, 573–582 (1959). Ch C H l T il i h i i i d lif i i i d i i i hi i ll hii 59. Cho, C. H. et al. Tailoring hot-exciton emission and lifetimes in semiconducting nanowires via whispering-gallery nanocavity plasmons. Nat. Mater. 10, 669–675 (2011). 59. Cho, C. H. et al. Tailoring hot-exciton emission and lifetimes in semiconducting nanowires via whispering-gallery nanoc plasmons. Nat. Mater. 10, 669–675 (2011). 0. Montazeri, M. et al. Transient Rayleigh Scattering: A New Probe of Picosecond Carrier Dynamics in a Single Semiconductor Nanowire. Nano Lett. 12, 5389–5395 (2012). 61. Xiao, Y. et al. Single-Nanowire Single-Mode Laser. Nano Lett. 11, 1122–1126 (2011). 61. Xiao, Y. et al. Single-Nanowire Single-Mode Laser. Nano Lett. 11, 1122–1126 (2011). Acknowledgementsh g This work was supported by the National Natural Science Foundation of China (Grant No: 11474297, 11274330), and Shanghai Pujiang Program (14PJ1409600). L. Sun acknowledges Youth Innovation Promotion Association CAS (2016221). R. Agarwal acknowledges the National Institutes of Health through the NIH Director’s New Innovator Award Program, 1-DP2-7251-01. X.Z acknowledges Shanghai Committee of Science and Technology, China (13ZR1446200). 8, 3412–3420 (2014). Piezoelectric nanogenerator using CdS nanowires. Appl. Phys. Lett g g pp y 24. Fu, Q. A. et al. Linear strain-gradient effect on the energy bandgap in bent CdS nanowires. Nano Res. 4, 308–314 (2011).i f 25. Feng, J. et al. Strain-engineered artificial atom as a broad-spectrum solar energy funnel. Nat. Photonics 6, 865–871 (2012) 26. He, K. et al. Experimental Demonstration of Continuous Electronic Structure Tuning via Strain in Atomically Thin Mo Lett. 13, 2931–2936 (2013). ( ) 27 Conley H J et al Bandgap Engineering of Strained Monolayer and Bilayer MoS2 Nano Lett 13 3626–3630 (2013) 27. Conley, H. J. et al. Bandgap Engineering of Strained Monolayer and Bilayer MoS2. Nano Lett. 13, 3626–3630 (2013). 8. Wu, W. Z. et al. Piezoelectricity of single-atomic-layer MoS2 for energy conversion and piezotronics. Nature 514, 470–474 (2014). 9. Sun, L. X. et al. Strain-Induced Large Exciton Energy Shifts in Buckled CdS Nanowires. Nano Lett. 13, 3836–3842 (2013). 28. Wu, W. Z. et al. Piezoelectricity of single-atomic-layer MoS2 for energy conversion and piezotronics. Nature 514, 470–474 (20 29. Sun, L. X. et al. Strain-Induced Large Exciton Energy Shifts in Buckled CdS Nanowires. Nano Lett. 13, 3836–3842 (2013). 28. Wu, W. Z. et al. Piezoelectricity of single atomic layer MoS2 for energy conversion and piezotronics. Nature 514, 470 29. Sun, L. X. et al. Strain-Induced Large Exciton Energy Shifts in Buckled CdS Nanowires. Nano Lett. 13, 3836–3842 (20 g gyt 30. Signorello, G. et al. Tuning the Light Emission from GaAs Nanowires over 290 meV with Uniaxial Strain. Nano Lett. 13, 917–924 (2013). Scientific Reports \| 6:26607 \| DOI: 10.1038/srep26607 Scientific Reports \| 6:26607 \| DOI: 10.1038/srep26607 6 www.nature.com/scientificreports/ 1. Khang, D. Y. et al. A stretchable form of single-crystal silicon for high-performance electronics on rubber substrates. Science 311 208–212 (2006). 208 212 (2006). 32. Sun, Y. G. & Rogers, J. A. Structural forms of single crystal semiconductor nanoribbons for high-performance stretchable electronics J Mater Chem 17 832–840 (2007) 32. Sun, Y. G. & Rogers, J. A. Structural forms of single crystal semiconductor nanoribbons for high-performance stretc electronics. J. Mater. Chem. 17, 832–840 (2007). 33. Kim, D. H. & Rogers, J. A. Stretchable Electronics: Materials Strategies and Devices. Adv. Mater. 20, 4887–4892 (2008). 33. Kim, D. H. & Rogers, J. A. Stretchable Electronics: Materials Strategies and Devices. Adv. Mater. 20, 4887–4892 (2008). 8, 3412–3420 (2014). 34 Khang, D Y et al Mechanical Buckling: Mechanics, Metrology, and Stretchable Electronics Adv Funct Mater 19, 1526–1536 33. Kim, D. H. & Rogers, J. A. Stretchable Electronics: Materials Strategies and Devices. Adv. Mater. 20, 4887 4892 (2008). 34. Khang, D. Y. et al. Mechanical Buckling: Mechanics, Metrology, and Stretchable Electronics. Adv. Funct. Mater. 19, 1526–1536 33. Kim, D. H. & Rogers, J. A. Stretchable Electronics: Materials Strategies and Devices. Adv. Mater. 20, 4887 4892 (2008). 34. Khang, D. Y. et al. Mechanical Buckling: Mechanics, Metrology, and Stretchable Electronics. Adv. Funct. Mater. 19, 1526–1536 (2009) 34. Khang, D. Y. et al. Mechanical Buckling: Mechanics, Metrology, and Stretchable Electronics. Adv. Funct. Mater. 19, 1526– (2009).i ( ) 35. Feng, X. et al. Stretchable Ferroelectric Nanoribbons with Wavy Configurations on Elastomeric Substrates. ACS Nano 5, 3326–3332 (2011). ( ) 6. Sun, Y. G. et al. Controlled buckling of semiconductor nanoribbons for stretchable electronics. Nat. Nanotechnol. 1, 201–207 (2006) 36. Sun, Y. G. et al. Controlled buckling of semiconductor nanoribb 36. Sun, Y. G. et al. Controlled buckling of semiconductor nanoribbons for stretchable electronics. Nat. Nanotechnol. 1, 201–207 (2006). 37. Qi, Y. et al. Enhanced Piezoelectricity and Stretchability in Energy Harvesting Devices Fabricated from Buckled PZT Ribbons. Nano Lett. 11, 1331–1336 (2011). g 37. Qi, Y. et al. Enhanced Piezoelectricity and Stretchability in Energy Harvesting Devices Fabricated from Buckled PZT Ribbons. Nano Lett. 11, 1331–1336 (2011). , ( ) 38. Law, M. et al. Nanoribbon waveguides for subwavelength photonics integration. Science 305, 1269–1273 (2004). 39. Yan, H. Q. et al. ZnO nanoribbon microcavity lasers. Adv. Mater. 15, 1907–1911 (2003). 40. Pan, A. L. et al. Optical waveguide through CdS nanoribbons. Small 1, 980–983 (2005). 40. Pan, A. L. et al. Optical waveguide through CdS nanoribbons. Small 1, 980–983 (2005). 1. Xu, J. Y. et al. Room-Temperature Dual-Wavelength Lasing from Single-Nanoribbon Lateral Heterostructures. J. Am. Chem. Soc 134, 12394–12397 (2012). , J R p 134, 12394–12397 (2012). 42. Sun, L. X. et al. Resolving Parity and Order of Fabry-Perot Modes in Semiconductor Nanostructure Waveguides and Lasers: Young’s Interference Experiment Revisited. Nano Lett. 14, 6564–6571 (2014). p 43. Zhang, J. et al. Laser cooling of a semiconductor by 40 kelvin. Nature 493, 504–508 (2013). 44. Li, D. H. et al. Strain-induced spatially indirect exciton recombination in zinc-blende/wurtzite CdS heterostructures. Nano R 3035–3044 (2015). 45. Desai, S. B. et al. Strain-Induced Indirect to Direct Bandgap Transition in Multi layer WSe2. Nano Lett. 14, 4592–4597 (2014). 46. Van Vugt, L. K. et al. One-dimensional polaritons with size-tunable and enhanced coupling strengths in semiconductor nano Proc. Natl. Acad. Sci. USA 108, 10050–10055 (2011). 47. Piccione, B. et al. All-optical active switching in individual semiconductor nanowires. Nat. Nanotechnol. 7, 640–645 (2012). , p g , ( ) 48. Agarwal, R. et al. Lasing in single cadmium sulfide nanowire optical cavities. Nano Lett. 5, 917–920 (2005). et al. Lasing in single cadmium sulfide nanowire optical cavities. N Agarwal, R. et al. Lasing in single cadmium sulfide nanowire optica i 49. Oulton, R. F. et al. Plasmon lasers at deep subwavelength scale. Nature 461, 629–632 (2009). 50. Ren, M. L. et al. Enhanced second-harmonic generation from metal-integrated semiconductor nanowires via high whispering gallery modes Nat Commun 5 5432 (2014) 50. Ren, M. L. et al. Enhanced second-harmonic generation f 50. Ren, M. L. et al. Enhanced second-harmonic generation from metal-integrated semiconductor nanowires via highly confined whispering gallery modes. Nat. Commun. 5, 5432 (2014).i whispering gallery modes. Nat. Commun. 5, 5432 (2014).i p g g y 51. Li, D. H. et al. Surface Depletion Induced Quantum Confinement in CdS Nanobelts. ACS Nano 6, 5283–5290 (2012). g g y H. et al. Surface Depletion Induced Quantum Confinement in CdS N 51. Li, D. H. et al. Surface Depletion Induced Quantum Confine i 52. Agarwal, R. et al. Real-Time Observation of Morphological Transformations in II–VI Semiconducting Nanobelts via En Transmission Electron Microscopy. Nano Lett. 15, 3303–3308 (2015).f py 53. Han, X. B. et al. Strain-Gradient Effect on Energy Bands in Bent ZnO Microwires. Adv. Mater. 24, 4707–4711 (2012). 54. Xu, S. G. et al. Piezotronic Effects on the Optical Properties of ZnO Nanowires. Nano Lett. 12, 5802–5807 (2012). y 53. Han, X. B. et al. Strain-Gradient Effect on Energy Bands in Bent ZnO Microwires. Adv. Mater. 24, 4707–4711 (2012). Additional Information upplementary information accompanies this paper at http://www.nature.com/srepi Supplementary information accompanies this paper at http://www.nature.com/srepihi 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: Wang, Q. et al. Emission energy, exciton dynamics and lasing properties of buckled CdS nanoribbons. Sci. Rep. 6, 26607; doi: 10.1038/srep26607 (2016). How to cite this article: Wang, Q. et al. Emission energy, exciton dynamics and lasing properties of buckled CdS nanoribbons. Sci. 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https://openalex.org/W2768958220	https://www.ecologyandsociety.org/vol22/iss4/art30/ES-2017-9480.pdf	English	null	Land-use change in a telecoupled world: the relevance and applicability of the telecoupling framework in the case of banana plantation expansion in Laos	Ecology and society	2,017	cc-by	15,831	Research, part of a Special Feature on Telecoupling: A New Frontier for Global Sustainability Land-use change in a telecoupled world: the relevance and applicability of the telecoupling framework in the case of banana plantation expansion in Laos Cecilie Friis 1,2 and Jonas Østergaard Nielsen 1,2 ABSTRACT. Land-use change is increasingly influenced by complex socioeconomic and environmental interactions that transcend spatial, institutional, and temporal scales. These interactions challenge classical place-based land system analysis and require new analytical approaches equipped for tackling processes, flows, and feedbacks over distance. The recently proposed telecoupling framework offers interesting perspectives for bringing place-based and process-oriented research together in the study of land-use change. However, few studies have explored the influence and implications of telecouplings in local land-use changes. One reason for this is that the framework still faces challenges for application in empirical research. Here, we offer a qualitative operationalization of the telecoupling framework to explore its relevance and applicability in a case of local land-use change. Investigating the case of a recent boom in commercial banana cultivation in Luang Namtha Province, Lao PDR, we use a grounded empirical approach starting with the observed land-use change at the village level. We then trace flows and distal processes influencing the conversion to banana cultivation from the perspectives and experiences of the local actors involved. The results identify four prominent material and immaterial telecouplings at various spatial and temporal scales, as well as some potential feedbacks. This complexity points to the need for interdisciplinary research because the processes involved in creating telecoupled land-use change transcend the boundaries of any one discipline. Overall, however, telecoupling presents a strong heuristic lens for examining and describing distal causal relations in land-use change in a manner that does not favor a specific analytical scale or type of interaction. Key Words: case-study research; Chinese investments; distal flows; feedbacks; land systems; land-use change; Laos; qualitative research; t l li Key Words: case-study research; Chinese investments; distal flows; feedbacks; land systems; land-use change; Laos; qualitative research; telecoupling al. 2012, Liu et al. 2013, Eakin et al. 2014). Telecoupling describes combined socioeconomic and environmental interactions, as well as potential feedbacks and spillover effects between two or more coupled human-environment systems (Liu et al. 2013, Eakin et al. 2014). A growing number of studies have advanced the understanding of telecouplings at regional and national scales (e.g., Liu 2014, Gasparri et al. 2016, Liu et al. 2015a, Chignell and Laituri 2016, Deines et al. 2016) and for material and trade-related interactions (e.g., Bruckner et al. 2015, Henders et al. 2015, Kastner et al. 2015, Schaffartzik et al. 2015, Fang et al. 2016, Schierhorn et al. Copyright © 2017 by the author(s). Published here under license by the Resilience Alliance. Friis, C., and J. Ø. Nielsen. 2017. Land-use change in a telecoupled world: the relevance and applicability of the telecoupling framework in the case of banana plantation expansion in Laos. Ecology and Society 22(4):30. https://doi.org/10.5751/ ES 09480 220430 Copyright © 2017 by the author(s). Published here under license by the Resilience Alliance. Friis, C., and J. Ø. Nielsen. 2017. Land-use change in a telecoupled world: the relevance and applicability of the telecoupling framework in the case of banana plantation expansion in Laos. Ecology and Society 22(4):30. https://doi.org/10.5751/ ES 09480 220430 Copyright © 2017 by the author(s). Published here under license by the Resilience Alliance. Friis, C., and J. Ø. Nielsen. 2017. Land-use change in a telecoupled world: the relevance and applicability of the telecoupling framework in the case of banana plantation expansion in Laos. Ecology and Society 22(4):30. https://doi.org/10.5751/ ES-09480-220430 Research, part of a Special Feature on Telecoupling: A New Frontier for Global Sustainability Land-use change in a telecoupled world: the relevance and applicability of the telecoupling framework in the case of banana plantation expansion in Laos 2016, Bicudo da Silva et al. 2017). However, less attention has been given to the immaterial dimensions of telecoupling, and to our knowledge, only two studies have engaged in empirical analysis of how telecouplings influence local land-use change (i.e., Baird and Fox 2015, Leisz et al. 2016). One reason for this lack of study is that the framework still faces a number of challenges for empirical application in case study research (Friis et al. 2016a, GLP 2016), and although there have been calls and emerging advancements for methodological operationalization through quantitative and modeling approaches (Liu et al. 2015b, Millington et al. 2017), there is still a need for critical qualitative engagement with the framework. 1IRI THESys, Humboldt-Universität zu Berlin, 2Geography Department, Humboldt-Universität zu Berlin INTRODUCTION That “places are processes too” (Massey 1991:29) is increasingly acknowledged by scholars working on urban dynamics and land- use change, who argue for a recognition of the mutually constitutive processes linking specific urban and rural places regardless of their geographical location (Seitzinger et al. 2012, Seto et al. 2012, Güneralp et al. 2013, Qureshi and Haase 2014). We next introduce the theoretical perspectives that frame our study, including the telecoupling framework, before we describe the methodology, fieldwork, and data. In the main section of the paper, we present the results of our study in two parts. In the first part, the details of the new banana land system is described, including the land-use change from rice paddies to banana, and the main actors involved. In the second part, we identify and outline the main flows and causal relations behind the expansion based on the accounts of our informants and situated in the context of agricultural transformation in northern Laos. Subsequently, we sketch out how these flows and relations constitute four telecouplings, as well as the first societal feedbacks of the banana expansion, and discuss the implications of this analysis. We then emphasize the strength of a qualitative operationalization of the telecoupling framework as a heuristic device for exploring and describing distal causal relations in local land-use change and discuss it in relation to other analytical approaches. We finish with a conclusion. These efforts toward the integration of place-based and processual analyses in LSS are captured in the telecoupling framework (Eakin et al. 2014, Liu et al. 2014). Conceptually, telecoupling presents a way to address “not only the ‘action at a distance’ but also the feedback between social processes and land outcomes in multiple interacting systems” (Eakin et al. 2014:143). Telecoupling arises when a so-called trigger sets in motion changes in, e.g., policy, consumer demand, or land use in one human-environment system that indirectly or unexpectedly cause change in another, distant, human-environment system. The unfolding change in the “receiving” land system then potentially gives rise to feedback processes returning the signal of change or to spillover processes affecting systems “outside” the main interaction. In a structured framework presented by Liu et al. (2013), five main analytical components of telecoupled human- environment systems are defined, i.e., systems, flows, agents, causes, and effects, and systems are classified as sending, receiving, or spillover systems, depending on their role in a particular interaction. INTRODUCTION Our study examines the questions: What are the main causes of the banana expansion, and how is this expansion influenced by telecouplings? We explore these questions through a qualitative case study, taking as an analytical entry point the “observed” land-use change and working progressively “outward” by tracing the actors, flows, and processes involved (Vayda 1983). This grounded empirical approach inspired by human geography and political ecology (e.g., Blaikie and Brookfield 1987, Walters and Vayda 2009) offers an advantageous qualitative operationalization of the telecoupling framework that allows us to outline the main distal interactions from the perspectives of the local actors. By using telecoupling as a flexible heuristic lens, it becomes possible to identify several causal linkages with varying spatial and temporal extents, as well as some regulatory feedbacks. We thus demonstrate the importance of qualitative analysis for telecoupling research, especially with regard to capturing immaterial interactions such as social relations, discourses, and information. 2013). Social-ecological systems scholars have made similar efforts to address teleconnected vulnerabilities in environment and livelihood change (Adger et al. 2009, Eakin et al. 2009). Others have begun to engage with flow-based analyses using global value chain and global production network approaches adopted from economic geography (Garrett et al. 2013, Rueda and Lambin 2013, Galvan-Miyoshi et al. 2015, le Polain de Waroux et al. 2016). Although these efforts have substantially advanced the understanding of complex and geographically disconnected dynamics between land systems, a more fundamental critique of the basic conceptualization of land systems as place-based bounded entities has emerged (see, e.g., Mansfield et al. 2010, Seto et al. 2012, Munroe et al. 2014). Scholars have emphasized the need for LSS to engage with relational understandings of place and space found in critical human and economic geography (e.g., Massey 1991, Henderson et al. 2002, Jessop et al. 2008) to move beyond, for example, hierarchically nested scale conceptualizations that often result in a conflation of spatial scale with agency (Munroe et al. 2014). Others have stressed that places and place- based change should always be analyzed as the result of the (social) relations, interactions, and processes that connect them to other places (Mansfield et al. 2010, Niewöhner et al. 2016). INTRODUCTION Change processes in local to global land systems are becoming ever more complex and intertwined (Turner et al. 2007, Meyfroidt et al. 2013, Seto and Reenberg 2014, Verburg 2014, Verburg et al. 2015). Long-distance movements of raw material, products, energy, and waste, as well as technology, information, discourses, capital, and people have increased the interconnectedness of places around the world. At the same time, distal flows are facilitating spatial, institutional, and temporal decoupling of the causes and outcomes of land-use change that challenge place- based land system analysis (Erb et al. 2009, Reenberg et al. 2010, Seto et al. 2012, Kastner et al. 2014a, van Vliet et al. 2016). The growth in global volumes of biomass trade, for example, attests to the increasing distance between the places of demand and places of supply for land-based products (Erb et al. 2009, Kastner et al. 2014). The intensified connectedness of places and people, cities and their hinterlands, and sites of production and consumption often manifests itself as rapid and unexpected land- use change, especially in forest and agricultural commodity frontiers, where economic, political, and sustainability agendas converge to create multiple overlapping and conflicting claims to land (van Vliet et al. 2012, Gasparri et al. 2016, Taylor 2016). Understanding contemporary land-use changes thus requires a conceptual framework geared toward capturing not only the place-based and site-specific factors of change, but also the multidirectional flows of capital, produce, and information linking it to processes in distal places. Here, we contribute to this agenda by exploring the relevance and applicability of the telecoupling framework in the case of banana plantation expansion in Luang Namtha Province, Laos. Since 2008, mono-cropped banana plantations have expanded rapidly in this area, resulting in widespread conversion of paddy rice fields and gardens to banana. The Chinese investors driving the banana production are leasing land from Lao farmers and Within the literature on coupled human-environment systems and land system science (LSS), the recently established telecoupling framework provides a basis for such integrative research (Seto et 1IRI THESys, Humboldt-Universität zu Berlin, 2Geography Department, Humboldt-Universität zu Berlin Ecology and Society 22(4): 30 https://www.ecologyandsociety.org/vol22/iss4/art30/ Ecology and Society 22(4): 30 andsociety.org/vol22/iss4/art30/ exporting the fruit to the Chinese market. This indicates a clear separation of origin of demand and supply of the banana that potentially involves telecoupling. INTRODUCTION Moreover, telecoupling alludes to the inherently networked and cross-scalar causal processes linking change in two or more distant human-environment systems in multiple material and immaterial ways (Eakin et al. 2014). In relation to a classical LSS approach focusing on proximate causes and underlying driving forces (Geist and Lambin 2002, Geist et al. 2006), telecoupling enhances the analytical attention to specific causal interactions between land systems rather than to localized factors or broader underlying drivers. METHODOLOGY “Conceptual frameworks are neither models nor theories [...] rather they help to think about phenomena, to order material, revealing patterns” (Rapoport 1985, as cited in Berkes and Folke 1998:15). As a conceptual framework, telecoupling presents a way to think about distal interactions between human-environment systems. This makes multiple analytical entry points and methodological approaches possible and allows for a focus on certain aspects of the telecoupling while maintaining an overview of the entire process (Eakin et al. 2014, Friis et al. 2016a). For this study, our starting point was the observed land-use change in the study area: the expansion of banana plantations on lowland rice fields. By working through the fundamental methodological position used by anthropologists and human geographers asking “What the hell is going on?” (Geertz, as cited in Olson 1991:248), we trace the actors involved, their networks of interaction, and the (distal) flows involved in the banana boom. Understanding what is going on, who is involved, and how a particular land-use change takes place allows us to explore why the situation unfolds at this particular place and in this particular social, political, and historical moment. Fig. 1. Map of the study area and the main sites linked to banana plantations in Luang Namtha Province, Lao PDR. Fig. 1. Map of the study area and the main sites linked to banana plantations in Luang Namtha Province, Lao PDR. This approach to telecoupling analysis thus builds on the legacy of well-established approaches in LSS, human geography, and political ecology, and in the wider field of human-environment research for tracing processes and causal explanations outward in space and time from specific place-based changes, events, and experiences (e.g., Vayda 1983, Blaikie and Brookfield 1987, de Groot 1992, Verburg et al. 2003, Zimmerer and Bassett 2003, Perz and Almeyda 2009, Walters and Vayda 2009, Nielsen and Reenberg 2012, Meyfroidt 2016). These fields have long histories of researching how global flows, exchanges, and networks influence local social and environmental change through grounded empirical case studies exploring, for example, the increasing globalization of agriculture and rural livelihoods (e.g., Bebbington and Batterbury 2001, Zimmerer 2007) and translocal or transnational migration and the role of remittances in changing land access and land use (see, e.g., Moran-Taylor and Taylor 2010, Piguet 2010, Barney 2012). THEORETICAL PERSPECTIVES Land systems are conventionally conceptualized as bounded place- based human-environment systems (Turner et al. 2003, GLP 2005, Liu et al. 2007) or social-ecological systems (Folke et al. 2005, Young et al. 2006, Fischer-Kowalski and Haberl 2007). A focal point of scientific exploration has therefore been studies that model and characterize patterns of land-use and land-cover change in particular regions, countries, or at the global level, as well as (case) studies analyzing the causal processes of land change, land-use decisions, and land management impacts in specific geographical locales (Rindfuss et al. 2004, Turner et al. 2007, Verburg et al. 2013, 2015, Müller and Munroe 2014). However, the growing prominence of socioeconomic and environmental flows connecting geographically distant land systems challenges place-based analyses. Recently, LSS researchers have therefore started to focus on distal drivers and indirect land-use changes such as displacements, leakages, and cascade effects (Lambin and Meyfroidt 2011, Meyfroidt et al. 2013, Chen et al. 2014, Friis et al. 2016b), “land teleconnections” (Nepstad et al. 2006, Haberl et al. 2009, Yu et al. 2013, Henders et al. 2015, Schaffartzik et al. 2015), and “urban land teleconnections” (Seto et al. 2012, Güneralp et al. While the telecoupling framework is gaining momentum in LSS, it still faces a number of challenges for operationalization and Ecology and Society 22(4): 30 https://www.ecologyandsociety.org/vol22/iss4/art30/ Ecology and Society 22(4): 30 yandsociety.org/vol22/iss4/art30/ application in empirical research (Friis and Nielsen 2014, Friis et al. 2016a). The complexity of telecoupling processes and the comprehensive scope of the framework challenge researchers by introducing a fundamental trade-off between analytical depth and temporal and spatial coverage. In addition, the ambiguity of demarcating system boundaries and attributing roles to the interacting systems is analytically challenging because spatial and temporal scales of analysis will invariably influence the nature and extent of the networks of actors, causes, and effects that are attributed to one system as opposed to another. Here, we meet these challenges by employing the framework as a flexible heuristic lens. As such, telecoupling presents a conceptual framework that takes the increasing global interconnectivity between people and places for granted while breaking this connectivity down into tractable units of analysis. This approach allows for processual and networked analysis without abandoning concrete and place-based land-use change research (Eakin et al. 2014, Friis et al. 2016a). Study area i ld k y Fieldwork for the study was carried out in Luang Namtha Province, Lao PDR (Fig. 1) in April–May and August–December 2014, as well as June 2015. Luang Namtha Province is located in the northern uplands of Laos in the borderland toward Myanmar and China, making the province an important gateway for trade in the region. An exploratory survey and key informant interviews with village authorities in 16 villages hosting banana plantations in Muang Sing and Muang Long districts provided an initial overview of the dynamics involved in the banana boom. These remote districts are characterized by a rugged mountainous terrain and narrow river valleys, and the district road connects Muang Long town with Muang Sing town, a main trading town and gateway to China, in the east (Fig. 1). THEORETICAL PERSPECTIVES concessions in Laos and Cambodia using a political ecology inspired “grounded approach, contextualizing from the local up to the global scale, land-use and forest-cover changes, and interrelations with political-economic dynamics.” Drawing on these approaches can therefore add qualitative methodological depth to telecoupling research aimed not only at understanding material flows, but immaterial interactions as well. Ecology and Society 22(4): 30 https://www.ecologyandsociety.org/vol22/iss4/art30/ Ecology and Society 22(4): 30 https://www.ecologyandsociety.org/vol22/iss4/art30/ Table 1. Synthesis of methods used, informants, data acquisition timeframe, place, and quantitative density. Method Informants Data acquisition timeframe Place Quantitative density Participant observation August–December 2014, June 2015 Ban Sirimoon Presence in the village Semistructured interviews Middlemen, land brokers August–December 2014, June 2015 Ban Sirimoon 12 interviews Government officials at the senior level April–May 2014, August– December 2014, June 2015 Luang Namtha Province, Long District 27 interviews Banana investors, plantation managers November and December 2014, June 2015 Long District 8 investors, 3 plantation managers Villagers June 2015 Ban Sirimoon 12 interviews with 17 villagers (including two small groups) Household questionnaire survey Heads of household and their wives September–November 2014 Ban Sirimoon 48 of 66 households interviewed; randomly sampled based on list of households in village † Focus-group discussions Villagers September–December 2014, June 2015 Ban Sirimoon 12 groups; 3 to 8 participants; differentiated according to age, gender, and main agricultural activities Informal conversations Village authorities, villagers, banana contract laborers, banana buyers, Chinese agro- traders April–May 2014, August– December 2014, June 2015 Long District, Ban Sirimoon †A sample of 50 households was selected, but 2 households were unavailable for the survey. managers, and banana buyers working in the area. In addition, repeated semistructured interviews with senior officials at five governmental departments (Agricultural and Forestry Department, Department of Environment and Natural Resources, Department of Planning and Investment, Department of Trade and Industry, and Department of Social Welfare and Labor) at district and provincial levels provided contextual information. All semistructured interviews and focus-group discussions contained questions related to the role of the various stakeholders in banana plantation development, their perception of why it was taking place, and their perspectives on the future implications of the plantations. Furthermore, interviews with investors and plantation managers included questions related to their prior experience with banana cultivation, their reasons for investing in banana cultivation, and the process of exporting the bananas. minority of Samtao people (several villagers, however, also referred to themselves as Doi Samtao); however, as Buddhists, they share cultural traits with the larger group of Lue people in the area, and the villagers speak Lue in addition to their main Samtao language. The village is located on the district road approximately 30 km East of Muang Long town and 20 km West of Muang Sing town. Ecology and Society 22(4): 30 https://www.ecologyandsociety.org/vol22/iss4/art30/ Its territory therefore includes a narrow strip of lowland in the valley between the road and the Nam Ma River, as well as the hills on both sides of the valley. Many households combine lowland paddy rice production with rotational shifting cultivation of upland rice, and while the villagers still engage in subsistence agriculture, cash-crop production of crops such as maize, cassava, and sugarcane has been on the rise in recent years. Most recently, agricultural change accelerated when the village was incorporated into the banana boom and started leasing land to two Chinese banana investment companies in 2010. All interviews were conducted in the Lao, Lue, or Chinese language. The same translator, a local Lue man from Muang Sing District, with a background in environmental sciences, was used for all interviews. The interviews were digitally recorded and subsequently transcribed and translated into English before being coded and analyzed qualitatively, as were field notes taken during participant observation. Direct quotes used here have been corrected grammatically for readability. For describing the village, we use the unofficial name used by the villagers themselves, whereas all names of informants and companies have been changed to ensure anonymity. Methods During fieldwork, participant observation and informal conversations enabled us to gain insights into daily activities in the village and create trust for discussions of sensitive topics, including the processes around leasing out land for banana plantations. A household questionnaire survey provided background information about general livelihood strategies and land-use activities, as well as household participation in the plantation developments. In addition, semistructured and focus- group interviews with villagers provided information on the establishment of the banana plantations (Table 1). METHODOLOGY Within the telecoupling literature, Baird and Fox (2015:440) take a first inspiring step in such directions by investigating nearby, opportunistic, and transnational telecouplings associated with large-scale land Based on the village survey, Ban Sirimoon, a small rural community in Muang Long District hosting two banana plantations since the end of 2010, was selected as the starting point for the empirical investigation using a set of qualitative criteria, including the ratio of land occupied by the plantations, the proportion of households involved in the schemes and that perceived reported effects of the plantations should be both positive and negative. Ban Sirimoon has 323 people in 66 households (August 2014), mainly belonging to the small ethnic Based on the village survey, Ban Sirimoon, a small rural community in Muang Long District hosting two banana plantations since the end of 2010, was selected as the starting point for the empirical investigation using a set of qualitative criteria, including the ratio of land occupied by the plantations, the proportion of households involved in the schemes and that perceived reported effects of the plantations should be both positive and negative. Ban Sirimoon has 323 people in 66 households (August 2014), mainly belonging to the small ethnic THE BANANA LAND SYSTEM Area of banana harvested (A) and production of banana (B) in Lao PDR between 1975 and 2013. Data: FAO (2016). Fig. 2. Area of banana harvested (A) and production of banana (B) in Lao PDR between 1975 and 2013. Data: FAO (2016). The expansion of banana plantations in Muang Long District began in approximately 2008, and by November 2014, 820.75 ha of banana were planted in the district, according to official records from the District Agriculture and Forestry Office (DAFO). These records include 13 legally registered banana investment companies holding between 16.63 and 269.83 ha on individual plantation plots ranging from < 1 to 61.87 ha. The investors are predominantly small Chinese companies and private businessmen from the borderland region and/or with long term The case of Ban Sirimoon illustrates how the majority of investors gain access to land. In the village, two banana companies successfully leased approximately 35 and 46 ha of land at the end of 2010. One of the companies (LFA Company) is a joint venture between five Chinese investors of mixed ethnicity, including a Tai Lue Chinese partner (who could communicate with the villagers in the Lue language) and others who had previously traded in forest and agricultural products in the province. The other investor (XG Company) is owned by a Han Chinese investor with > 20 years of business experience in the district. In the land acquisition process, both companies made extensive use of their personal relationships and referrals by contacts to identify and employ well-connected local middlemen tasked with finding suitable land, making contact in the targeted villages, and engaging village land brokers. In Ban Sirimoon, the LFA and XG middlemen hired various old acquaintances as land brokers to facilitate the negotiations with the landholders, and the land brokers also acted as first movers in accepting the contracts. These informal land acquisition strategies made it possible for the investors to circumvent any official involvement of the government authorities until the contracts had been set up. Subsequently, official investment permissions were obtained, and both companies held these for the plantations in Ban Sirimoon. However, the XG Company actually operated as an intermediary for a private Chinese investor, who subsequently sold the plantation to another private Chinese investor, and the LFA Company sold its plantation to a Chinese joint venture in February 2015 (see also Friis and Nielsen 2016). THE BANANA LAND SYSTEM Since the mid-2000s, rapid and widespread conversion of land to mono-cropped banana plantations has taken place in Luang Namtha Province as well as several other provinces in northern Laos (see also Higashi 2015, Ling 2015, Friis and Nielsen 2016). At the national level, statistics from the Food and Agriculture Organization of the United Nations show that the area of banana The insights gained in the village informed our interviews with key banana stakeholders. Using snowball sampling techniques (Bernard 2002) whereby stakeholders were identified sequentially by information provided by other informants, we identified middlemen, land brokers, other banana investors, plantation Ecology and Society 22(4): 30 https://www.ecologyandsociety.org/vol22/iss4/art30/ Ecology and Society 22(4): 30 andsociety.org/vol22/iss4/art30/ harvested per year in Laos began increasing in the mid-1990s, but especially accelerated since 2008–2009 (see Fig. 2; historical data at the district and provincial levels were not available). This expansion of banana plantations has produced a new banana land system consisting of the fields of banana plantations as the place-based component of the system, and the networks of actors and governance arrangements influencing this land-use change as the processual component (see also Friis and Nielsen 2017). harvested per year in Laos began increasing in the mid-1990s, but especially accelerated since 2008–2009 (see Fig. 2; historical data at the district and provincial levels were not available). This expansion of banana plantations has produced a new banana land system consisting of the fields of banana plantations as the place-based component of the system, and the networks of actors and governance arrangements influencing this land-use change as the processual component (see also Friis and Nielsen 2017). investors, the initial investment capital came from personal saving and loans from friends and family. Because the sales transactions take place in China, revenues are only partly reinvested or paid in taxes in Laos. For cultivating the bananas, several investors explained that they have experience in banana production in China or bring in technicians and plantation managers who do. While some investors noted that they used such previous experience and personal contacts in China for selling the crops, most of the sales are facilitated by Chinese trading agencies. For the most part, the banana investors carry the costs and paperwork involved in exporting the bananas, whereas the buyers or their trading agencies assist with importing the bananas to China and organizing the transport from plantations to market. Fig. 2. THE BANANA LAND SYSTEM For both plantations, the contracts are 6 years and the land leasing fee is 10 million LAK ha−1 yr−1 (1 USD ≈ 8078 LAK based on the Bureau of Fiscal Services, U.S. Department of Treasury exchange rate on 31 December 2014; https://www.fiscal.treasury.gov/ fsreports/rpt/treasRptRateExch/itin-12-31-2014.pdf). Elsewhere in the district, contracts varied between 3 and 6 years and renting fees between 8 million and 20 million LAK ha−1 yr−1 depending on the quality of the land and the timing of the contracts. The expansion of banana plantations in Muang Long District began in approximately 2008, and by November 2014, 820.75 ha of banana were planted in the district, according to official records from the District Agriculture and Forestry Office (DAFO). These records include 13 legally registered banana investment companies holding between 16.63 and 269.83 ha on individual plantation plots ranging from < 1 to 61.87 ha. The investors are predominantly small Chinese companies and private businessmen from the borderland region and/or with long-term business relations in the area. Both investors and government officers emphasized that setting up and maintaining banana plantations involve substantial costs. For most of the interviewed The expansion of banana plantations in Muang Long District began in approximately 2008, and by November 2014, 820.75 ha of banana were planted in the district, according to official records from the District Agriculture and Forestry Office (DAFO). These records include 13 legally registered banana investment companies holding between 16.63 and 269.83 ha on individual plantation plots ranging from < 1 to 61.87 ha. The investors are predominantly small Chinese companies and private businessmen from the borderland region and/or with long-term business relations in the area. Both investors and government officers emphasized that setting up and maintaining banana plantations involve substantial costs. For most of the interviewed Because of the specific cultivation requirements for banana, investors primarily target accessible and fertile lowland areas along the main roads and rivers. Consequently, there has been an extensive conversion of rice paddies to banana plantations throughout the district, as well as in neighboring Muang Sing District. This conversion entails complete field transformations, including the destruction of traditional irrigation channels and plot borders, and cultivation of banana in monocultures with seedlings imported from China and with heavy application of Ecology and Society 22(4): 30 https://www.ecologyandsociety.org/vol22/iss4/art30/ Ecology and Society 22(4): 30 andsociety.org/vol22/iss4/art30/ chemical inputs. Dynamic land use intensification and frontier development in northern Laos The expansion of banana plantations in Luang Namtha Province represents the latest boom crop in an area that has long been influenced by external actors and distal drivers of land-use change. Historically, the province prospered from its strategic location on the trade routes from Yunnan, China to Siam (now Thailand) bringing flows of goods and people (Thongmanivong et al. 2009, Sturgeon 2013a); however, these interactions were largely disrupted during the two Indochina Wars (1945–1954 and 1959–1975) and in the politically tense post-war period, turning the region into a remote and “underdeveloped” frontier (Lagerqvist 2013). Following a number of political and economic reforms carried out by the Government of Laos since the late 1980s, including an ongoing transformation from planned to socialist market economy, a relaxation of political tensions with its neighboring countries, and a reopening of the regional borders in the mid-1990s (Lund 2011, Lestrelin et al. 2012), Luang Namtha Province has gradually been opened up to foreign investors and incorporated into the national and regional economy. Coinciding with the growing economic strength and demand for natural resources in Laos’ big neighboring countries (China, Thailand, and Vietnam) and with a general improvement in road infrastructure (Lyttleton et al. 2004, Sturgeon 2013b), these developments have resulted in substantial increases in transnational investments in land and agricultural production (Schönweger et al. 2012, Messerli et al. 2015). In addition to large- scale formal investment in, for example, infrastructure and rubber production (Shi 2008, Dwyer 2011, Vongvisouk and Dwyer 2016), the borderland region is characterized by informal economic interactions between people with close ethnic and kinship relations across the border (Lyttleton et al. 2004, Sturgeon 2010, 2013a, Lagerqvist 2013), as well as by returning refugees who, among other things, introduced smallholder rubber in the early 1990s (Manivong and Cramb 2008b, Baird and Vue 2017). The These historical flows of governmental development interventions, land management, and policy narratives of upland underdevelopment emanating from the central government in Vientiane, the capital, underpins the ongoing agricultural intensification embedded in the banana plantation expansion. A strong agricultural modernization discourse was prevalent among the informants, both villagers and government authorities, as well as Chinese investors. In Ban Sirimoon, such discourses found expression in the villagers’ main reasons for leasing out land. Many people commented that it was necessary to “follow society’s development” and noted that banana plantations helped them to achieve this. TRACING DISTAL INTERACTIONS AND POTENTIAL FEEDBACKS From the accounts of our informants, we identified a number of historical, social, economic, environmental, and political interactions behind the plantation expansion linking the banana land system to distal places and processes. In the following, we briefly contextualize the banana boom before sketching out these interactions. THE BANANA LAND SYSTEM In Ban Sirimoon, approximately one-third of the households (19 of 66) were involved, and the 16 households participating in the household survey on average leased out 0.93 ha of land (ranging from 0.2 to 1.44 ha). For the majority of these households, this land constituted their only paddy rice fields, and a few plots had previously been used for sugarcane, vegetable gardens, or young fallow. The rest of the leased land belonged to people in neighboring villages. Although the villagers indicated that the land had been subject to water shortage for rice production in the past, the production of rice from these fields had constituted a considerable part of their rice supplies. The continued spread of banana in the district, including on some very productive rice paddies in three neighboring villages in 2015, caused considerable concern in the village with regard to the generally increasing rice prices. result was a series of crop booms of, aside from rubber, sugarcane, watermelon, pumpkin, cassava, and maize, causing massive land- use change and spurring the ongoing transition from subsistence to market-oriented livelihood strategies (Manivong and Cramb 2008a, Thongmanivong et al. 2009, Baird and Vue 2017, Cramb et al. 2017). In Muang Long District, the improved political and economic relations between Laos and China, including the increasing call for foreign investments by the Government of Laos, were also mentioned by several interviewed banana investors who explained that they saw these calls as an opportunity to move away from trade in forest products and local crops toward commodity crop investment and production. Reinforcing the agricultural transformations in this area are substantial governmental efforts to control and territorialize the northern borderlands, as well as its populations of “unruly” and “marginal” ethnic minorities (Lagerqvist 2013). Using arguments of poverty alleviation and socioeconomic development, the Government of Laos has carried out a host of land reforms, land- use zoning schemes, and forced resettlements of ethnic minorities from the uplands to the lowlands (Vandergeest 2003, Evrard and Goudineau 2004, Lestrelin et al. 2012). A pivotal objective has been to stabilize or eradicate shifting cultivation practices that are deemed undesirable and environmentally destructive (despite substantial contradictory evidence; Lestrelin 2010), and instead promote stable and economically “rational” lowland agriculture (Fujita and Phanvilay 2008, Lestrelin et al. 2012). Dynamic land use intensification and frontier development in northern Laos Moreover, the villagers stressed that to do so, increasing amounts of cash income was needed for the rising costs of electricity, medicine, schooling, and taxes, as well as to purchase rice because more and more land was converted to cash-crop production. Information about rising incomes and economic prosperity among other minority farmers engaged in “new” and “desirable” agricultural activities across the borders (e.g., Sturgeon 2010, 2013a), as well as in other areas of Laos, flowing into the village were influential in the villagers’ desire to engage with investors. Entering into new forms of agricultural activities was emphasized as being central to achieve “household improvement,” just as they had seen people in other villages in the area do. In general, villagers explained that the land leasing fees were higher than the possible income from the sale of surplus rice, and the leasing fees were viewed as “earning money without working.” The fees, along with other cash-crop income, had enabled households to invest in house improvements, small business ventures, motorbikes, and other consumer goods. Many stated that if there had been no Chinese investors for banana, as well as for other cash-crop activities, they would still be “poor.” Some of Ecology and Society 22(4): 30 https://www.ecologyandsociety.org/vol22/iss4/art30/ Ecology and Society 22(4): 30 yandsociety.org/vol22/iss4/art30/ the investors used parallel arguments to emphasize their role in “bringing development” and income to poor villagers with few economic opportunities. Fang, a Chinese banana investor, for example, stressed this by commenting, “The first time I drove through this area, I saw that these villagers were very poor, but that their land was very fertile and there was labor available to work in agriculture. Therefore, I decided to come to plant banana to make a profit and to help the local people“ (Mr. Fang, 11 December 2014). the Lao ones and they are cheaper too, so if China opens up for banana import from this country again, it will impact the Lao bananas [...]” (Mr. Sang, 12 December 2014). Recently, commentators have noted how a rapprochement between China and the Philippines has led to improved trade relations between the two countries, and for banana, in particular, this resulted in a suspension of import restrictions (Associated Press 2016, Perlez 2016, Phillips 2016, Simeon 2016). The local impacts of this change are yet to be seen. Distal market linkage, political spillover effects, and environmental push-pull factors The emphasis on the combined potential for profit and the suitability of the northern Lao landscape for banana expressed by Fang above were prominent in the narratives of all of the interviewed banana stakeholders. Overall, the investors emphasized the increasing demand for banana in China (see also Prowse 2015), especially among urban consumers, as the main driver for entering into banana investments in Laos. Several investors noted that the Chinese government was promoting fresh fruit consumption, especially to elderly people, and that a general increase in purchasing power among Chinese consumers influenced the demand. The strategic role of fresh fruit in China was stressed by some investors explaining that the banana trucks were rarely stopped for unexpected checks or nontransparent tax collection once inside China, something often encountered on the Lao side of the border. Buyers encountered during fieldwork were shipping bananas as far as Shanghai, Beijing, and several of the northern regions bordering Mongolia and Russia, and the bananas harvested on the XG plantation in Ban Sirimoon in 2015 were sold to buyers from Tianjin and Sichuan provinces. Some informants also indicated how the incentive to expand banana plantations in Laos was linked to the geopolitical dispute between China and the Philippines. A few investors, as well as district officials, recounted that China had restricted the import of bananas from the Philippines a couple of years before 2014, creating a decrease in the supply of bananas to the Chinese market. Although none of the interviewed local actors could provide substantial details of these events, it was noted that the two countries “have a problem state to state” and “a national relationship conflict.” The Philippines and China have long been in a dispute over the territorial rights to marine and island resources in parts of the South China Sea (see Associated Press 2012a,b, Branigan and Watts 2012, Reuters 2012, 2016). In 2012, the Chinese government imposed a series of import restrictions on Philippine bananas after the discovery of bacteria and infectious pests; these restrictions, however, were attributed by several commentators to the territorial conflict as part of a Chinese strategy to test the Philippines’ reaction to potential trade sanctions (Branigan and Watts 2012, Cuneta and Hookway 2012, Ravindran 2013). Dynamic land use intensification and frontier development in northern Laos In addition to market price factors and despite some complaints about the cost of setting up plantations in Laos, the banana investors highlighted that relatively low land prices compared to southern China, as well as biophysical and climatic conditions in Laos making it possible to take advantage of off-season production, constituted important drivers behind the plantations. Tao, a Han Chinese plantation manager, explained, “In Laos, we can plant banana the whole year round, which is not possible during the winter in China. Therefore, the market demand for bananas is higher in the winter, and the price is better. So we wanted to come to Southeast Asia for investing in banana plantations” (Mr. Tao, 30 November 2014). In addition, growing cultivation constraints and rising land prices in the banana- producing regions of China caused by land degradation, typhoon-related disasters, and general land-use competition made investments in Laos attractive. The main banana-producing region of Hainan in China was severely hit by Typhoon Nesat in 2011 (Xinhua 2011a,b), and huge areas of banana plantations have been destroyed by typhoons across Hainan, Guangxi, and Guangdong provinces since then (An 2014). Banana investors also mentioned that large areas of plantations in the Philippines, one of the world’s largest banana exporters (FAO 2015), had been subject to typhoon disasters in recent years (Agence France- Presse 2012, FAO 2013), providing further incentives to cultivate bananas elsewhere. Distal market linkage, political spillover effects, and environmental push-pull factors Although the diplomatic trade dispute between China and the Philippines occurred after the initial onset of the banana boom in Luang Namtha Province, banana investors in Muang Long District explained that it had created a spike in the price of Lao bananas, giving them incentives to expand plantations further. The strong position of Philippine bananas on the Chinese market was, for example, noted by Sang, a Tai Lue Chinese investor, who explained, “The bananas imported from the Philippines are very popular with the consumers. In fact, the bananas imported from the Philippines are a better quality than In turn, the image of Laos as a virgin and fertile resource frontier was prominent in the investors’ narratives, as summed up by the XG plantation manager, Yang: “They [Chinese investors] used to plant banana in Hainan, Xishuangbanna, Guangdong, and Guangxi, but many of them failed because of typhoon disasters. Also, in Xishuangbanna, the land is currently becoming unfertile because it has had banana on it for more than 10 years. That is why the investors come to Laos, because there is no risk of natural disasters and the land is virgin” (Mr. Yang, 25 September 2014). Deterioration of soil quality and consequent decreases in banana yields in long-term monoculture banana cultivation represents a substantial problem in Chinese banana plantations (see Zhong et al. 2014, 2015), as does the rapidly spreading Fusarium Wilt Tropical Race 4 or “the Panama disease,” a soil pathogenic fungus causing irreversible infection in banana roots (Ordonez et al. 2015). Because the spread of banana cultivation in Luang Namtha Province mirrors that of previous expansion across the border in the southern districts of Xishuangbanna (see, e.g., Sturgeon 2010, Zhang et al. 2014), these environmental problems are likely to start flowing into the plantations in Laos as well. In fact, several informants noted increasing problems with the Panama disease, including in the XG plantation in Ban Sirimoon, where 60 trees were infected by 2014. The villagers in Ban Sirimoon were highly Ecology and Society 22(4): 30 https://www.ecologyandsociety.org/vol22/iss4/art30/ Ecology and Society 22(4): 30 yandsociety.org/vol22/iss4/art30/ aware of these environmental risks and noted, for example, “They do not have any fertile land in China anymore because all their lands are polluted,” and, “There is no land but more people in China now, and that is why they are coming to Laos to make investments” (Ms. Souk and Ms. Tor, 08 November 2014). Distal market linkage, political spillover effects, and environmental push-pull factors This awareness also translated into a substantial fear among the involved households, who expressed concern that the banana roots, the extensive use of chemical inputs, plastic waste, and gravel stones used on roads in the plantations would damage the soil to the extent that it would be impossible, or at least very costly, for them to return the land to productive uses after the contracts end. A lack of clear responsibility for land restoration in the contracts contributed to these concerns both among villagers and government officials. yield important insights because it directs attention to the cross- scalar networks and flows that link spatially, institutionally, and socially distant land systems while maintaining a focus on the causes and effects embedded within a particular place (Eakin et al. 2014, Friis et al. 2016a). Driven by foreign investments in land and with export-oriented production, the banana plantations represent a land system with an inherently strong spatial and institutional disconnection between the drivers and outcomes of land-use change. As such, we can characterize and analyze the banana plantations as a telecoupled land system. Concretely, a number of flows, including economic, political, environmental, and developmental or discursive flows, form four types of telecouplings between the banana land system and several other places at various spatial and temporal scales (Fig. 3). In addition to these environmental issues, the rapid and extensive conversion of paddy rice fields to banana plantations worried the Lao authorities in relation to food security. It was stressed that Luang Namtha Province is appointed a strategic rice producing province for the northern region of Laos, and the banana investments interfere and threaten several donor-sponsored irrigation projects aimed at increasing rice production. One DAFO officer summed up these concerns, “Each year the district government needs to ensure that the rice quantities match the ‘master production expectation plan.’ So, in order to make sure we meet expectations, we have to work hard to support all the targeted villages. But banana is not in the strategic plan, so we need to restrict banana on paddy land” (Mr. Laeng, DAFO Sing District, 09 June 2015). To curb banana expansion, the Provincial Governor therefore issued a moratorium on banana plantations on paddy fields in 2011, and in 2014, a Prime Ministerial Decree placed a national ban on banana cultivation on paddy fields. Distal market linkage, political spillover effects, and environmental push-pull factors The accounts of the informants, however, also illustrate how this relatively straightforward market or production network interaction is intensified by both political and environmental flows. First, a seemingly unrelated geopolitical dispute regarding territorial rights in the South Chinese Sea is indirectly and through spillover effects influencing the demand for banana from Laos. Capturing this spillover effect is particularly important in the context of the reestablished banana-trade relationship between China and the Philippines (Simeon 2016) that could influence Distal market linkage, political spillover effects, and environmental push-pull factors A parallel ban aimed at stopping villagers from converting mature rubber gardens to banana, a trend taking off in 2014 and 2015 in response to very low rubber latex prices, was also instated (Vongvisouk and Dwyer 2016). However, both provincial and district officers acknowledged that these bans were very difficult to enforce, and largely unsuccessful. A main reason quoted was that the short-term economic gains for villagers and investors generally outweighed their concerns about the potential retribution of disobeying the regulations. Furthermore, district officers noted that it was difficult to enforce the bans because the banana plantations to some extent represent the type of agricultural intensification promoted heavily by the Lao authorities, just on the “wrong” type of land. In general, government officials complained that a lack of budget and trained staff made it difficult to implement the regulations, as did the investors’ widespread use of intermediaries and the sales and resales of the plantations. Problems with corruption and patron- client relationships between several investors and high-ranking district officials also contributed to the continued banana expansion. In November 2016, the Lao media reported that the Lao Prime Minister had reinstated the ban on banana plantation development in the northern provinces (Laotian Times 2016, Vaenkeo 2016). Fig. 3. Sketch of the flows involved in the four telecouplings driving the expansion of banana plantations in Luang Namtha Province, Lao PDR. Province, Lao PDR. The economic telecoupling consisting of capital and material flows of seedlings and chemicals into Laos and flows of harvested fruit out of the system links the Lao banana system to increasing demand for and economic growth in different urban centers in China. The accounts of the informants, however, also illustrate how this relatively straightforward market or production network interaction is intensified by both political and environmental flows. First, a seemingly unrelated geopolitical dispute regarding territorial rights in the South Chinese Sea is indirectly and through spillover effects influencing the demand for banana from Laos. Capturing this spillover effect is particularly important in the context of the reestablished banana-trade relationship between China and the Philippines (Simeon 2016) that could influence The economic telecoupling consisting of capital and material flows of seedlings and chemicals into Laos and flows of harvested fruit out of the system links the Lao banana system to increasing demand for and economic growth in different urban centers in China. Reflections on methodology At the same time, the study demonstrates the need to situate some of the more recent economic and political telecouplings in a longer temporal perspective, taking the history of state territorialization and upland development into account. Approaching the case study through the lens of telecoupling hence reveals the diverse interactions that link the Lao banana land system to diverse human-environment systems, near and far. Depending on the interaction in question, the banana land system represents, for example, a sending system for the produce, linking it to urban land systems in China; a receiving system for investors and investments as well as for the environmental pressures that link it to degrading banana land systems in China and the Philippines; and a spillover system of the Chinese-Philippine banana trade dispute. By breaking up these various relations into separate units of analysis, the telecoupling approach puts into focus the complexity of the directions of exchanges and interactions that are important for understanding the causal relations behind the banana boom. This insight is important because allocating or limiting a particular role to a system or a place in a production network can have implications for how agency and consequently power between actors involved in a telecoupling are understood. Often, sending systems are attributed key agency by triggering the initial interaction (McKinney 2014, Chignell and Laituri 2016, Leisz et al. 2016). Finally, the qualitative exploratory approach allowed us to overcome some of the identified challenges of telecoupling research such as setting system boundaries and defining spatial and temporal scales of analysis prior to empirical investigation (Friis et al. 2016a). Defining the focal system empirically by beginning in Ban Sirimoon and focusing on the two banana plantations there, the distal temporal and spatial processes identified by the informants determined where we traced the other ends of the different flows. The system boundaries were thus determined by scale choices and analytical interests reflecting the observed empirical setting, rather than a priori theoretical decisions (Richards and Clifford 2008, Abson et al. 2017, Friis and Nielsen 2017). As such, our study shows the analytical potential of telecoupling research when exploring local land-use change, something only few other studies have attempted thus far (Baird and Fox 2015, Leisz et al. 2016). Reflections on methodology future development of the banana system in Laos. Second, increasing land constraints and soil degradation, rising land prices, and climatic hazards in the banana-producing regions of China, as well as in the Philippines, combined with the conception of the Lao landscape as virgin and underused constitute an environmental telecoupling that strengthens the economic incentives for banana expansion into Laos. Finally, the case study shows how these contemporary flows of economic, political, and environmental nature need to be understood in a historical context of national development policies and discourses. The presence of a strong modernization discourse in the villagers’ aspirations to improve their economic status and in the investors’ self-promoted image as agents of development thus ground the distal international interactions in the local social and political context. gy Although telecoupling research is still an emerging and not yet consolidated research field, substantial advancements have been made in both empirical and methodological directions (Liu et al. 2016, Schierhorn et al. 2016, Fragkias et al. 2017, Millington et al. 2017, Prell et al. 2017). However, few studies thus far have tackled local land-use change from a telecoupling perspective, and calls have been made for more interdisciplinary work in this direction (Eakin et al. 2014, Liu et al. 2014, 2015a). The methodological approach adopted here lends a number of important insights to these ongoing discussions. Our qualitative operationalization of the framework, drawing on the legacy of empirical human geography and political ecology approaches that start with the observed land-use change and trace the change processes outwards in time and space by way of progressive contextualization (Vayda 1983), enabled us to capture both material and nonmaterial flows. Whereas economic and trade- related telecouplings have been analyzed with increasing success using trade statistics, land footprint accounting, and input-output models (e.g., Kastner et al. 2014b, Bruckner et al. 2015, Schaffartzik et al. 2015), ethnographic fieldwork and qualitative inquiry enables analyses of important political, environmental, and cultural interactions through the narratives and experience of the involved actors. The interview-based analysis thus allows us to get to some of the more elusive and immaterial interactions that are not easily captured by more readily available data sources. As the results show, this strategy permitted us to capture all the various components of a telecoupled system, including triggering events and potential feedbacks. DISCUSSION: A TELECOUPLED BANANA SYSTEM The case study we have presented here demonstrates how multiple distal drivers influence and create banana plantation expansion in Ban Sirimoon. Adopting the telecoupling framework as a heuristic tool for understanding how these drivers interact can Ecology and Society 22(4): 30 https://www.ecologyandsociety.org/vol22/iss4/art30/ Reflections on methodology Moreover, we identified the first “societal feedbacks” from the plantation developments, i.e., the governmental bans on conversion of rice and rubber to banana, and showed how they have had limited effects. Despite considerable concerns about the bananas’ long-term environmental and food security impacts, the regulatory responses are generally failing. A major reason for this lack of success is the networked and informal land acquisition strategies pursued by the Chinese investors that enable them to mediate the local context and the distant demand by relying on well-established economic and social ties in Laos and across the border, thus responding quickly to the new market opportunity while circumventing formal involvement of the government authorities. This highlights the challenges facing traditional territorial land governance arrangements in relation to rapidly unfolding and institutionally unexpected telecouplings, as is currently widely discussed in the LSS literature (Sikor et al. 2013, Eakin et al. 2014, Gentry et al. 2014, Eriksson et al. 2015, Gasparri and le Polain de Waroux 2015, Lenschow et al. 2016). It also shows the importance of an integration of place-based and processual analysis embedded in the telecoupling framework to capture how the various flows of, for example, capital investment, economic incentives, and environmental impacts are transmitted through networks of actors in a specific place. Remaining challenges and ways forward As such, telecoupling offers an integrative systemic perspective for thinking about the distal causal relations of a particular land-use change while avoiding a holistic trap whereby everything gets linked to everything else. Depending on the specific research objective or social-ecological change in question, and with a flexible analytical entry point, LSS researchers can thus tackle various aspects of global interconnectivity while maintaining an overview over the entire process. For this (ambitious) agenda to be fulfilled, however, continued collaborative work across disciplinary boundaries is required. qualitative research in a case study comes a long way in terms of capturing and assessing this diversity, in the wider telecoupling literature, the analytical category of “flows” continues to hide often very diverse dynamics and logics of exchange. This presents a question of how to continue to engage with and integrate theoretical insights and discussions from different fields. The need to embrace other theoretical approaches dealing with global connectedness has also clearly been highlighted in the literature (Eakin et al. 2014, Friis et al. 2016a, see also Baumann and Kuemmerle 2016). With the banana case in mind, we could, for example, have turned to the global production network framework from economic geography (Henderson et al. 2002, Challies 2008, Coe and Yeung 2015) for analyzing the economic telecoupling identified, as is increasingly done in LSS (e.g., Galvan-Miyoshi et al. 2015, Hauge 2016). The global production network offers a comprehensive framework for understanding flows and distribution of power and value, as well as the social, institutional, and territorial embeddedness of actors in transnational production networks (Coe and Yeung 2015). Similarly, one could look to advancements in political ecology that draw on actor-network theory to assist the understanding of how specific transnational networks of actors produce specific land-use changes (Rocheleau and Roth 2007, Birkenholtz 2012). Both the global production network and political ecology can thus aid the analysis of power relations between actors, not only in relation to economic processes, but also to the production of environmental and discursive telecouplings. Political ecology has indeed been shown to facilitate a deep understanding of how various constellations of Lao state actors, foreign investors, and local middlemen assemble to gain and maintain access to land by invoking images of virgin land, underdevelopment, and the need for agricultural modernization (Lestrelin et al. 2013), as also hinted at in our analysis. Remaining challenges and ways forward The Southeast Asian crop boom literature, in turn, presents valuable insights for analyzing the processes leading to rising values of land for a particular cash crop and the practices that allow various actors to exercise and maintain control over land and crop production (Hall 2011, Hall et al. 2011, Taylor 2016). Such insights can facilitate a deeper understanding of how and why certain regulatory responses might be failing (see also Friis and Nielsen 2016). Lastly, the spillover processes associated with the political telecoupling attest to the geopolitical aspects of the current banana boom in Laos, and a historical geopolitical analysis of the position of China with respect to the political- economic position of Laos, the Philippines, and other banana producers in the global banana market could potentially have fostered a deeper understanding of the insights we have presented here (see Dwyer 2014). Remaining challenges and ways forward Remaining challenges and ways forward Despite its potential, challenges remain for integrated telecoupling research. One prominent outstanding issue relates to the trade-off between scope and depth of analysis when multiple and complex interactions are identified for the same case study (Eakin et al. 2014, Friis et al. 2016a). Ultimately, the telecouplings identified here represent substantially different causal processes, including market integration and geopolitical disputes, environmental degradation, and the exchange of information, ideas, and discourses. Whereas our study shows how Ecology and Society 22(4): 30 https://www.ecologyandsociety.org/vol22/iss4/art30/ Ecology and Society 22(4): 30 yandsociety.org/vol22/iss4/art30/ agency between such firms while lacking a deeper engagement with social and environmental aspects of change outside the activity of firms in the production network (Coe et al. 2008, Kelly 2013). In contrast, the strength of the telecoupling framework lies exactly in the manner in which it combines an in-depth focus on both place-based systemic and process-oriented relational aspects of change in human-environment systems. The potential of telecoupling, we would argue, is the open and flexible way that our analytical attention is drawn to cross-scalar flows and feedbacks without favoring specific scales of analysis (local, national, international relations, etc.), types of interactions (economic, political, environmental, etc.), or particular analytical or theoretical approaches. As such, telecoupling offers an integrative systemic perspective for thinking about the distal causal relations of a particular land-use change while avoiding a holistic trap whereby everything gets linked to everything else. Depending on the specific research objective or social-ecological change in question, and with a flexible analytical entry point, LSS researchers can thus tackle various aspects of global interconnectivity while maintaining an overview over the entire process. For this (ambitious) agenda to be fulfilled, however, continued collaborative work across disciplinary boundaries is required. agency between such firms while lacking a deeper engagement with social and environmental aspects of change outside the activity of firms in the production network (Coe et al. 2008, Kelly 2013). In contrast, the strength of the telecoupling framework lies exactly in the manner in which it combines an in-depth focus on both place-based systemic and process-oriented relational aspects of change in human-environment systems. The potential of telecoupling, we would argue, is the open and flexible way that our analytical attention is drawn to cross-scalar flows and feedbacks without favoring specific scales of analysis (local, national, international relations, etc.), types of interactions (economic, political, environmental, etc.), or particular analytical or theoretical approaches. LITERATURE CITED Bernard, H. R. 2002. Nonprobability sampling and choosing informants. Research methods in anthropology: qualitative and quantitative approaches. Third edition. Altamira, Lanham, Maryland, USA. Abson, D. J., J. Fischer, J. Leventon, J. Newig, T. Schomerus, U. Vilsmaier, H. von Wehrden, P. Abernethy, C. D. Ives, N. W. Jager, and D. J. Lang. 2017. Leverage points for sustainability transformation. Ambio 46(1):30-39. http://dx.doi.org/10.1007/ s13280-016-0800-y Bicudo da Silva, R. F., M. Batistella, Y. Dou, E. Moran, S. McMillan Torres, and J. Liu. 2017. The Sino-Brazilian telecoupled soybean system and cascading effects for the exporting country. Land 6(3):53. http://dx.doi.org/10.3390/ land6030053 Adger, W. N., H. Eakin, and A. Winkels. 2009. Nested and teleconnected vulnerabilities to environmental change. Frontiers in Ecology and the Environment 7(3):150-157. http://dx.doi. org/10.1890/070148 Birkenholtz, T. 2012. Network political ecology: method and theory in climate change vulnerability and adaptation research. Progress in Human Geography 36(3):295-315. http://dx.doi. org/10.1177/0309132511421532 Agence France-Presse. 2012. Typhoon destroys quarter of Philippine banana crop. Hurriyet Daily News 06 December 2012. Agence France-Presse. 2012. Typhoon destroys quarter of Philippine banana crop. Hurriyet Daily News 06 December 2012. [online] URL: http://www.hurriyetdailynews.com/typhoon-destroys- quarter-of-philippine-banana-crop.aspx?pageID=238&nid=36245 [online] URL: http://www.hurriyetdailynews.com/typhoon-destroys- quarter-of-philippine-banana-crop.aspx?pageID=238&nid=36245 Blaikie, P., and H. Brookfield. 1987. Land degradation and society. Methuen, London, UK. An. 2014. Banana trees affected by typhoon Rammasun in China’s Guangxi. Xinhuanet Englishnews.cn 24 July 2014. [online] URL h // hi / i /2014 07/25/ An. 2014. Banana trees affected by typhoon Rammasun in China’s Guangxi. Xinhuanet Englishnews.cn 24 July 2014. [online] URL: http://www.china.org.cn/environment/2014-07/25/ content_33058546_2.htm g y [ ] URL: http://www.china.org.cn/environment/2014-07/25/ content_33058546_2.htm Branigan, T., and J. Watts. 2012. Philippines accuses China of deploying ships in Scarborough shoal. The Guardian 23 May 2012. [online] URL: https://www.theguardian.com/world/2012/may/23/ philippines-china-ships-scarborough-shoal Associated Press. 2012a. Philippine warship in standoff with China vessels. The Guardian 11 April 2012. [online] URL: https:// www.theguardian.com/world/2012/apr/11/philippines-china-stand- off-south-china-sea Bruckner, M., G. Fischer, S. Tramberend, and S. Giljum. 2015. Measuring telecouplings in the global land system: a review and comparative evaluation of land footprint accounting methods. Ecological Economics 114:11-21. http://dx.doi.org/10.1016/j. ecolecon.2015.03.008 Associated Press. 2012b. Philippines reignites row with China over oil exploration rights. The Guardian 29 February 2012. [online] URL: https://www.theguardian.com/world/2012/feb/29/philippines- row-china-oil-exploration Challies, E. R. T. 2008. Commodity chains, rural development and the global agri-food system. Geography Compass 2 (2):375-394. http://dx.doi.org/10.1111/j.1749-8198.2008.00095.x Associated Press. 2016. Philippines to ‘set aside’ South Chinese Sea tribunal ruling to avoid imposing on Beijing. The Guardian 17 December 2016. [online] URL: https://www.theguardian.com/ world/2016/dec/17/philippines-to-set-aside-south-china-sea-tribunal- ruling-to-avoid-imposing-on-beijing Chen, R., C. Ye, Y. Cai, X. CONCLUSION The global production network framework has, for example, been criticized for giving analytical preference to transnational firms and the distribution of economic power and Ecology and Society 22(4): 30 https://www.ecologyandsociety.org/vol22/iss4/art30/ Ecology and Society 22(4): 30 https://www.ecologyandsociety.org/vol22/iss4/art30/ Ecology and Society 22(4): 30 andsociety.org/vol22/iss4/art30/ Responses to this article can be read online at: http://www.ecologyandsociety.org/issues/responses. php/9480 Baird, I. G., and J. Fox. 2015. How land concessions affect places elsewhere: telecoupling, political ecology, and large-scale plantations in southern Laos and northeastern Cambodia. Land 4(2):436-453. http://dx.doi.org/10.3390/land4020436 Baird, I. G., and P. Vue. 2017. The ties that bind: the role of Hmong social networks in developing small-scale rubber cultivation in Laos. Mobilities 12(1):136-154. http://dx.doi.org/10.1080/17450 101.2015.1016821 Acknowledgments: This study was funded by the German Excellence Initiative. The fieldwork was carried out as part of the EU-funded project, “Impacts of Reducing Emissions from Deforestation and Forest Degradation and Enhancing Carbon Stocks (I-REDD+)” (EU FP7-ENV-2010, Project 265286) in collaboration with and with extensive support from the Faculty of Forestry, the National University of Laos, Lao PDR. Thanks are extended to Provincial Authorities in Luang Namtha Province and the District Authorities in Muang Long and Muang Sing districts for their support throughout the fieldwork. We thank the villagers in the study site for their great hospitality, patience, and contributions to the study; Mr. Soukkaseum for invaluable research assistance and translations; and Eric Lambin, Yann le Polain de Waroux, and Iago Otero for helpful comments. Any remaining shortcomings or mistakes are our own. Barney, K. 2012. Land, livelihoods, and remittances: a political ecology of youth out-migration across the Lao-Thai Mekong border. Critical Asian Studies 44(1):57-83. http://dx.doi. org/10.1080/14672715.2012.644887 Baumann, M., and T. Kuemmerle. 2016. The impacts of warfare and armed conflict on land systems. Journal of Land Use Science 11(6):672-688. http://dx.doi.org/10.1080/1747423X.2016.1241317 Bebbington, A. J., and S. P. J. Batterbury. 2001. Transnational livelihoods and landscapes: political ecologies of globalization. Cultural Geographies 8(4):369-380. https://doi.org/10.1177/0967 46080100800401 Berkes, F., and C. Folke. 1998. Linking social and ecological systems: management practices and social mechanisms for building resilience. Cambridge University Press, Cambridge, UK. CONCLUSION Here, we have explored the influence of distal causal relations involved in a land-use transformation from rice paddies to mono- cropped banana plantations in northern Laos. Our study shows how banana expansion in Muang Long District, Luang Namtha Province is influenced by economic, environmental, political, and discursive interactions that create telecouplings to several spatially and institutionally distant land systems. The case study of two banana plantations in the small rural community of Ban Sirimoon demonstrates how these multiple distant interactions are interlinked and co-constitutive and thus highlights how the study of localized land-use change requires attention to telecouplings. However, the complex role of banana investors as mediators between distal flows and the local setting also demonstrates the importance of maintaining a place-based perspective to understand the network of actors and contextual factors that ground such telecouplings in a particular location. As a flexible heuristic tool, the telecoupling framework allowed us to sketch out both aspects of land-use change and explore how global phenomena such as market-driven land-use change manifests in a particular place. The framework enabled us to move beyond a distinction between the local and the global in a manner that transcends the need for nested spatial hierarchies and adds specificity to a diffuse set of underlying driving forces without favoring a specific scale of analysis or type of interaction. However, the fundamentally different types of flows identified here illustrate the continued need for research that transcends disciplinary boundaries when seeking to understand the full complexity of telecoupling in local land-use change. Future work should thus continue to bring the research agenda on telecoupling forward with interdisciplinary methodological and empirical studies. Using qualitative and ethnographic methods to capture some of the more diffuse and immaterial flows, as well as potential feedbacks, our study presents a small step in this direction. This is undoubtedly by no means an exhaustive list of potential analytical perspectives that could deepen our understanding of the causal processes involved in the expansion of banana cultivation in northern Laos, or for telecoupling research in general. However, while each of these theoretical perspectives provides good starting points for analysis, sticking to any one of them would potentially limit the explanatory scope of what is going on. LITERATURE CITED Xing, and Q. Chen. 2014. The impact of rural out-migration on land use transition in China: past, present and trend. Land Use Policy 40:101-110. http://dx.doi. org/10.1016/j.landusepol.2013.10.003 Ecology and Society 22(4): 30 https://www.ecologyandsociety.org/vol22/iss4/art30/ Ecology and Society 22(4): 30 https://www.ecologyandsociety.org/vol22/iss4/art30/ Economics 69(2):328-334. http://dx.doi.org/10.1016/j. ecolecon.2009.06.025 Chignell, S. M., and M. J. Laituri. 2016. Telecoupling, urbanization, and the unintended consequences of water development aid in Ethiopia. Pages 125-135 in G. R. Wessel and J. K. Greenberg, editors. Geoscience for the public good and global development: toward a sustainable future. Geological Society of America, Boulder, Colorado, USA. http://dx.doi.org/10.1130/2016.2520 (13) Eriksson, H., H. Österblom, B. Crona, M. Troell, N. Andrew, J. Wilen, and C. Folke. 2015. Contagious exploitation of marine resources. Frontiers in Ecology and the Environment 13(8):435-440. htt //d d i /10 1890/140312 Evrard, O., and Y. Goudineau. 2004. Planned resettlement, unexpected migrations and cultural trauma in Laos. Development and Change 35(5):937-962. http://dx.doi.org/10.1111/ Coe, N. M., P. Dicken, and M. Hess. 2008. Global production networks: realizing the potential. Journal of Economic Geography 8(3):271-295. http://dx.doi.org/10.1093/jeg/lbn002 Coe, N. M., and H. W.-C. Yeung. 2015. Global production networks: theorizing economic development in an interconnected world. Oxford University Press, Oxford, UK. Fang, B., Y. Tan, C. Li, Y. Cao, J. Liu, P.-J. Schweizer, H. Shi, B. Zhou, H. Chen, and Z. Hu. 2016. Energy sustainability under the framework of telecoupling. Energy 106:253-259. http://dx.doi. org/10.1016/j.energy.2016.03.055 Cramb, R., V. Manivong, J. Newby, K. Sothorn, and P. Sujang. 2015. Alternatives to land grabbing: smallholder engagement in commodity booms in Southeast Asia. Conference Paper 44. Land grabbing, conflict and agrarian-environmental transformations: perspectives from East and Southeast Asia. Chiang Mai University, Chiang Mai, Thailand. https://www.iss.nl/fileadmin/ ASSETS/iss/Research_and_projects/Research_networks/BICAS/ CMCP_44-Cramb_et_al.pdf Fischer-Kowalski, M., and H. Haberl, editors. 2007. Socioecological transition and global change: trajectories of social metabolism and land use. Edward Elgar, Cheltenham, UK. http:// dx.doi.org/10.4337/9781847209436 Fischer-Kowalski, M., and H. Haberl, editors. 2007. Socioecological transition and global change: trajectories of social metabolism and land use. Edward Elgar, Cheltenham, UK. http:// dx.doi.org/10.4337/9781847209436 Folke, C., T. Hahn, P. Olsson, and J. Norberg. 2005. Adaptive governance of social-ecological systems. Annual Review of Environment and Resources 30:441-473. http://dx.doi.org/10.1146/ annurev.energy.30.050504.144511 Cuneta, J., and J. Hookway. 2012. China dispute threatens Philippine industries. Wall Street Journal 16 May 2012. [online] URL: http://www.wsj.com/articles/SB100014240527023038796 04577407730408858666 Food and Agriculture Organization (FAO). 2013. The Philippines: strong typhoon Haiyan severely affected the agriculture sector in central regions. GIEWS update. FAO, Rome, Italy. LITERATURE CITED [online] URL: https://reliefweb.int/report/philippines/giews-update-philippines- strong-typhoon-haiyan-severely-affected-agriculture Food and Agriculture Organization (FAO). 2013. The Philippines: strong typhoon Haiyan severely affected the agriculture sector in central regions. GIEWS update. FAO, Rome, Italy. [online] URL: De Groot, W. T. 1992. Environmental science theory: concepts and methods in a one-world, problem-oriented paradigm. Elsevier, Amsterdam, The Netherlands. Food and Agriculture Organization (FAO). 2015. Banana market review 2013–2014. FAO, Rome, Italy. Deines, J. M., X. Liu, and J. Liu. 2016. Telecoupling in urban water systems: an examination of Beijing’s imported water supply. Water International 41(2):251-270. http://dx.doi. org/10.1080/02508060.2015.1113485 Food and Agriculture Organization (FAO). 2016. FAOStat crops. FAO, Rome, Italy. [online] URL: http://www.fao.org/faostat/en/ #data/QC Dwyer, M. B. 2011. Territorial affairs: turning battlefields into marketplaces in postwar Laos. Dissertation. University of California, Berkeley, California, USA. [online] URL: https:// escholarship.org/uc/item/67m1169x Fragkias, M., S. Islam, and C. Sprague. 2017. Modeling teleconnected urban social-ecological systems: opportunities and challenges for resilience research. International Journal of Urban Sustainable Development 9(2):207-225. http://dx.doi. Dwyer, M. B. 2014. Micro-geopolitics: capitalising security in Laos’s Golden Quadrangle. Geopolitics 19(2):377-405. http://dx. doi.org/10.1080/14650045.2013.780033 org/10.1080/19463138.2017.1324455 Friis, C., and J. Ø. Nielsen. 2014. Exploring the potential of the telecoupling framework for understanding land change. THESys Discussion Papers. IRI THESys, Berlin, Germany. http://dx.doi. org/10.18452/3128 Eakin, H., R. DeFries, S. Kerr, E. F. Lambin, J. Liu, P. J. Marcotullio, P. Messerli, A. Reenberg, X. Rueda, S. R. Swaffield, B. Wicke, and K. Zimmerer. 2014. Significance of telecoupling for exploration of land-use change. In K. C. Seto and A. Reenberg, editors. Rethinking global land use in an urban era. MIT Press, Cambridge, Massachusetts, USA. http://dx.doi.org/10.7551/ mitpress/9780262026901.003.0008 Friis, C., and J. Ø. Nielsen. 2016. Small-scale land acquisitions, large-scale implications: exploring the case of Chinese banana investments in northern Laos. Land Use Policy 57:117-129. http:// dx.doi.org/10.1016/j.landusepol.2016.05.028 Friis, C., and J. Ø. Nielsen. 2017. On the system. Boundary choices, implications, and solutions in telecoupling land use change research. Sustainability 9(6):974. http://dx.doi.org/10.3390/ su9060974 Eakin, H., A. Winkels, and J. Sendzimir. 2009. Nested vulnerability: exploring cross-scale linkages and vulnerability teleconnections in Mexican and Vietnamese coffee systems. Environmental Science and Policy 12(4):398-412. http://dx.doi. org/10.1016/j.envsci.2008.09.003 su9060974 Friis, C., J. Ø. Nielsen, I. Otero, H. Haberl, J. Niewöhner, and P. Hostert. 2016a. From teleconnection to telecoupling: taking stock of an emerging framework in land system science. Journal of Land Use Science 11(2):131-153. http://dx.doi.org/10.1080/1747423X .2015.1096423 Erb, K.-H., F. Krausmann, W. Lucht, and H. Haberl. 2009. Embodied HANPP: mapping the spatial disconnect between global biomass production and consumption. Ecological Ecology and Society 22(4): 30 https://www.ecologyandsociety.org/vol22/iss4/art30/ Ecology and Society 22(4): 30 andsociety.org/vol22/iss4/art30/ Ecology and Society 22(4): 30 https://www.ecologyandsociety.org/vol22/iss4/art30/ Friis, C., A. Reenberg, A. Heinimann, and O. Schönweger. 2016b. Changing local land systems: implications of a Chinese rubber plantation in Nambak District, Lao PDR. Singapore Journal of Tropical Geography 37(1):25-42. http://dx.doi.org/10.1111/ sjtg.12137 Güneralp, B., K. C. Seto, and M. Ramachandran. 2013. Evidence of urban land teleconnections and impacts on hinterlands. Current Opinion in Environmental Sustainability 5(5):445-451. http://dx.doi.org/10.1016/j.cosust.2013.08.003 Haberl, H., K.-H. Erb, F. Krausmann, S. Berecz, N. Ludwiczek, J. Martínez-Alier, A. Musel, and A. Schaffartzik. 2009. Using embodied HANPP to analyze teleconnections in the global land system: conceptual considerations. Geografisk Tidsskrift-Danish Journal of Geography 109(2):119-130. http://dx.doi. org/10.1080/00167223.2009.10649602 Fujita, Y., and K. Phanvilay. 2008. Land and forest allocation in Lao People’s Democratic Republic: comparison of case studies from community-based natural resource management research. Society and Natural Resources 21(2):120-133. http://dx.doi. org/10.1080/08941920701681490 Hall, D. 2011. Land grabs, land control, and Southeast Asian crop booms. Journal of Peasant Studies 38(4):837-857. http://dx. doi.org/10.1080/03066150.2011.607706 Galvan-Miyoshi, Y., R. Walker, and B. Warf. 2015. Land change regimes and the evolution of the maize-cattle complex in neoliberal Mexico. Land 4(3):754-777. http://dx.doi.org/10.3390/ land4030754 Hall, D., P. Hirsch, and T. M. Li. 2011. Powers of exclusion: land dilemmas in Southeast Asia. University of Hawai’i Press, Honolulu, Hawai’i, USA. Garrett, R. D., X. Rueda, and E. F. Lambin. 2013. Globalization’s unexpected impact on soybean production in South America: linkages between preferences for non-genetically modified crops, eco-certifications, and land use. Environmental Research Letters 8(4):044055. http://dx.doi.org/10.1088/1748-9326/8/4/044055 Hauge, M. M. 2016. Mind the GAP: Vietnamese rice farmers and distal markets. Pages 75-89 in J. Niewöhner, A. Bruns, P. Hostert, T. Krueger, J. Ø. Nielsen, H. Haberl, C. Lauk, J. Lutz, and D. Müller, editors. Land use competition: ecological, economic and social perspectives. Springer, Cham, Switzerland. http://dx.doi. org/10.1007/978-3-319-33628-2_5 Gasparri, N. I., T. Kuemmerle, P. Meyfroidt, Y. le Polain de Waroux, and H. Kreft. 2016. The emerging soybean production frontier in southern Africa: conservation challenges and the role of South-South telecouplings. Conservation Letters 9(1):21-31. http://dx.doi.org/10.1111/conl.12173 Henders, S., U. M. su9060974 Persson, and T. Kastner. 2015. Trading forests: land-use change and carbon emissions embodied in production and exports of forest-risk commodities. Environmental Research Letters 10(12):125012. http://dx.doi.org/10.1088/1748-9326/10/12/125012 Gasparri, N. I., and Y. le Polain de Waroux. 2015. The coupling of South American soybean and cattle production frontiers: new challenges for conservation policy and land change science. Conservation Letters 8(4):290-298. http://dx.doi.org/10.1111/ conl.12121 Henderson, J., P. Dicken, M. Hess, N. Coe, and H. W.-C. Yeung. 2002. Global production networks and the analysis of economic development. Review of International Political Economy 9 (3):436-464. http://dx.doi.org/10.1080/09692290210150842 Geist, H. J., and E. F. Lambin. 2002. Proximate causes and underlying driving forces of tropical deforestation: tropical forests are disappearing as the result of many pressures, both local and regional, acting in various combinations in different geographical locations. Bioscience 52(2):143-150. http://dx.doi.org/10.1641/0006-3568 (2002)052[0143:PCAUDF]2.0.CO;2 Higashi, S. 2015. Impacts on regional land use from investment in banana contract farming by Chinese companies: case studies in Oudomxay Province, northern Laos. Mekong Watch, Vientiane, Laos. Jessop, B., N. Brenner, and M. Jones. 2008. Theorizing sociospatial relations. Environment and Planning D: Society and Space 26(3):389-401. http://dx.doi.org/10.1068/d9107 Geist, H., W. McConnell, E. F. Lambin, E. Moran, D. Alves, and T. Rudel. 2006. Causes and trajectories of land-use/cover change. Pages 41-70 in E. F. Lambin and H. Geist, editors. Land-use and land-cover change: local processes and global impacts. Springer, Berlin, Germany. http://dx.doi.org/10.1007/3-540-32202-7_3 Kastner, T., K.-H. Erb, and H. Haberl. 2014a. Rapid growth in agricultural trade: effects on global area efficiency and the role of management. Environmental Research Letters 9(3):034015. https://doi.org/10.1088/1748-9326/9/3/034015 Gentry, B. S., T. Sikor, G. Auld, A. J. Bebbington, T. A. Benjaminsen, C. A. Hunsberger, A.-M. Izac, M. E. Margulis, T. Plieninger, H. Schroeder, and C. Upton. 2014. Changes in land- use governance in an urban era. In K. C. Seto and A. Reenberg, editors. Rethinking global land use in an urban era. MIT Press, Cambridge, Massachussetts, USA. http://dx.doi.org/10.7551/ mitpress/9780262026901.003.0013 Kastner, T., K.-H. Erb, and H. Haberl. 2015. Global human appropriation of net primary production for biomass consumption in the European Union, 1986–2007. Journal of Industrial Ecology 19(5):825-836. http://dx.doi.org/10.1111/ jiec.12238 Kastner, T., A. Schaffartzik, N. Eisenmenger, K.-H. Erb, H. Haberl, and F. Krausmann. 2014b. Cropland area embodied in international trade: contradictory results from different approaches. Ecological Economics 104:140-144. https://doi. org/10.1016/j.ecolecon.2013.12.003 Global Land Programme (GLP). 2016. Global land programme: science plan and implementation strategy 2016–2021. Global Land Programme, Bern, Switzerland. [online] URL: https://glp.earth/ sites/default/files/uploads/glpscienceplan_25_10_16.pdf Global Land Project (GLP). su9060974 2005. Global land project: science plan and implementation strategy. IGBP Report 53/IHDP Report 19. IGBP Secretariat, Stockholm, Sweden. [online] URL: http:// www.globalcarbonproject.org/global/pdf/pep/GLP_FINAL_IGBP- IHDP.pdf Kelly, P. F. 2013. Production networks, place and development: thinking through global production networks in Cavite, Philippines. Geoforum 44:82-92. http://dx.doi.org/10.1016/j. geoforum.2011.10.003 Ecology and Society 22(4): 30 https://www.ecologyandsociety.org/vol22/iss4/art30/ Ecology and Society 22(4): 30 https://www.ecologyandsociety.org/vol22/iss4/art30/ Ecology and Society 22(4): 30 andsociety.org/vol22/iss4/art30/ Lagerqvist, Y. F. 2013. Imagining the borderlands: contending stories of a resource frontier in Muang Sing. Singapore Journal of Tropical Geography 34(1):57-69. http://dx.doi.org/10.1111/ sjtg.12013 Liu, J., V. Hull, M. Batistella, R. DeFries, T. Dietz, F. Fu, T. W. Hertel, R. C. Izaurralde, E. F. Lambin, S. Li, L. A. Martinelli, W. J. McConnell, E. F. Moran, R. Naylor, Z. Ouyang, K. R. Polenske, A. Reenberg, G. de Miranda Rocha, C. S. Simmons, P. H. Verburg, P. M. Vitousek, F. Zhang, and C. Zhu. 2013. Framing sustainability in a telecoupled world. Ecology and Society 18 (2):26. http://dx.doi.org/10.5751/ES-05873-180226 Lambin, E. F., and P. Meyfroidt. 2011. Global land use change, economic globalization, and the looming land scarcity. Proceedings of the National Academy of Sciences 108 (9):3465-3472. http://dx.doi.org/10.1073/pnas.1100480108 Liu, J., V. Hull, J. Luo, W. Yang, W. Liu, A. Viña, C. Vogt, Z. Xu, H. Yang, J. Zhang, L. An, X. Chen, S. Li, Z. Ouyang, W. Xu, and H. Zhang. 2015a. Multiple telecouplings and their complex interrelationships. Ecology and Society 20(3):44. http://dx.doi. org/10.5751/ES-07868-200344 Laotian Times. 2016. Banana farms ordered to stop. Laotian Times 12 December 2016. [online] URL: https://laotiantimes. com/2016/12/12/banana-farms-ordered-stop/ le Polain de Waroux, Y., R. D. Garrett, R. Heilmayr, and E. F. Lambin. 2016. Land-use policies and corporate investments in agriculture in the Gran Chaco and Chiquitano. Proceedings of the National Academy of Sciences 113(15):4021-4026. http://dx. doi.org/10.1073/pnas.1602646113 Liu, J., V. Hull, E. Moran, H. Nagendra, S. R. Swaffield, and B. L. Turner II. 2014. Applications of the telecoupling framework to land-change science. In K. C. Seto and A. Reenberg, editors. Rethinking global land use in an urban era. MIT Press, Cambridge, Massachusetts, USA. http://dx.doi.org/10.7551/mitpress/97802 62026901.003.0007 Leisz, S. J., E. Rounds, N. The An, N. Thi Bich Yen, T. Nguyen Bang, S. Douangphachanh, and B. Ninchaleune. 2016. Telecouplings in the East-West economic corridor within borders and across. Remote Sensing 8(12):1012. http://dx.doi.org/10.3390/ rs8121012 Liu, J., H. Mooney, V. Hull, S. J. Davis, J. Gaskell, T. Hertel, J. Lubchenco, K. C. Seto, P. Gleick, C. Kremen, and S. Li. 2015b. su9060974 Systems integration for global sustainability. Science 347 (6225):963. http://dx.doi.org/10.1126/science.1258832 Lenschow, A., J. Newig, and E. Challies. 2016. Globalization’s limits to the environmental state? Integrating telecoupling into global environmental governance. Environmental Politics 25 (1):136-159. http://dx.doi.org/10.1080/09644016.2015.1074384 Liu, J., W. Yang, and S. Li. 2016. Framing ecosystem services in the telecoupled Anthropocene. Frontiers in Ecology and the Environment 14(1):27-36. http://dx.doi.org/10.1002/16-0188.1 Lund, C. 2011. Fragmented sovereignty: land reform and dispossession in Laos. Journal of Peasant Studies 38(4):885-905. http://dx.doi.org/10.1080/03066150.2011.607709 Lestrelin, G. 2010. Land degradation in the Lao PDR: discourses and policy. Land Use Policy 27(2):424-439. http://dx.doi. org/10.1016/j.landusepol.2009.06.005 Lyttleton, C., H. Rattanavong, B. Thongkhamhane, and S. Sisaengrat. 2004. Watermelons, bars and trucks: dangerous intersections in northwest Lao PDR. Institute of Cultural Research of Laos and Macquarie University, Vientiane, Laos. Lestrelin, G., J.-C. Castella, and J. Bourgoin. 2012. Territorialising sustainable development: the politics of land-use planning in Laos. Journal of Contemporary Asia 42(4):581-602. http://dx.doi. org/10.1080/00472336.2012.706745 Manivong, V., and R. A. Cramb 2008a. The adoption of smallholder rubber production by shifting cultivators in northern Laos: a village case study. Pages 117-137 in D. J. Snelder and R. D. Lasco, editors. Smallholder tree growing for rural development and environmental services: lessons from Asia. Springer, Dordrecht, The Netherlands. Manivong, V., and R. A. Cramb 2008a. The adoption of smallholder rubber production by shifting cultivators in northern Lestrelin, G., J.-C. Castella, and J. Fox. 2013. Forest transitions in Southeast Asia: synergies and shortcomings in land change science and political ecology. Pages 48-65 in C. Brannstrom and J. M. Vadjunec, editors. Land change science, political ecology, and sustainability: synergies and divergences. Earthscan, London, UK. Laos: a village case study. Pages 117-137 in D. J. Snelder and R. D. Lasco, editors. Smallholder tree growing for rural development and environmental services: lessons from Asia. Springer, Dordrecht, The Netherlands. Manivong, V., and R. A. Cramb 2008b. Economics of smallholder rubber expansion in northern Laos. Agroforestry Systems 74 (2):113-125. https://doi.org/10.1007/s10457-008-9136-3 Ling, S. 2015. The use of remittances by circular Hmong migrants to Chinese banana plantations in Bokeo, Lao PDR. Thesis. University of New England, Armidale, Australia. [online] URL: Mansfield, B., D. K. Munroe, and K. McSweeney. 2010. Does economic growth cause environmental recovery? Geographical explanations of forest regrowth. Geography Compass 4 (5):416-427. http://dx.doi.org/10.1111/j.1749-8198.2010.00320.x http://rightslinklao.org/wp-content/uploads/downloads/2016/03/ Dissertation_-_Use_of_remittances_by_migrants_to_banana_p lantations in Bokeo pdf Dissertation_-_Use_of_remittances_by_migrants_to_banana_p lantations_in_Bokeo.pdf Liu, J. 2014. Forest sustainability in China and implications for a telecoupled world. Asia and the Pacific Policy Studies 1 (1):230-250. http://dx.doi.org/10.1002/app5.17 Massey, D. 1991. su9060974 A global sense of place. Marxism Today 38:24-29. McKinney, L. A. 2014. Foreign direct investment, development, and overshoot. Social Science Research 47:121-133. http://dx.doi. org/10.1016/j.ssresearch.2014.04.003 Liu, J., T. Dietz, S. R. Carpenter, M. Alberti, C. Folke, E. Moran, A. N. Pell, P. Deadman, T. Kratz, J. Lubchenco, E. Ostrom, Z. Ouyang, W. Provencher, C. L. Redman, S. H. Schneider, and W. W. Taylor. 2007. Complexity of coupled human and natural systems. Science 317(5844):1513-1516. http://dx.doi.org/10.1126/ science.1144004 Messerli, P., A. Peeters, O. Schönweger, V. Nanhthavong, and A. Heinimann. 2015. Marginal land or marginal people? Analysing patterns and processes of large-scale land acquisitions in South- Ecology and Society 22(4): 30 https://www.ecologyandsociety.org/vol22/iss4/art30/ East Asia. Pages 136-171 in C. Gironde, C. Golay, and P. Messerli, editors. Large-scale land acquisitions: focus on South-East Asia. Brill Nijhoff, Leiden, The Netherlands. http://dx.doi. org/10.1163/9789004304758_007 Perlez, J. 2016. Rodrigo Duterte gets closer to China, and the neighbours notice. New York Times 24 October 2016. [online] URL: https://www.nytimes.com/2016/10/25/world/asia/rodrigo- duterte-philippines-china.html Perz, S. G., and A. M. Almeyda. 2009. A tri-partite framework of forest dynamics: hierarchy, panarchy, and heterarchy in the study of secondary growth. Pages 59-84 in H. Nagendra and J. Southworth, editors. Reforesting landscapes: linking pattern and process. Springer, Dordrecht, The Netherlands. http://dx.doi. org/10.1007/978-1-4020-9656-3_4 Meyfroidt, P. 2016. Approaches and terminology for causal analysis in land systems science. Journal of Land Use Science 11 (5):501-522. http://dx.doi.org/10.1080/1747423X.2015.1117530 Meyfroidt, P., E. F. Lambin, K.-H. Erb, and T. W. Hertel. 2013. Globalization of land use: distant drivers of land change and geographic displacement of land use. Current Opinion in Environmental Sustainability 5(5):438-444. http://dx.doi.org/10.1016/ j.cosust.2013.04.003 Phillips, T. 2016. Rodrigo Duterte arrives in China with ‘make friends, not war’ message. The Guardian 18 October 2016. [online] URL: https://www.theguardian.com/world/2016/oct/18/rodrigo- duterte-philippines-president-china-make-friends-not-war Phillips, T. 2016. Rodrigo Duterte arrives in China with ‘make friends, not war’ message. The Guardian 18 October 2016. [online] i i URL: https://www.theguardian.com/world/2016/oct/18/rodrigo- duterte-philippines-president-china-make-friends-not-war Millington, J. D. A., H. Xiong, S. Peterson, and J. Woods. 2017. Integrating modelling approaches for understanding telecoupling: global food trade and local land use. Land 6(3):56. http://dx.doi. org/10.3390/land6030056 Piguet, E. 2010. Linking climate change, environmental degradation, and migration: a methodological overview. Wiley Interdisciplinary Reviews: Climate Change 1(4):517-524. http://dx. doi.org/10.1002/wcc.54 Moran-Taylor, M. J., and M. J. Taylor. 2010. Land and leña: linking transnational migration, natural resources, and the environment in Guatemala. Population and Environment 32 (2-3):198-215. [online] URL: http://www.jstor.org/stable/40984176 Prell, C., L. Sun, K. Feng, J. He, and K. Hubacek. 2017. su9060974 Uncovering the spatially distant feedback loops of global trade: a network and input-output approach. Science of the Total Environment 586:401-408. https://doi.org/10.1016/j.scitotenv.2016.11.202 Müller, D., and D. K. Munroe. 2014. Current and future challenges in land-use science. Journal of Land Use Science 9 (2):133-142. http://dx.doi.org/10.1080/1747423X.2014.883731 Prowse, W. 2015. China’s fruit imports grow 23 per cent. AsiaFruit 25 February 2015. [online] URL: http://www.fruitnet.com/ asiafruit/article/164372/chinas-fruit-imports-grow-23-per-cent Munroe, D. K., K. McSweeney, J. L. Olson, and B. Mansfield. 2014. Using economic geography to reinvigorate land-change science. Geoforum 52:12-21. https://doi.org/10.1016/j. f 2013 12 005 Qureshi, S., and D. Haase. 2014. Compact, eco-, hybrid or teleconnected? Novel aspects of urban ecological research seeking compatible solutions to socio-ecological complexities. Ecological Indicators 42:1-5. https://doi.org/10.1016/j.ecolind.2014.04.017 Nepstad, D. C., C. M. Stickler, and O. T. Almeida. 2006. Globalization of the Amazon soy and beef industries: opportunities for conservation. Conservation Biology 20 (6):1595-1603. http://dx.doi.org/10.1111/j.1523-1739.2006.00510. Ravindran, M. S. 2013. China’s potential for economic coercion in the South China Sea disputes: a comparative study of the Philippines and Vietnam. Journal of Current Southeast Asian Affairs 31(3):105-132. [online] URL: https://journals.sub.uni- hamburg.de/giga/jsaa/article/view/572 Nielsen, J. Ø., and A. Reenberg. 2012. Exploring causal relations: the societal effects of climate change. Geografisk Tidsskrift- Danish Journal of Geography 112(2):89-92. http://dx.doi. org/10.1080/00167223.2012.741884 Reenberg, A., T. Langanke, S. B. P. Kristensen, and T. S. Colding. 2010. Globalisation of agricultural landscapes a land systems approach. Pages 31-56 in J. Primdahl and S. Swaffield, editors. Globalisation and agricultural landscapes: change patterns and policy trends in developed countries. Cambridge University Press, Cambridge, UK. https://doi.org/10.1017/CBO9780511844928.004 Niewöhner, J., J. Ø. Nielsen, I. Gasparri, Y. Gou, M. Hauge, N. Joshi, A. Schaffartzik, F. Sejersen, K. C. Seto, and C. Shughrue. 2016. Conceptualizing distal drivers in land use competition. Pages 21-40 in J. Niewöhner, A. Bruns, P. Hostert, T. Krueger, J. Ø. Nielsen, H. Haberl, C. Lauk, J. Lutz, and D. Müller, editors. Land use competition: ecological, economic and social perspectives. Springer, Cham, Switzerland. http://dx.doi.org/10.1007/978-3-3 19-33628-2_2 Reuters. 2012. Philippines seeks new markets amid sea dispute with China. Reuters 17 May 2012. [online] URL: http://www. reuters.com/article/philippines-china-idUSL4E8GH53R20120517 Reuters. 2016. South China Sea: ‘We have no fear of trouble,’ Chinese admiral warns. The Guardian 05 June 2016. [online] URL: Olson, G. A. 1991. The social scientist as author: Clifford Geertz on ethnography and social construction. Journal of Advanced Composition 11(2):245-268. [online] URL: http://www.jstor.org/ stable/20865794 https://www.theguardian.com/world/2016/jun/05/south-china-sea- we-have-no-fear-of-trouble-chinese-admiral-warns Richards, K., and N. Clifford. 2008. Science, systems and geomorphologies: why LESS may be more. su9060974 Earth Surface Processes and Landforms 33(9):1323-1340. http://dx.doi. org/10.1002/esp.1718 Ordonez, N., M. F. Seidl, C. Waalwijk, A. Drenth, A. Kilian, B. P. H. J. Thomma, R. C. Ploetz, and G. H. J. Kema. 2015. Worse comes to worst: bananas and Panama disease—when plant and pathogen clones meet. Plos Pathogens 11(11):e1005197. https:// doi.org/10.1371/journal.ppat.1005197 Rindfuss, R. R., S. J. Walsh, B. L. Turner II, J. Fox, and V. Mishra. 2004. Developing a science of land change: challenges and Ecology and Society 22(4): 30 https://www.ecologyandsociety.org/vol22/iss4/art30/ Ecology and Society 22(4): 30 https://www.ecologyandsociety.org/vol22/iss4/art30/ Ecology and Society 22(4): 30 andsociety.org/vol22/iss4/art30/ methodological issues. Proceedings of the National Academy of Sciences 101(39):13976-13981. http://dx.doi.org/10.1073/pnas.0401545101 Sturgeon, J. C. 2010. Governing minorities and development in Xishuangbanna, China: Akha and Dai rubber farmers as entrepreneurs. Geoforum 41(2):318-328. https://doi.org/10.1016/ j.geoforum.2009.10.010 Rocheleau, D., and R. Roth. 2007. Rooted networks, relational webs and powers of connection: rethinking human and political ecologies. Geoforum 38(3):433-437. http://dx.doi.org/10.1016/j. geoforum 2006 10 003 Sturgeon, J. C. 2013a. Cross-border rubber cultivation between China and Laos: regionalization by Akha and Tai rubber farmers. Singapore Journal of Tropical Geography 34(1):70-85. http://dx. doi.org/10.1111/sjtg.12014 Rueda, X., and E. F. Lambin. 2013. Linking globalization to local land uses: how eco-consumers and gourmands are changing the Colombian coffee landscapes. World Development 41:286-301. http://dx doi org/10 1016/j worlddev 2012 05 018 Sturgeon, J. C. 2013b. Introduction - Regionalization at the margins: ethnic minority cross-border dynamics in the Greater Mekong subregion. Singapore Journal of Tropical Geography 34 (1):3-8. http://dx.doi.org/10.1111/sjtg.12009 Schaffartzik, A., H. Haberl, T. Kastner, D. Wiedenhofer, N. Eisenmenger, and K.-H. Erb. 2015. Trading land: a review of approaches to accounting for upstream land requirements of traded products. Journal of Industrial Ecology 19(5):703-714. http://dx.doi.org/10.1111/jiec.12258 Taylor, P. 2016. Frontier commoditisation in post-socialist Southeast Asia. Asia Pacific Viewpoint 57(2):145-153. http://dx. doi.org/10.1111/apv.12125 Schierhorn, F., P. Meyfroidt, T. Kastner, T. Kuemmerle, A. V. Prishchepov, and D. Müller. 2016. The dynamics of beef trade between Brazil and Russia and their environmental implications. Global Food Security 11:84-92. https://doi.org/10.1016/j. gfs.2016.08.001 Thongmanivong, S., Y. Fujita, K. Phanvilay, and T. Vongvisouk. 2009. Agrarian land use transformation in northern Laos: from swidden to rubber. Southeast Asian Studies 47(3):330-347. Turner, B. L. II, R. E. Kasperson, P. A. Matson, J. J. McCarthy, R. W. Corell, L. Christensen, N. Eckley, J. X. Kasperson, A. Luers, M. L. Martello, C. Polsky, A. Pulsipher, and A. Schiller. 2003. A framework for vulnerability analysis in sustainability science. su9060974 Proceedings of the National Academy of Sciences 100 (14):8074-8079. http://dx.doi.org/10.1073/pnas.1231335100 Schönweger, O., A. Heinimann, M. Epprecht, J. Lu, and P. Thalongsengchanh. 2012. Concessions and leases in the Lao PDR: taking stock of land investments. Centre for Development and Environment (CDE), University of Bern Vientiane, Laos. Seitzinger, S. P., U. Svedin, C. L. Crumley, W. Steffen, S. A. Abdullah, C. Alfsen, W. J. Broadgate, F. Biermann, N. R. Bondre, J. A. Dearing, L. Deutsch, S. Dhakal, T. Elmqvist, N. Farahbakhshazad, O. Gaffney, H. Haberl, S. Lavorel, C. Mbow, A. J. McMichael, J. M. F. deMorais, P. Olsson, P. F. Pinho, K. C. Seto, P. Sinclair, M. Stafford Smith, and L. Sugar. 2012. Planetary stewardship in an urbanizing world: beyond city limits. Ambio 41 (8):787-794. http://dx.doi.org/10.1007/s13280-012-0353-7 Seitzinger, S. P., U. Svedin, C. L. Crumley, W. Steffen, S. A. Abdullah, C. Alfsen, W. J. Broadgate, F. Biermann, N. R. Bondre, Turner, B. L. II, E. F. Lambin, and A. Reenberg. 2007. The emergence of land change science for global environmental change and sustainability. Proceedings of the National Academy of Sciences 104(52):20666-20671. http://dx.doi.org/10.1073/ pnas.0704119104 Vaenkeo, S. 2016. Govt bans banana plantations. Vientiane Times 12 November 2016. [online] URL: http://www.vientianetimes.org. la/FreeContent/FreeConten_Govt_265.htm Seto, K. C., and A. Reenberg. 2014. Rethinking global land use in an urban era. MIT Press, Cambridge, Massachusetts, USA. http:// dx.doi.org/0.7551/mitpress/9780262026901.001.0001 van Vliet, J., N. R. Magliocca, B. Büchner, E. Cook, J. M. Rey Benayas, E. C. Ellis, A. Heinimann, E. Keys, T. M. Lee, J. Liu, O. Mertz, P. Meyfroidt, M. Moritz, C. Poeplau, B. E. Robinson, R. Seppelt, K. C. Seto, and P. H. Verburg. 2016. Meta-studies in land use science: current coverage and prospects. Ambio 45(1):15-28. http://dx doi org/10 1007/s13280-015-0699-8 Mertz, P. Meyfroidt, M. Moritz, C. Poeplau, B. E. Robinson, R. Seppelt, K. C. Seto, and P. H. Verburg. 2016. Meta-studies in land use science: current coverage and prospects. Ambio 45(1):15-28. http://dx.doi.org/10.1007/s13280-015-0699-8 Seto, K. C., A. Reenberg, C. G. Boone, M. Fragkias, D. Haase, T. Langanke, P. Marcotullio, D. K. Munroe, B. Olah, and D. Simon. 2012. Urban land teleconnections and sustainability. Proceedings of the National Academy of Sciences 109 (20):7687-7692. http://dx.doi.org/10.1073/pnas.1117622109 van Vliet, N., O. Mertz, A. Heinimann, T. Langanke, U. Pascual, B. Schmook, C. Adams, D. Schmidt-Vogt, P. Messerli, S. Leisz, J.-C. Castella, L. Jørgensen, T. Birch-Thomsen, C. Hett, T. Bech- Bruun, A. Ickowitz, K. C. Vu, K. Yasuyuki, J. Fox, C. Padoch, W. Dressler, and A. D. su9060974 Ziegler. 2012. Trends, drivers and impacts of changes in swidden cultivation in tropical forest-agriculture frontiers: a global assessment. Global Environmental Change 22 (2):418-429. https://doi.org/10.1016/j.gloenvcha.2011.10.009 Shi, W. 2008. Rubber boom in Luang Namtha: a transnational perspective. GTZ, Vientiane, Laos. [online] URL: https://www2. giz.de/wbf/4tDx9kw63gma/ShiW_Rubber_Luang_Namtha_0802_final. pdf Sikor, T., G. Auld, A. J. Bebbington, T. A. Benjaminsen, B. S. Gentry, C. Hunsberger, A.-M. Izac, M. E. Margulis, T. Plieninger, H. Schroeder, and C. Upton. 2013. Global land governance: from territory to flow? Current Opinion in Environmental Sustainability 5(5):522-527. https://doi.org/10.1016/j.cosust.2013.06.006 Vandergeest, P. 2003. Land to some tillers: development-induced displacement in Laos. International Social Science Journal 55 (175):47-56. http://dx.doi.org/10.1111/1468-2451.5501005 Vayda, A. P. 1983. Progressive contextualization: methods for research in human ecology. Human Ecology 11(3):265-281. http:// dx.doi.org/10.1007/BF00891376 Simeon, L. M. 2016. China lifts import ban on Philippine bananas. Philippine Star 07 October 2016. [online] URL: http:// www.philstar.com/business/2016/10/07/1630958/china-lifts-import- ban-philippine-bananas Simeon, L. M. 2016. China lifts import ban on Philippine bananas. Philippine Star 07 October 2016. [online] URL: http:// hil /b i /2016/10/07/1630958/ hi lif i Verburg, P. H. 2014. The representation of human-environment interactions in land change research and modelling. Pages Ecology and Society 22(4): 30 https://www.ecologyandsociety.org/vol22/iss4/art30/ Ecology and Society 22(4): 30 https://www.ecologyandsociety.org/vol22/iss4/art30/ based rotations in the tropics. Pedosphere 25(6):868-877. http:// dx.doi.org/10.1016/S1002-0160(15)30067-9 161-177 in M. J. Manfredo, J. J. Vaske, A. Rechkemmer, and E. A. Duke, editors. Understanding society and natural resources: forging new strands of integration across the social sciences. Springer, Dordrecht, The Netherlands. http://dx.doi. Zimmerer, K. S. 2007. Agriculture, livelihoods, and globalization: the analysis of new trajectories (and avoidance of just-so stories) of human-environment change and conservation. Agriculture and Human Values 24(1):9-16. http://dx.doi.org/10.1007/s10460-006-9028- y Verburg, P. H., N. Crossman, E. C. Ellis, A. Heinimann, P. Hostert, O. Mertz, H. Nagendra, T. Sikor, K.-H. Erb, N. Golubiewski, R. Grau, M. Grove, S. Konaté, P. Meyfroidt, D. C. Parker, R. R. Chowdhury, H. Shibata, A. Thomson, and L. Zhen. 2015. Land system science and sustainable development of the earth system: a global land project perspective. Anthropocene 12:29-41. https://doi.org/10.1016/j.ancene.2015.09.004 Zimmerer, K. S., and T. J. Bassett, editors. 2003. Political ecology: an integrative approach to geography and environment-development studies. Guilford Press, New York, New York, USA. Zimmerer, K. S., and T. J. Bassett, editors. 2003. Political ecology: an integrative approach to geography and environment-development studies. Guilford Press, New York, New York, USA. Verburg, P. H., W. T. de Groot, and A. J. Veldkamp. 2003. Zimmerer, K. S., and T. J. Bassett, editors. 2003. Political ecology: an integrative approach to geography and environment-development studies. Guilford Press, New York, New York, USA. su9060974 Methodology for multi-scale land-use change modelling: concepts and challenges. Pages 17-51 in A. J. Dolman, A. Verhagen, and C. A. Rovers, editors. Global environmental change and land use. Springer, Dordrecht, The Netherlands. http://dx.doi. org/10.1007/978-94-017-0335-2_2 Verburg, P. H., K.-H. Erb, O. Mertz, and G. Espindola. 2013. Land system science: between global challenges and local realities. Current Opinion in Environmental Sustainability 5(5):433-437. https://doi.org/10.1016/j.cosust.2013.08.001 Vongvisouk, T., and M. B. Dwyer. 2016. Falling rubber prices in northern Laos: local responses and policy options. Helvetas, Vientiane, Laos. Walters, B. B., and A. P. Vayda. 2009. Event ecology, causal historical analysis, and human-environment research. Annals of the Association of American Geographers 99(3):534-553. http://dx. doi.org/10.1080/00045600902931827 Xinhua. 2011a. S China braces for strong tropical storm Nalgae. China Daily 03 October 2011. [online] URL: http://www. chinadaily.com.cn/china/2011-10/03/content_13834880.htm Xinhua. 2011b. Typhoon Nesat causes heavy damage in S China. China Daily 01 October 2011. [online] URL: http://www. chinadaily.com.cn/china/2011-10/01/content_13831889.htm Young, O. R., F. Berkhout, G. C. Gallopin, M. A. Janssen, E. Ostrom, and S. van der Leeuw. 2006. The globalization of socio- ecological systems: an agenda for scientific research. Global Environmental Change 16(3):304-316. https://doi.org/10.1016/j. gloenvcha.2006.03.004 Yu, Y., K. Feng, and K. Hubacek. 2013. Tele-connecting local consumption to global land use. Global Environmental Change 23 (5):1178-1186. https://doi.org/10.1016/j.gloenvcha.2013.04.006 Zhang, L., Y. Kono, and S. Kobayashi. 2014. The process of expansion in commercial banana cropping in tropical China: a case study at a Dai village, Mengla County. Agricultural Systems 124:32-38. http://dx.doi.org/10.1016/j.agsy.2013.10.006 Zhong, S., Y. Mo, G. Guo, H. Zeng, and Z. Jin. 2014. Effect of continuous cropping on soil chemical properties and crop yield in banana plantation. Journal of Agricultural Science and Technology 16(1):239-250. [online] URL: http://jast.modares.ac. ir/article 10314 5044 html Zhong, S., H. Zeng, and Z. Jin. 2015. Soil microbiological and biochemical properties as affected by different long-term banana-
https://openalex.org/W2985289457	https://escholarship.org/content/qt5zf6499d/qt5zf6499d.pdf?t=q9hugg	English	null	Effect of Brief Biofeedback via a Smartphone App on Stress Recovery: Randomized Experimental Study	JMIR serious games	2,019	cc-by	9,648	UC Irvine UC Irvine Previously Published Works Title Effect of Brief Biofeedback via a Smartphone App on Stress Recovery: Randomized Experimental Study Permalink https://escholarship.org/uc/item/5zf6499d Journal JMIR Serious Games, 7(4) ISSN 2291-9279 Authors Hunter, John F Olah, Meryl S Williams, Allison L et al. Publication Date 2019 DOI 10.2196/15974 Peer reviewed UC Irvine UC Irvine Previously Published Works Title Effect of Brief Biofeedback via a Smartphone App on Stress Recovery: Randomized Experimental Study Permalink https://escholarship.org/uc/item/5zf6499d Journal JMIR Serious Games, 7(4) ISSN 2291-9279 Authors Hunter, John F Olah, Meryl S Williams, Allison L et al. Publication Date 2019 DOI 10.2196/15974 Peer reviewed UC Irvine UC Irvine Previously Published Works Title Effect of Brief Biofeedback via a Smartphone App on Stress Recovery: Randomized Experimental Study Permalink https://escholarship.org/uc/item/5zf6499d Journal JMIR Serious Games, 7(4) ISSN 2291-9279 Authors Hunter, John F Olah, Meryl S Williams, Allison L et al. Publication Date 2019 DOI 10.2196/15974 Peer reviewed UC Irvine UC Irvine Previously Published Title Effect of Brief Biofeedback via a Smartphon Experimental Study Permalink https://escholarship.org/uc/item/5zf6499d Journal JMIR Serious Games, 7(4) ISSN 2291-9279 Authors Hunter, John F Olah, Meryl S Williams, Allison L et al. Publication Date 2019 DOI 10.2196/15974 Peer reviewed Effect of Brief Biofeedback via a Smartphone App on Stress Recovery: Randomized Experimental Study John F Hunter1, PhD; Meryl S Olah1, BA; Allison L Williams2, PhD; Acacia C Parks2, PhD; Sarah D Pressman1, PhD 1Department of Psychological Science, University of California, Irvine, Irvine, CA, United States 2Happify Health, New York, NY, United States Corresponding Author: John F Hunter, PhD Department of Psychological Science University of California, Irvine 4201 Social & Behavioral Sciences Gateway Irvine, CA, 92697 United States Phone: 1 9493459987 Email: jhunter1@uci.edu Abstract Background: Smartphones are often vilified for negatively influencing well-being and contributing to stress. However, these devices may, in fact, be useful in times of stress and, in particular, aid in stress recovery. Mobile apps that deliver evidence-based techniques for stress reduction, such as heart rate variability biofeedback (HRVB) training, hold promise as convenient, accessible, and effective stress-reducing tools. Numerous mobile health apps that may potentially aid in stress recovery are available, but very few have demonstrated that they can influence health-related physiological stress parameters (eg, salivary biomarkers of stress). The ability to recover swiftly from stress and reduce physiological arousal is particularly important for long-term health, and thus, it is imperative that evidence is provided to demonstrate the effectiveness of stress-reducing mobile health apps in this context. Objective: The purpose of this research was to investigate the physiological and psychological effects of using a smartphone app for HRVB training following a stressful experience. The efficacy of the gamified Breather component of the Happify mobile health app was examined in an experimental setting. Methods: In this study, participants (N=140) underwent a laboratory stressor and were randomly assigned to recover in one of three ways: no phone present, phone present, with the HRBV game. Those in the no phone condition had no access to their phone. Those in the phone present condition had their phone but did not use it. Those in the HRVB game condition used the serious game Breather on the Happify app. Stress recovery was assessed via repeated measures of salivary alpha amylase, cortisol, and self-reported acute stress (on a 1-100 scale). Results: Participants in the HRVB game condition had significantly lower levels of salivary alpha amylase during recovery than participants in the other conditions (F2,133=3.78, P=.03). There were no significant differences among the conditions during recovery for salivary cortisol levels or self-reported stress. Conclusions: These results show that engaging in a brief HRVB training session on a smartphone reduces levels of salivary alpha amylase following a stressful experience, providing preliminary evidence for the effectiveness of Breather in improving physiological stress recovery. Given the known ties between stress recovery and future well-being, this study provides a possible mechanism by which gamified biofeedback apps may lead to better health. (JMIR Serious Games 2019;7(4):e15974) doi: 10.2196/15974 Powered by the California Digital Library University of California Powered by the California Digital Library University of California eScholarship.org eScholarship.org JMIR SERIOUS GAMES Hunter et al Original Paper JMIR Serious Games 2019 \| vol. 7 \| iss. 4 \| e15974 \| p. 1 (page number not for citation purposes) http://games.jmir.org/2019/4/e15974/ Background When undergoing a stressor, the typical response is for our sympathetic nervous system to activate and parasympathetic activity to decline (indicating low HRV). However, an adaptive response to a stressor would be for an individual to exhibit higher HRV. This is because greater fluctuations in heart rhythm (higher HRV) indicate greater adaptability to physiological needs than fewer fluctuations (lower HRV) [24]. When HRV is higher, it is a sign that our cardiovascular system (and multiple associated systems) is responding appropriately to environmental demands (eg, a stressor). Thus, using HRVB to increase HRV may be helpful when recovering from a stressor because it activates our parasympathetic nervous system and allows us to more quickly reduce physiological arousal. It is also important to note that high HRV is considered a protective factor against cardiovascular disease and is generally associated with good health and well-being [26]. Additionally, low HRV has numerous negative implications for long-term health outcomes, such as increased risk for mortality and morbidity [27,28]. HRVB training has been successfully utilized in a variety of acute stress settings and is well-validated technique for reducing stress [29,30]. The goal of undergoing a 5-minute guided session on Breather is for the user to increase HRV and recover effectively from a stressful experience. Smartphones provide a range of possibilities for helping individuals recover from a stressful experience and may even do so when not actively used. Even when merely present, smartphones serve as symbols that can cause cognitive distraction [10] or activate representations of social connections [11]. Distraction induced by smartphones has generally been viewed as detrimental [12]; however, such distraction can be beneficial when faced with a stressor, because it can draw attention away from the negative stimuli at hand and help circumvent rumination [13]. In addition, symbolic representations of social connections can elicit perceptions of social support [14], which, when perceived passively, is the most effective form of support for stress alleviation [15]. By providing distraction and perceived social support, the mere presence of a smartphone may aid in stress recovery by serving as a “digital security blanket” in instances of social stress [16]. Thus, it is important to investigate more fully how merely having a smartphone in one’s presence may aid in stress alleviation. Research has demonstrated that actually using one’s smartphone can be beneficial or detrimental for stress recovery, depending on how and when the device is used. Background Although smartphones are often criticized for contributing to ill-being [1-4], these devices hold great potential for improving one’s well-being if utilized properly in specific contexts. Smartphones may be particularly useful as tools that provide gamified apps to deliver stress-buffering interventions. Stress is prevalent in many peoples’ lives, and its accumulated effects can lead to various undesirable physical and mental health outcomes, such as an increased risk for mortality [5]. Many of these negative outcomes are due to prolonged activation of one’s stress systems [6]. However, if individuals employ strategies that promote more efficient recovery from stressors, some of the negative long-term impacts may be mitigated. One promising way to use a smartphone to aid in stress reduction is by engaging with a mobile health (mHealth) app. mHealth apps can utilize technological capabilities (eg, phone sensors, interactive displays) and draw on the ubiquity of smartphones in everyday life to deliver functional and convenient interventions [22]. By combining evidence-based stress-reduction techniques with an engaging and ever-present medium, mHealth apps hold great promise for mitigating the negative effects of stress. Why do smartphones present a promising opportunity for altering stress recovery? Psychologists have developed a variety of evidence-backed strategies that aid in stress reduction [7], such as biofeedback training, which directs individuals to monitor and attempt to alter their physiological arousal pattern [8]. Smartphones are ideally to be used as a tool for biofeedback training and to combat the negative effects of stress because they are popular, conveniently accessible, and have an array of technological capabilities [9]. Since these devices are nearly omnipresent in daily life, they can deliver interventions and assistance wherever and whenever needed. Happify is an mHealth app that provides gamified activities aimed at improving well-being and reducing stress [23]. Happify is representative of multiple aspects of other mHealth apps because it employs various smartphone technological capabilities (eg, sensors, visual and audio components, engaging interface) and incorporates empirically validated strategies to deliver training in a self-contained package. Within the Happify suite of activities, the Breather function delivers heart rate variability biofeedback (HRVB) training (Multimedia Appendix 1). HRVB is a particularly effective stress-reducing activity that targets changes in heart rate variability (HRV) by regulating breathing and bringing awareness to physiological function [24]. HRV is an index of beat-to-beat changes in heart rate and is an indicator of parasympathetic nervous system activity [25]. KEYWORDS heart rate variability biofeedback; stress recovery; salivary alpha amylase; smartphone; mHealth http://games.jmir.org/2019/4/e15974/ http://games.jmir.org/2019/4/e15974/ XSL•FO RenderX JMIR SERIOUS GAMES Hunter et al beneficial for stress recovery. In fact, when looked at more broadly, greater use of smartphones is associated with higher levels of physiological stress [21]. Therefore, if we hope to highlight the most effective ways to use a phone to reduce stress, it may be important to go beyond natural phone use habits and, instead, provide structured apps that are specifically designed for stress reduction. JMIR Serious Games 2019 \| vol. 7 \| iss. 4 \| e15974 \| p. 2 (page number not for citation purposes) http://games.jmir.org/2019/4/e15974/ Background The app is designed to increase HRV and reduce stress if the users adhere to the directions properly for a 5-minute session. rement and calibrating breathing guidance. After calibration is complete, the interface changes into a calming nature scene (eg, underwater coral bed, tropical beach, mountaintops). The user then travels through the natural environment while he/she continues to breathe along with the meter (Figure 2A). As they breathe deeply and regularly, their HRV increases and the scene becomes more complex and beautiful (eg, coral polyps bloom, flowers grow; Figure 2B). After calibration is complete, the interface changes into a calming nature scene (eg, underwater coral bed, tropical beach, mountaintops). The user then travels through the natural environment while he/she continues to breathe along with the meter (Figure 2A). As they breathe deeply and regularly, their HRV increases and the scene becomes more complex and beautiful (eg, coral polyps bloom, flowers grow; Figure 2B). By visually monitoring the changes in the scene, individuals are undergoing HRVB; this process is analogous to how individuals monitor electrocardiogram signals in more traditional “nongamified” HRVB trainings. The app is designed to increase HRV and reduce stress if the users adhere to the directions properly for a 5-minute session. Figure 2. Example of how the full display unfolds when an individual is using Breather. g multimodal approach that combines subjective and objective assessments [34]. multimodal approach that combines subjective and objective assessments [34]. http://games.jmir.org/2019/4/e15974/ JMIR Serious Games 2019 \| vol. 7 \| iss. 4 \| e15974 \| p. 3 (page number not for citation purposes) Background For example, research has shown that using social media sites such as Facebook can provide social resources that sometimes help buffer acute stress [17], but at other times, fail to do so [18]. In some instances, social support gleaned via text message can reduce cardiovascular responses to stress [19]. However, sending and receiving text messages can also increase physiological indicators of stress such as heart rate, respiration, and skin conductance [20]. These mixed findings concerning how phone use influences stress underscore the fact that the ways in which we commonly interact with our devices are not universally Delivering HRVB through a smartphone app provides many advantages over traditional training. Breather overcomes barriers of nondigital HRVB interventions (eg, bulky and expensive equipment, lengthy sessions) because it is quick to administer, is portable and readily accessible, and has all the hardware and http://games.jmir.org/2019/4/e15974/ XSL•FO RenderX JMIR SERIOUS GAMES Hunter et al a simple signal that is visible to the user. The accuracy of this technology for determining HRV has been recently validated in a series of experiments that compared simultaneously obtained HRV metrics from Happify Breather and traditional electrocardiogram techniques using electrodes [31]. software integrated into a single device. Breather has taken advantage of mobile technology affordances to package an HRVB product in ways that should allow it to be used across a variety of stressful contexts. Users of Breather generate HRV observations by placing their index finger over the camera of their smartphone (Figure 1A). The light from the camera can be used to monitor blood volume changes within the finger. This process (ie, photoplethysmography) relies on measuring changes in light absorption on the skin of the finger. Algorithms programmed by Happify software engineers then transform those data into After calibrating the heart rate of the individual, a circular meter directs the individual to follow the breathing patterns on screen (Figure 1B). The meter directs the individual to breathe in for 4 seconds and then breathe out for 6 seconds. This 10-second breathing cycle is ideal for creating a resonant frequency (ie, breathing and heart rate align) that should maximize HRV [32]. Figure 1. On-screen instructions for obtaining heart rate variability measurement and calibrating breathing guidance. ement and calibrating breathing guidance. By visually monitoring the changes in the scene, individuals are undergoing HRVB; this process is analogous to how individuals monitor electrocardiogram signals in more traditional “nongamified” HRVB trainings. This Study This study investigated the effectiveness of using an HRVB smartphone app to aid in stress recovery. In order to account for the potential stress-buffering effects of simply having a phone [16], we included a condition in which individuals had a phone in their presence. Thus, use of the HRVB app was compared to two control conditions, one in which no phone was present and one in which individuals had their smartphones present when recovering from a stressor. To examine how these different types of smartphone interactions influence stress recovery, a laboratory experiment was conducted in which participants underwent a standardized stressor, used their phone in a particular way depending on their assigned condition, and were assessed on a range of psychological and physiological stress indicators. We hypothesized that those in the HRVB game condition would recover from the stressor more effectively than those who had their phones present or had no phone at all. Participants then underwent a shortened version of the Trier Social Stress Task (TSST) [40,41] to induce psychological and physiological stress. The TSST consists of participants undergoing a public speaking task and arithmetic task in front of a panel of critically evaluative judges. The TSST has been shown to be a valid and reliable instrument for inducing physiological and psychological stress responses [42]. Immediately after the conclusion of the TSST, participants collected another saliva sample and self-reported their feelings of stress. For the next 5 minutes, participants were left alone in the room and interacted with their phone in a particular way depending on condition. Those in the no phone condition did not have their phone returned and were told to sit quietly for the next 5 minutes while the next portion of the study was prepared. Those in the phone present condition were given their phone but told “please do not use your phone for the remainder of the study.” Those in HRVB game condition were told to open the Happify app, navigate to Breather, and “follow the instructions on the application.” After the 5-minute phone manipulation period, the researcher returned to the room and instructed the participant to continue answering a series of questionnaires. Twenty minutes after completion of the TSST, a third saliva sample was collected. Forty minutes after the completion of the TSST, a fourth saliva sample was collected. At the conclusion of the study, the researcher and both judges debriefed the participant. This Study This study is one of the first empirical investigations to assess the effects of smartphone app usage on salivary biomarkers of physiological stress [39]. To our knowledge, it is also the first to examine the stress-buffering effects of an HRVB intervention delivered via a smartphone without any external equipment. In addition, since simply having a phone in your presence has been shown to aid in stress recovery, the inclusion of separate experimental conditions for a HRVB game and mere phone presence enabled us to differentiate their effects on stress. The results of this study will help our understanding of why smartphones might be helpful in times of stress, which may inform future recommendations about the most effective way to use a smartphone following a stressful experience. Participants The study was approved by the University of California, Irvine Institutional Review Board, and participants were recruited via the University of California, Irvine undergraduate psychology subject pool. These data were drawn from a larger project that included additional research questions outside the scope of this study. For this particular study, a total of 140 participants were examined (mean age 20.28, SD 2.68; 77.1% female; 45.7% Asian; 27.9% Hispanic/Latino; 15.7% Caucasian; 6.4% African American). Participants were screened for eligibility and excluded from participation if they were diagnosed with a cardiovascular disease, were regularly taking mood altering or cardiovascular altering medication, regularly smoked cigarettes, Procedures Participants underwent an approximately 90-minute laboratory session. All participant phones were confiscated at the beginning of the study under the pretext of measuring the external physical properties of the phone, which allowed the experimenter to later manipulate the phone conditions without arousing suspicion and ensure that all participants experienced similar circumstances of having their phone taken away. For participants randomly assigned to the HRVB game condition, the Happify app was installed on their phone and the experimenter guided them through the calibration settings of the Breather function while carefully concealing any indication that the purpose of using Breather was to reduce stress. Participants in the other conditions filled out surveys during this time. After participants completed a series of questionnaires and acclimated to the laboratory environment (approximately 25 minutes), the experimenter returned to the laboratory room and collected a baseline saliva sample. Participants were instructed in the passive drool technique of collecting their own saliva sample. Hunter et al were not fluent in English, or did not have an iPhone. All participants were University of California, Irvine, students and consented to participate. Data collection took place from July 2018 through February 2019. were not fluent in English, or did not have an iPhone. All participants were University of California, Irvine, students and consented to participate. Data collection took place from July 2018 through February 2019. physiologically aroused, sAA is released via the salivary glands and indicates an immediate stress response. In some cases, sAA is more strongly tied to stress and anxiety than cortisol [37,38], and sAA (but not cortisol) has also been shown to be influenced by smartphones while recovering from a stressor [16]. Thus, sAA is considered our primary outcome of interest. These salivary assessments of hypothalamic-pituitary-adrenal and autonomic nervous system activities combined with self-report give researchers a comprehensive understanding of physiological responses to stress. http://games.jmir.org/2019/4/e15974/ JMIR Serious Games 2019 \| vol. 7 \| iss. 4 \| e15974 \| p. 4 (page number not for citation purposes) Assessment of Stress In this paper, we used a multimethodological approach to determine the effects of smartphones on stress recovery. Stress can be assessed in a variety of ways, and each method provides unique insight into the complex dynamics of how stress impacts our bodies and brains. One of the most common ways to assess stress is to ask individuals to subjectively rate their stress level. Although self-report is advantageous for assessing perceived stress, there are problems of bias (eg, self-presentation concerns) that limit the accuracy and generalizability of these assessments [33]. Due to its complexity, the most appropriate and comprehensive manner in which stress is assessed is a In this paper, we used a multimethodological approach to determine the effects of smartphones on stress recovery. Stress can be assessed in a variety of ways, and each method provides unique insight into the complex dynamics of how stress impacts our bodies and brains. One of the most common ways to assess stress is to ask individuals to subjectively rate their stress level. Although self-report is advantageous for assessing perceived stress, there are problems of bias (eg, self-presentation concerns) that limit the accuracy and generalizability of these assessments [33]. Due to its complexity, the most appropriate and comprehensive manner in which stress is assessed is a One of the most effective, reliable, and efficient ways to capture physiological measurements of stress is to analyze salivary biomarkers. Salivary cortisol is a downstream output of hypothalamic-pituitary-adrenal axis system activation and is one of the most widely used and reliable measures of physiological stress [35]. Higher levels of salivary cortisol indicate greater physiological stress. Another emerging indicator of physiological stress is salivary alpha amylase (sAA), which is an indicator of autonomic nervous system activity and is most strongly tied with sympathetic nervous system activity, the system responsible for the “fight-or-flight” response [36]. When XSL•FO RenderX XSL•FO RenderX XSL•FO RenderX JMIR SERIOUS GAMES Hunter et al Manipulation checks Analysis of sAA from baseline (mean 85.31, SD 59.40) to post-TSST (mean 128.58, SD 88.53) revealed that participants displayed significant increases in sAA following the TSST (t276=–4.78, P<.001). In addition, analysis of cortisol from baseline (mean 0.21, SD 0.11) to post-TSST (mean 0.34, SD 0.24) showed that participants displayed significant increases in cortisol following the TSST, (t271=-5.99, P<.001). Finally, analysis of self-reported stress from baseline (mean 22.73, SD 21.48) to post-TSST (mean 47.31, SD 29.37) demonstrated that participants displayed significant increases in self-reported stress following the TSST (t276=–7.96, P<.001). These results indicate that TSST reliably increased psychological and physiological stress. Salivettes were stored at –80°C until batch analysis at the end of data collection at the laboratory of the Institute for Interdisciplinary Salivary Bioscience Research (University of California Irvine, Irvine, California). Before assaying, the samples were thawed for an hour to return them to room temperature. For cortisol, all samples were assayed in duplicate by using an expanded-range high-sensitivity salivary cortisol enzyme immunoassay kit (Salimetrics, LLC, State College, Pennsylvania). The assay range of sensitivity was 0.007 to 3.0 µg/dL, and the average intraassay coefficient of variation was 5.5%. For sAA, samples were tested in duplicate using a commercially available kinetic enzyme reaction assay kit (Salimetrics, LLC). The assay range of sensitivity was 0.4-400 U/mL, and the average intraassay coefficient of variation was 3.3.%. Differences in Salivary Alpha Amylase Recovery Between Conditions Between-subject comparisons indicated that there was a significant main effect of condition on sAA recovery (F2,133=3.78, P=.03; no phone: mean 10.078, SE 0.271; phone presence: mean 10.007, SE 0.280; HRVB game: mean 9.132, SE 0.266). Post-hoc comparisons revealed that those in the HRVB game condition displayed significantly less sAA during recovery than those in the no phone condition (t93=2.48, P=.02) and the phone present condition (t90=2.26, P=.03). The no phone and phone present conditions did not differ (t88=–0.19, P=.85; Multimedia Appendix 2). Within-subject analyses revealed that there was no significant main effect of time for sAA recovery (F1,133=.003, P=.96) and no significant interaction between time and condition (F2,133=0.081, P=.92). Hunter et al were therefore controlled for in cortisol analyses. Baseline sAA was associated with sAA recovery and was therefore controlled for in sAA analyses. Baseline self-reported stress was associated with self-reported stress recovery and was therefore controlled for in self-reported stress analyses. Self-reported Stress Participants were asked to indicate, “How stressed do you feel right now?” on a visual analog scale from 1-100. This simple one-item scale has been shown to be valid and reliable for assessing perceptions of acute stress [43]. Self-reported stress was assessed at three time points (baseline, post-TSST, +20 minutes recovery). Independent sample t tests were used to conduct manipulation checks and ensure that exposure to the TSST reliably increased self-reported stress, sAA, and cortisol from baseline (time 1) to post-TSST stress (time 2). Repeated-measures mixed analysis of covariance was used to analyze the effect of condition on each dependent variable. Since the phone manipulation occurred after the TSST, analyses focused on differences in recovery and therefore used time (post-TSST stress at time 2 and +20 minute recovery at time 3) as the within-subject factor. Condition was included as a between-subject factor, and appropriate covariates were controlled for depending on the outcome of interest. Baseline values were controlled for to provide a more conservative and unbiased estimate of between-subject differences in composite recovery values [44]. Post-hoc comparisons were conducted to examine specific pairwise differences when a significant effect of condition was found. Physiological Stress Salivary cortisol and sAA were both collected to provide a broad assessment of the physiological stress response. Since these salivary biomarkers indicate activity of different physiological stress systems and have different secretion times, the inclusion of both gives us a more comprehensive understanding of stress effects. Salivary cortisol and sAA were collected using a passive drool technique with polypropylene cryovial salivettes at four time points. The first three samples were assayed for sAA, and the last three samples were assayed for cortisol to accommodate for the differing secretion times of each analyte (ie, an approximately 20-minute lag time for salivary cortisol secretion into saliva compared to immediate secretion of sAA) and ensure that the collection timing aligned to capture measures of baseline, post-TSST, and +20 minute recovery time points. Experimental sessions were conducted in the afternoon (between 1 PM to 6 PM) to account for the diurnal rhythm of sAA and cortisol. Demographics and Covariates Demographic information and potential covariates, including age, sex, ethnicity, socioeconomic status, perceived psychological stress, measures of daily phone use, time since waking, use of hormonal contraceptives, and caffeine intake, were collected via self-report. JMIR Serious Games 2019 \| vol. 7 \| iss. 4 \| e15974 \| p. 4 (page number not for citation purposes) JMIR Serious Games 2019 \| vol. 7 \| iss. 4 \| e15974 \| p. 4 (page number not for citation purposes) XSL•FO RenderX JMIR SERIOUS GAMES JMIR Serious Games 2019 \| vol. 7 \| iss. 4 \| e15974 \| p. 5 (page number not for citation purposes) http://games.jmir.org/2019/4/e15974/ Limitations There are several limitations that limit the generalizability of these results. First, our sample is not representative of the population at large. The majority of our participants were healthy young Asian women, all of whom were iPhone users and college educated. Since we drew our sample from a university population, our participants were likely wealthier, younger, more dependent on their phone, and more educated than the average person. Thus, these conclusions cannot be extrapolated to all populations. In addition, the effectiveness of Breather for influencing sAA recovery compared to the other conditions may have been statistically limited by differences in baseline values. Those in the HRVB game condition had significantly lower levels of sAA at baseline. These differences may have been due to a methodological inconsistency, as the individuals in the HRVB game condition had a slightly different experience during the baseline period before undergoing the TSST; they spent approximately 2 minutes receiving training on the HRVB app. Per methodological recommendations, these baseline values were controlled for, to provide a more conservative and unbiased estimate of between-subject differences in recovery [43]. Without the inclusion of baseline sAA as a covariate in the models, there would have been greater statistical differences between HRVB game use and the other conditions. Although this statistical decision does limit the magnitude of our sAA recovery findings, these differences in baseline raise an interesting point about the ways in which Breather influences reactions to a stressor. If, indeed, the brief training period reduced baseline sAA and sAA reactivity to the stressor, then using an HRVB serious game like Breather could possibly be an effective method for buffering stress reactivity as well as recovery and may be an advisable activity to engage in prior to a major stressor. Before any recommendations can be made, future studies should explore the optimal timing for HRVB implementation and determine whether it is most effective before or after a stressor. Our study design did not allow us to conclusively determine the mechanisms responsible for the stress-buffering effect, but there are several possibilities for why Breather effectively aided in stress recovery. The most obvious explanation is that undergoing HRVB training increases parasympathetic activity, which is typically inversely related to sympathetic indicators such as sAA. Thus, the low levels of sAA for those in the HRVB game condition may be indicative of direct physiological alterations induced by the use of Breather. Differences in Cortisol Recovery Between Conditions Although the cortisol levels declined during recovery for all conditions, there was no main effect of condition on cortisol recovery (F2,126=1.19, P=.31). Interestingly, those who had their phones present during stress recovery did not glean any additional stress-buffering benefits beyond those with no phone. Previous work has demonstrated that having a phone present, but not using it, leads to steep declines in sAA during physiological recovery from a stressor [16]. We failed to replicate this outcome. In the study by Hunter et al [16], participants had their phones with them while undergoing the stressor. In this study, participants only had their phones immediately after the study. This difference in timing implies that it may be helpful to have a phone present while experiencing a stressor, but it provides little to no benefit when present during recovery. In addition, a phone may serve as a “digital security blanket” in mildly stressful situations like social exclusion but may not exert similarly beneficial effects under more potent stressors such as the TSST. Discussion In this study, we examined the effect of using or having a smartphone on psychological and physiological stress reduction during recovery. We found that those who used an HRVB training app exhibited the lowest levels of sAA during recovery. Specifically, those in the HRVB game condition released less sAA during recovery than those who had their phones present or had no phone at all. These results indicate that engaging in a brief 5-minute HRVB training session on a smartphone can effectively reduce stress-related sympathetic activity, as assessed by levels of sAA. Although the magnitude of the effect for the change in sAA was only small to medium (η2=0.05) [45], it was similar to previous studies [16,46]. The sAA findings are particularly important because high levels of sAA are associated with a range of deleterious health-related outcomes such as asthma, frequency of illness, and chronic fatigue [47-49]; therefore, lower levels of sAA are desirable from a health perspective. Our findings provide health-related information about the use of mHealth interventions on a smartphone. Since delayed physiological recovery can be predictive of risk for long-term health issues [50], we can infer that using a serious game such as Breather when recovering from a stressful experience may provide long-term health benefits. Differences in Self-Reported Stress Recovery Between Conditions Although self-reported stress declined during recovery for all conditions, there was no main effect of condition on self-reported stress recovery (F2,133=1.42, P=.24). http://games.jmir.org/2019/4/e15974/ JMIR Serious Games 2019 \| vol. 7 \| iss. 4 \| e15974 \| p. 6 (page number not for citation purposes) Analytic Strategy All dependent variables (self-reported stress, sAA, and cortisol) were checked for skewness and kurtosis and transformed accordingly. No transformation was performed for values of self-reported stress. Values of sAA were moderately skewed, and a square root transformation was used to transform the values to approximate a normal distribution. Values of cortisol were moderately skewed, and a logarithmic transformation was used to transform the values to approximate a normal distribution. Outlying values above or below three SDs from the mean were removed. No outliers were removed for self-reported stress, three outliers (2.1%) were removed for sAA, and six outliers (2.8%) were removed for cortisol. Models controlled for covariates that were significantly associated with the dependent variable. Sex, time since waking, and baseline cortisol were associated with cortisol recovery and XSL•FO RenderX JMIR SERIOUS GAMES Hunter et al for the lack of cortisol and self-reported stress, as we did not find significant effects on these measures. Future studies should further investigate the mechanisms for why HRVB delivered via a smartphone influences stress recovery. Differences in Cortisol Recovery Between Conditions Although the cortisol levels declined during recovery for all conditions, there was no main effect of condition on cortisol recovery (F2,126=1.19, P=.31). Conclusions and Implications Based on these results, one can conclude that completing HRVB training on an app such as Happify may be a practical and effective strategy for reducing acute physiological stress. It is often not feasible to use a smartphone to buffer stress while undergoing a stressor, but it is practical and ecologically valid to use a phone immediately after one has experienced a stressful experience. Our smartphones are conveniently with us at most times, and thus, we have this effective stress-reducing tool at our disposal anytime and anywhere we need it. To further examine how smartphones can aid in stress recovery, future research should investigate the mechanisms underlying how a gamified stress-reducing app may buffer stress and how it compares to other ways of using a phone. This will inform future interventions and provide recommendations for the development of other stress-buffering tools that can be delivered through smartphone apps. When considering a more comprehensive assessment of stress, conclusions from this study must be tempered by the lack of significant group differences for salivary cortisol and self-reported stress. Based on these discrepancies, we can only conclude that the HRVB game had a targeted effect on autonomic nervous system recovery as opposed to a general effect on all types of biological and psychological stress recovery. These inconsistencies in stress outcomes may be due to a variety of reasons. First, cortisol and sAA represent activity in different arms of the stress system and are not correlated at a 1:1 level [36,53]. Numerous studies have discovered significant sAA results, but not cortisol, during stress recovery [37,38]. The one study that examined both biomarkers in the context of phone usage only found significant sAA effects [16]. Our findings indicate that the HRVB training had a more robust impact on autonomic nervous system activity (indicated by sAA measures), which makes sense because HRVB training specifically targets fluctuations in cardiovascular activity that is intricately tied to autonomic activity [36]. In addition, the intervention period was short (about 5 minutes), which may not have been enough time to impact cortisol, often viewed as a chronic stress marker with a delayed release [53]. The discrepancy between self-reported and physiological stress is quite common in studies that assess both constructs [17,34,42] and one of the reasons many researchers argue for the Results such as these are beginning to change the narrative about the effect of smartphones on our well-being. Limitations In addition, psychological factors may have played a role in explaining sAA recovery. The simple distraction induced by diverting cognitive attention away from ruminating thoughts about the stressor may have positively contributed to the effects. Furthermore, parasympathetic activity has been associated with increases in positive valence and low arousal in emotions such as calmness [24], which suggests that feelings of calm may have also played a role in stress recovery. Finally, it is possible that the ability to monitor stress responses via the visual interface of Breather increased perceptions of control, which subsequently alleviated feelings of stress. This is due to the fact that acute stress is often induced by a perceived lack of control [51], and when that perceived control is increased, it can inhibit autonomic arousal [52]. It should be noted that no mechanisms can be determined XSL•FO RenderX XSL•FO RenderX JMIR SERIOUS GAMES Hunter et al importance of assessing both when contemplating health relevance of stress or psychological outcomes [54]. Additionally, there is substantial variation based on individual factors, such as demographics, in the association of subjective and objective measures of stress [55]. Furthermore, studies examining the convergence of self-report and physiological measures of stress have found that the assessments are highly correlated during the TSST, but not before or after [42]. Thus, it is not surprising that sAA was the only metric that yielded significant results. The significant sAA finding provides valuable information about how a HRVB training game via an mHealth app may aid in stress recovery; however, future studies should consider a wider range of health-related outcomes. Additionally, the effectiveness of Breather may have been hindered by the way in which the participant interacted with the app and understood the directions. During this training period, information about the purpose of using this app (eg, this activity makes you more relaxed and less stressed) was hidden from the participant in order to reduce demand characteristics and maintain internal validity across conditions. However, the success of biofeedback training hinges on the individuals’ perception that they are actively controlling their physiological functions in an effort to reduce stress [8]. Without this understanding about the purpose of the activity, the biofeedback exercise was likely less effective for the participants. In these ways, methodological constraints may have led to a more conservative effect of Breather compared to the other conditions. Limitations Finally, it should be acknowledged that the scope of this study did not allow us to conclusively determine whether using the HRVB app was more or less effective than performing other actions on one’s phone. Although past research is mixed on how phone use influences stress recovery [17,18], there is great potential for future researchers to explore how unstructured phone use (eg, listening to music, browsing social media) could impact physiological and psychological stress. Given the wide variety of potential ways in which people can use their phones, future studies should further investigate the effects of various types of phone interactions on stress recovery. Furthermore, user error issues that occurred within the app may have limited the effectiveness of Breather. The program requires the user’s finger to be placed very precisely on the light sensor to monitor heart rate change. It is sometimes difficult to maintain this position, and warnings pop up on the screen each time a finger is placed incorrectly. Based on participant feedback, these warnings made individuals feel as if they were performing poorly, which may have induced further stress rather than alleviate it. To investigate whether user error played a role, adherence to finger placement was assessed using metrics provided from the app’s database. Data showed that users had their fingers placed correctly for approximately 96% of the time; however, that still means that for 4% of the session, they were getting warnings telling them, “please place your finger on the sensor.” This may have been bothersome and unduly reduced the effectiveness of Breather, which is important to consider in future efficacy tests and real-life applications. JMIR Serious Games 2019 \| vol. 7 \| iss. 4 \| e15974 \| p. 7 (page number not for citation purposes) http://games.jmir.org/2019/4/e15974/ Multimedia Appendix 1 A brief demo video of Breather on the Happify app. pp y pp [MP4 File (MP4 Video), 157169 KB-Multimedia Appendix 1] Conflicts of Interest ACP and ALW are full-time employees of Happify Health. However, all study design decisions, data collection, and analyses were performed by JFH, MSO, and SDP at the University of California, Irvine. References 1. Ralph BCW, Thomson DR, Cheyne JA, Smilek D. Media multitasking and failures of attention in everyday life. Psychol Res 2014 Sep;78(5):661-669. [doi: 10.1007/s00426-013-0523-7] [Medline: 24178629] 1. Ralph BCW, Thomson DR, Cheyne JA, Smilek D. Media multitasking and failures of attention in everyday life. Psychol Res 2014 Sep;78(5):661-669. [doi: 10.1007/s00426-013-0523-7] [Medline: 24178629] 2. Roberts JA, Yaya LHP, Manolis C. The invisible addiction: cell-phone activities and addiction among male and female college students. J Behav Addict 2014 Dec;3(4):254-265 [FREE Full text] [doi: 10.1556/JBA.3.2014.015] [Medline: 25595966] 3. Rosen LD, Lim AF, Felt J, Carrier LM, Cheever NA, Lara-Ruiz JM, et al. Media and technology use predicts ill-being among children, preteens and teenagers independent of the negative health impacts of exercise and eating habits. Comput Human Behav 2014 Jun;35:364-375 [FREE Full text] [doi: 10.1016/j.chb.2014.01.036] [Medline: 25717216] 3. Rosen LD, Lim AF, Felt J, Carrier LM, Cheever NA, Lara-Ruiz JM, et al. Media and technology use predicts ill-being among children, preteens and teenagers independent of the negative health impacts of exercise and eating habits. Comput Human Behav 2014 Jun;35:364-375 [FREE Full text] [doi: 10.1016/j.chb.2014.01.036] [Medline: 25717216] 4. Twenge JM, Joiner TE, Rogers ML, Martin GN. Increases in Depressive Symptoms, Suicide-Related Outcomes, and Suicide Rates Among U.S. Adolescents After 2010 and Links to Increased New Media Screen Time. Clinical Psychological Science 2017 Nov 14;6(1):3-17. [doi: 10.1177/2167702617723376] 4. Twenge JM, Joiner TE, Rogers ML, Martin GN. Increases in Depressive Symptoms, Suicide-Related Outcomes, and Suicide Rates Among U.S. Adolescents After 2010 and Links to Increased New Media Screen Time. Clinical Psychological Science 2017 Nov 14;6(1):3-17. [doi: 10.1177/2167702617723376] 5. McEwen BS, Gianaros PJ. Stress- and allostasis-induced brain plasticity. Annu Rev Med 2011;62:431-445 [FREE Full text] [doi: 10.1146/annurev-med-052209-100430] [Medline: 20707675] 5. McEwen BS, Gianaros PJ. Stress- and allostasis-induced brain plasticity. Annu Rev Med 2011;62:431-445 [FREE Full text] [doi: 10.1146/annurev-med-052209-100430] [Medline: 20707675] 6. McEwen BS. Stress, adaptation, and disease. Allostasis and allostatic load. Ann N Y Acad Sci 1998 May 01;840:33-44. [doi: 10.1111/j.1749-6632.1998.tb09546.x] [Medline: 9629234] 6. McEwen BS. Stress, adaptation, and disease. Allostasis and allostatic load. Ann N Y Acad Sci 1998 May 01;840:33-44. [doi: 10.1111/j.1749-6632.1998.tb09546.x] [Medline: 9629234] 7. Goldenberg DL, Kaplan KH, Nadeau MG, Brodeur C, Smith S, Schmid CH. A Controlled Study of a Stress-Reduction, Cognitive-Behavioral Treatment Program in Fibromyalgia. Journal of Musculoskeletal Pain 2010 Jan 16;2(2):53-66. [doi: 10.1300/j094v02n02_05] 8. Moss D. Heart rate variability and biofeedback. Psychophysiology Today: The Magazine for Mind-Body Medicine 2004;1:4-11. 9. Conclusions and Implications Although it is important to recognize the deleterious effects of these devices on our lives, it may be even more critical to recognize the positive potential of smartphones and begin to develop and use technology in ways that augment well-being. Instead of simply hoping that individuals use technology in a beneficial manner, it is imperative that the hardware and software are designed in a way that facilitates positive behavior, thoughts, and interactions. Designing tools that take advantage of the technological affordances and ubiquity of smartphones to put stress-reducing tools in the palm of one’s hand is a promising strategy for finding ways for smartphones to maximize well-being. http://games.jmir.org/2019/4/e15974/ JMIR Serious Games 2019 \| vol. 7 \| iss. 4 \| e15974 \| p. 7 (page number not for citation purposes) JMIR Serious Games 2019 \| vol. 7 \| iss. 4 \| e15974 \| p. 7 (page number not for citation purposes) XSL•FO RenderX JMIR SERIOUS GAMES Hunter et al Acknowledgments This project was funded by Happify Health. Multimedia Appendix 2 Levels of salivary alpha amylase during recovery by experimental conditions. [PNG File , 40 KB-Multimedia Appendix 2] References Free C, Phillips G, Felix L, Galli L, Patel V, Edwards P. The effectiveness of M-health technologies for improving health and health services: a systematic review protocol. BMC Res Notes 2010;3:250 [FREE Full text] [doi: 10.1186/1756-0500-3-250] [Medline: 20925916] 10. Ward AF, Duke K, Gneezy A, Bos MW. Brain Drain: The Mere Presence of One’s Own Smartphone Reduces Available Cognitive Capacity. Journal of the Association for Consumer Research 2017 Apr;2(2):140-154. [doi: 10.1086/691462] , , y , p Cognitive Capacity. Journal of the Association for Consumer Research 2017 Apr;2(2):140-154. [doi: 10.1086/691462] 11. Misra S, Cheng L, Genevie J, Yuan M. The iPhone Effect. Environment and Behavior 2014 Jul;48(2):275-298. [doi: 10.1177/0013916514539755] 11. Misra S, Cheng L, Genevie J, Yuan M. The iPhone Effect. Environment and Behavior 2014 Jul;48(2):275-298. [doi: 10.1177/0013916514539755] 12. Horrey WJ, Wickens CD. Examining the impact of cell phone conversations on driving using meta-analytic techniques. Hum Factors 2006;48(1):196-205. [doi: 10.1518/001872006776412135] [Medline: 16696268] 13. Gerin W, Davidson KW, Christenfeld NJS, Goyal T, Schwartz JE. The role of angry rumination and distraction in blood pressure recovery from emotional arousal. Psychosom Med 2006;68(1):64-72. [doi: 10.1097/01.psy.0000195747.12404.aa] [Medline: 16449413] JMIR SERIOUS GAMES Am Heart J 2004 Mar;147(3):E11. [doi: 10.1016/j.ahj.2003.08.013] [Medline: 14999213] y y j j 28. Kleiger RE, Miller J, Bigger J, Moss AJ. Decreased heart rate variability and its association with increased mortality after acute myocardial infarction. The American Journal of Cardiology 1987 Feb;59(4):256-262. [doi: 10.1016/0002-9149(87)90795-8] 29. Dillon A, Kelly M, Robertson IH, Robertson DA. Smartphone Applications Utilizing Biofeedback Can Aid Stress Reduction. Front Psychol 2016;7:832 [FREE Full text] [doi: 10.3389/fpsyg.2016.00832] [Medline: 27378963] 30. Prinsloo GE, Rauch HGL, Lambert MI, Muench F, Noakes TD, Derman WE. The effect of short duration heart rate variability (HRV) biofeedback on cognitive performance during laboratory induced cognitive stress. Appl Cognit Psychol 2010 Oct 17;25(5):792-801. [doi: 10.1002/acp.1750] 31. Stein P. Washington University in St. Louis, School of Medicine. 2018. Validation of the Happify breather biofeedback exercise to track heart rate variability using an optical sensorURL: https://happify.com/health/results/HLS-05.pdf [accessed 2019-11-11] 32. Vaschillo E, Lehrer P, Rishe N, Konstantinov M. Heart rate variability biofeedback as a method for assessing baroreflex function: A preliminary study of resonance in the cardiovascular system. Applied Psychophysiology and Biofeedback 2002;27(1). [doi: 10.1023/A:1014587304314] 33. Robins R, Fraley R, Krueger R. The self-report method. In: Handbook Of Research Methods In Personality Psychology. New York: The Guilford Press; 2019. 34. Campbell J, Ehlert U. Acute psychosocial stress: does the emotional stress response correspond with p Psychoneuroendocrinology 2012 Aug;37(8):1111-1134. [doi: 10.1016/j.psyneuen.2011.12.010] [M 34. Campbell J, Ehlert U. Acute psychosocial stress: does the emotional stress response correspond with physiological responses? Psychoneuroendocrinology 2012 Aug;37(8):1111-1134. [doi: 10.1016/j.psyneuen.2011.12.010] [Medline: 22260938] 35. Hellhammer DH, Wüst S, Kudielka BM. Salivary cortisol as a biomarker in stress research. Psychoneuroendocrinology 2009 Feb;34(2):163-171. [doi: 10.1016/j.psyneuen.2008.10.026] y gy g j p y 35. Hellhammer DH, Wüst S, Kudielka BM. Salivary cortisol as a biomarker in stress research. Psychoneuroendocrinology 2009 Feb;34(2):163-171. [doi: 10.1016/j.psyneuen.2008.10.026] 36. Nater UM, Rohleder N. Salivary alpha-amylase as a non-invasive biomarker for the sympathetic nervous system: current state of research. Psychoneuroendocrinology 2009 May;34(4):486-496. [doi: 10.1016/j.psyneuen.2009.01.014] [Medline: 19249160] 37. Payne LA, Hibel LC, Granger DA, Tsao JCI, Zeltzer LK. Relationship of Salivary Alpha Amylase and Cortisol to Social Anxiety in Healthy Children Undergoing Laboratory Pain Tasks. J Child Adolesc Behav 2014;2 [FREE Full text] [doi: 10.4172/jcalb.1000129] [Medline: 25525630] 38. van Veen JF, van Vliet IM, Derijk RH, van Pelt J, Mertens B, Zitman FG. Elevated alpha-amylase but not cortisol in generalized social anxiety disorder. Psychoneuroendocrinology 2008 Nov;33(10):1313-1321. [doi: 10.1016/j.psyneuen.2008.07.004] [Medline: 18757137] 39. JMIR SERIOUS GAMES JMIR SERIOUS GAMES Hunter et al 17. Rus HM, Tiemensma J. Social media as a shield: Facebook buffers acute stress. Physiol Behav 2018 Mar 01;185:46-54. [doi: 10.1016/j.physbeh.2017.12.021] [Medline: 29273455] 18. Rus HM, Tiemensma J. Social Media under the Skin: Facebook Use after Acute Stress Impairs Cortisol Recovery. Front Psychol 2017 Sep 19;8. [doi: 10.3389/fpsyg.2017.01609] y p p yg 19. Hooker ED, Campos B, Pressman SD. It just takes a text: Partner text messages can reduce cardiovascular responses to stress in females. Computers in Human Behavior 2018 Jul;84:485-492. [doi: 10.1016/j.chb.2018.02.033] p j 20. Lin I, Peper E. Psychophysiological patterns during cell phone text messaging: a preliminary study Biofeedback 2009 Mar;34(1):53-57. [doi: 10.1007/s10484-009-9078-1] [Medline: 19199025] 20. Lin I, Peper E. Psychophysiological patterns during cell phone text messaging: a preliminary study. Appl Psychophysiol Biofeedback 2009 Mar;34(1):53-57. [doi: 10.1007/s10484-009-9078-1] [Medline: 19199025] 21. Afifi TD, Zamanzadeh N, Harrison K, Acevedo Callejas M. WIRED: The impact of media and technology use on stress (cortisol) and inflammation (interleukin IL-6) in fast paced families. Computers in Human Behavior 2018 Apr;81:265-273. [doi: 10.1016/j.chb.2017.12.010] 22. Fiordelli M, Diviani N, Schulz PJ. Mapping mHealth research: a decade of evolution. J Med Internet Res 2013;15(5):e95 [FREE Full text] [doi: 10.2196/jmir.2430] [Medline: 23697600] 23. Parks AC, Williams AL, Tugade MM, Hokes KE, Honomichl RD, Zilca RD. Testing a scalable web and smartphone based intervention to improve depression, anxiety, and resilience: A randomized controlled trial. Intnl J Wellbeing 2018 Dec 08;8(2):22-67. [doi: 10.5502/ijw.v8i2.745] ( ) [ j ] 24. Lehrer PM, Gevirtz R. Heart rate variability biofeedback: how and why does it work? Front Psychol 2014;5:756 [FREE Full text] [doi: 10.3389/fpsyg.2014.00756] [Medline: 25101026] 25. Lacey BC, Lacey JI. Two-way communication between the heart and the brain. Significance of time within the cardiac cycle. Am Psychol 1978 Feb;33(2):99-113. [Medline: 637402] 26. Karemaker J, Lie KI. Heart rate variability: a telltale of health or disease. Eur Heart J 2000 Mar 15;21(6):435-437. [doi: 10.1053/euhj.1999.1969] [Medline: 10681483] j ] [ ] 27. Del Pozo JM, Gevirtz RN, Scher B, Guarneri E. Biofeedback treatment increases heart rate variability in patients with known coronary artery disease. Am Heart J 2004 Mar;147(3):E11. [doi: 10.1016/j.ahj.2003.08.013] [Medline: 14999213] 28. Kleiger RE, Miller J, Bigger J, Moss AJ. Decreased heart rate variability and its association with increased mortality after 27. Del Pozo JM, Gevirtz RN, Scher B, Guarneri E. Biofeedback treatment increases heart rate variability in patients with known coronary artery disease. [Medline: 16449413] 14. Master SL, Eisenberger NI, Taylor SE, Naliboff BD, Shirinyan D, Lieberman MD. A picture's worth: partner photographs reduce experimentally induced pain. Psychol Sci 2009 Nov;20(11):1316-1318. [doi: 10.1111/j.1467-9280.2009.02444.x] [Medline: 19788531] 15. Brown SL, Nesse RM, Vinokur AD, Smith DM. Providing social support may be more beneficial than receiving it: results from a prospective study of mortality. Psychol Sci 2003 Jul;14(4):320-327. [Medline: 12807404] 15. Brown SL, Nesse RM, Vinokur AD, Smith DM. Providing social support may be more beneficial than receiving it: results from a prospective study of mortality. Psychol Sci 2003 Jul;14(4):320-327. [Medline: 12807404] 16. Hunter JF, Hooker ED, Rohleder N, Pressman SD. The Use of Smartphones as a Digital Security Blanket: The Influence of Phone Use and Availability on Psychological and Physiological Responses to Social Exclusion. Psychosom Med 2018 May;80(4):345-352. [doi: 10.1097/PSY.0000000000000568] [Medline: 29521885] 16. Hunter JF, Hooker ED, Rohleder N, Pressman SD. The Use of Smartphones as a Digital Security Blanket: The Influence of Phone Use and Availability on Psychological and Physiological Responses to Social Exclusion. Psychosom Med 2018 May;80(4):345-352. [doi: 10.1097/PSY.0000000000000568] [Medline: 29521885] JMIR Serious Games 2019 \| vol. 7 \| iss. 4 \| e15974 \| p. 8 (page number not for citation purposes) http://games.jmir.org/2019/4/e15974/ JMIR SERIOUS GAMES Winslow BD, Chadderdon GL, Dechmerowski SJ, Jones DL, Kalkstein S, Greene JL, et al. Development and Clinical Evaluation of an mHealth Application for Stress Management. Front Psychiatry 2016 Jul;7:130 [FREE Full text] [doi: 10.3389/fpsyt.2016.00130] [Medline: 27507949] 40. Kirschbaum C, Pirke KM, Hellhammer DH. The 'Trier Social Stress Test'--a tool for investigating psychobiological stress responses in a laboratory setting. Neuropsychobiology 1993;28(1-2):76-81. [doi: 10.1159/000119004] [Medline: 8255414] JMIR Serious Games 2019 \| vol. 7 \| iss. 4 \| e15974 \| p. 9 (page number not for citation purposes) http://games.jmir.org/2019/4/e15974/ JMIR SERIOUS GAMES JMIR SERIOUS GAMES Hunter et al 41. Allen AP, Kennedy PJ, Dockray S, Cryan JF, Dinan TG, Clarke G. The Trier Social Stress Test: Principles and practice. Neurobiol Stress 2017 Feb;6:113-126 [FREE Full text] [doi: 10.1016/j.ynstr.2016.11.001] [Medline: 28229114] 41. Allen AP, Kennedy PJ, Dockray S, Cryan JF, Dinan TG, Clarke G. The Trier Social Stress Test: Principles and practice. Neurobiol Stress 2017 Feb;6:113-126 [FREE Full text] [doi: 10.1016/j.ynstr.2016.11.001] [Medline: 28229114] 42. Hellhammer J, Schubert M. The physiological response to Trier Social Stress Test relates to subjective measures of stress during but not before or after the test. Psychoneuroendocrinology 2012 Jan;37(1):119-124. [doi: 42. Hellhammer J, Schubert M. The physiological response to Trier Social Stress Test relates to subjective measures of stress during but not before or after the test. Psychoneuroendocrinology 2012 Jan;37(1):119-124. [doi: 10.1016/j.psyneuen.2011.05.012] [Medline: 21689890] 43. Lesage F, Berjot S, Deschamps F. Clinical stress assessment using a visual analogue scale. Occup M Dec;62(8):600-605 [FREE Full text] [doi: 10.1093/occmed/kqs140] [Medline: 22965867] F, Berjot S, Deschamps F. Clinical stress assessment using a visual analogue scale. Occup Med (Lond) 20 (8):600-605 [FREE Full text] [doi: 10.1093/occmed/kqs140] [Medline: 22965867] q 44. Overall JE, Doyle SR. Implications of chance baseline differences in repeated measurement designs. J Biopharm Stat 1994 Jul;4(2):199-216. [doi: 10.1080/10543409408835083] [Medline: 7951275] 45. Richardson JT. Eta squared and partial eta squared as measures of effect size in educational research. Educational Research Review 2011 Jan;6(2):135-147. [doi: 10.1016/j.edurev.2010.12.001] 46. Stroud LR, Foster E, Papandonatos GD, Handwerger K, Granger DA, Kivlighan KT, et al. Stress response and the adolescent transition: performance versus peer rejection stressors. Dev Psychopathol 2009;21(1):47-68 [FREE Full text] [doi: 10 1017/S0954579409000042] [Medline: 19144222] 47. Granger DA, Kivlighan KT, Blair C, El-Sheikh M, Mize J, Lisonbee JA, et al. Integrating the measurement of salivary α-amylase into studies of child health, development, and social relationships. Journal of Social and Personal Relationships 2016 Jun 29;23(2):267-290. [doi: 10.1177/0265407506062479] 48. Granger DA, Kivlighan KT, el-Sheikh M, Gordis EB, Stroud LR. Salivary alpha-amylase in biobehavioral research: recent developments and applications. Ann N Y Acad Sci 2007 Mar;1098:122-144. [doi: 10.1196/annals.1384.008] [Medline: 17332070] developments and applications. Ann N Y Acad Sci 2007 Mar;1098:122-144. [doi: 10.1196/annals.1384.008] [Medline: 17332070] 49. Wolf JM, Nicholls E, Chen E. Chronic stress, salivary cortisol, and alpha-amylase in children with asthma and healthy children. Biol Psychol 2008 Apr;78(1):20-28. [doi: 10.1016/j.biopsycho.2007.12.004] [Medline: 18243483] 50. Stewart JC, France CR. Cardiovascular recovery from stress predicts longitudinal changes in blood pressure. JMIR SERIOUS GAMES Biol Psychol 2001 Nov;58(2):105-120. [doi: 10.1016/s0301-0511(01)00105-3] [Medline: 11600240] 51. Eysenck M. Anxiety: The cognitive perspective. London, UK: Psychology Press; 2013. 52. Leotti LA, Iyengar SS, Ochsner KN. Born to choose: the origins and value of the need for control. Trends Cogn Sci 2010 Oct;14(10):457-463 [FREE Full text] [doi: 10.1016/j.tics.2010.08.001] [Medline: 20817592] 53. Engert V, Vogel S, Efanov SI, Duchesne A, Corbo V, Ali N, et al. Investigation into the cross-correlation of salivary cortisol and alpha-amylase responses to psychological stress. Psychoneuroendocrinology 2011 Oct;36(9):1294-1302. [doi: 10.1016/j.psyneuen.2011.02.018] [Medline: 21470780] 54. Diener E, Pressman SD, Hunter J, Delgadillo-Chase D. If, Why, and When Subjective Well-Being Influences Health, and Future Needed Research. Appl Psychol Health Well-Being 2017 Jul 14;9(2):133-167. [doi: 10.1111/aphw.12090] 55. Föhr T, Tolvanen A, Myllymäki T, Järvelä-Reijonen E, Rantala S, Korpela R, et al. Subjective stress, objective heart rate variability-based stress, and recovery on workdays among overweight and psychologically distressed individuals: a cross-sectional study. J Occup Med Toxicol 2015;10:39 [FREE Full text] [doi: 10.1186/s12995-015-0081-6] [Medline: 26504485] ] 49. Wolf JM, Nicholls E, Chen E. Chronic stress, salivary cortisol, and alpha-amylase in children with asthma and healthy children. Biol Psychol 2008 Apr;78(1):20-28. [doi: 10.1016/j.biopsycho.2007.12.004] [Medline: 18243483] 50. Stewart JC, France CR. Cardiovascular recovery from stress predicts longitudinal changes in blood pressure. Biol Psychol 2001 Nov;58(2):105-120. [doi: 10.1016/s0301-0511(01)00105-3] [Medline: 11600240] ( ) [ ( ) ] [ ] 51. Eysenck M. Anxiety: The cognitive perspective. London, UK: Psychology Press; 2013. 52. Leotti LA, Iyengar SS, Ochsner KN. Born to choose: the origins and value of the need for control. Trends Cogn Sci 2010 Oct;14(10):457-463 [FREE Full text] [doi: 10.1016/j.tics.2010.08.001] [Medline: 20817592] 53. Engert V, Vogel S, Efanov SI, Duchesne A, Corbo V, Ali N, et al. Investigation into the cross-correlation of salivary cortisol and alpha-amylase responses to psychological stress. Psychoneuroendocrinology 2011 Oct;36(9):1294-1302. [doi: 10.1016/j.psyneuen.2011.02.018] [Medline: 21470780] 53. Engert V, Vogel S, Efanov SI, Duchesne A, Corbo V, Ali N, et al. Investigation into the cross-correlation of salivary cortisol and alpha-amylase responses to psychological stress. Psychoneuroendocrinology 2011 Oct;36(9):1294-1302. [doi: 10.1016/j.psyneuen.2011.02.018] [Medline: 21470780] j p y 54. Diener E, Pressman SD, Hunter J, Delgadillo-Chase D. If, Why, and When Subjective Well-Being Influences Health, and Future Needed Research. Appl Psychol Health Well-Being 2017 Jul 14;9(2):133-167. [doi: 10.1111/aphw.12090] 54. Diener E, Pressman SD, Hunter J, Delgadillo-Chase D. If, Why, and When Subjective Well-Being Influences Health, and Future Needed Research. Appl Psychol Health Well-Being 2017 Jul 14;9(2):133-167. [doi: 10.1111/aphw.12090] 55. JMIR SERIOUS GAMES Föhr T, Tolvanen A, Myllymäki T, Järvelä-Reijonen E, Rantala S, Korpela R, et al. Subjective stress, objective heart rate variability-based stress, and recovery on workdays among overweight and psychologically distressed individuals: a cross-sectional study. J Occup Med Toxicol 2015;10:39 [FREE Full text] [doi: 10.1186/s12995-015-0081-6] [Medline: 26504485] 55. Föhr T, Tolvanen A, Myllymäki T, Järvelä-Reijonen E, Rantala S, Korpela R, et al. Subjective stress, objective heart rate variability-based stress, and recovery on workdays among overweight and psychologically distressed individuals: a cross-sectional study. J Occup Med Toxicol 2015;10:39 [FREE Full text] [doi: 10.1186/s12995-015-0081-6] [Medline: 26504485] Edited by G Eysenbach; submitted 23.08.19; peer-reviewed by K Stasiak, D Banks, R Ciptaningtyas; comments to author 17.09.19; revised version received 10.10.19; accepted 31.10.19; published 23.11.19 Edited by G Eysenbach; submitted 23.08.19; peer-reviewed by K Stasiak, D Banks, R Ciptaningtyas; comments to author 17.09.19; revised version received 10.10.19; accepted 31.10.19; published 23.11.19 Please cite as: Hunter JF Olah MS Williams AL Parks AC Pressman SD Please cite as: Hunter JF, Olah MS, Williams AL, Parks AC, Pressman SD Effect of Brief Biofeedback via a Smartphone App on Stress Recovery: Randomized Experimental Study JMIR Serious Games 2019;7(4):e15974 URL: http://games.jmir.org/2019/4/e15974/ doi: 10.2196/15974 PMID: JMIR Serious Games 2019 \| vol. 7 \| iss. 4 \| e15974 \| p. 10 (page number not for citation purposes) http://games.jmir.org/2019/4/e15974/ XSL•FO RenderX Hunter et al ©John F Hunter, Meryl S Olah, Allison L Williams, Acacia C Parks, Sarah D Pressman. Originally published in JMIR Serious Games (http://games.jmir.org), 23.11.2019. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in JMIR Serious Games, is properly cited. The complete bibliographic information, a link to the original publication on http://games.jmir.org, as well as this copyright and license information must be included. JMIR Serious Games 2019 \| vol. 7 \| iss. 4 \| e15974 \| p. 11 (page number not for citation purposes) http://games.jmir.org/2019/4/e15974/ JMIR SERIOUS GAMES ©John F Hunter, Meryl S Olah, Allison L Williams, Acacia C Parks, Sarah D Pressman. Originally published in JMIR Serious Games (http://games.jmir.org), 23.11.2019. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in JMIR Serious Games, is properly cited. The complete bibliographic information, a link to the original publication on http://games.jmir.org, as well as this copyright and license information must be included. XSL•FO RenderX XSL•FO RenderX
https://openalex.org/W2023502853	https://hts.org.za/index.php/hts/article/download/2169/3983	Afrikaans	null	Wat is wetenskap vandag?	HTS teologiese studies	1,986	cc-by	4,993	What is science today? In this essay an attempt is made to answer the question as to what contemporary science is, by looking at science's main task, namely the sistematic methodical search for the truth. In this respect a cognitive model, which issued from radical change in recent science, is explicated; Attention is paid to a plural truth constitution of different sciences as well as to the problem of the existence and validity of different scientific paradigms in one and the same science. The importance as such, as well as the implications of radical reflection on science, is indicated. Wat is wetenskap vandag? AJ Antonites AJ Antonites 1. INLEIDEND Wanneer 'n chemikus besig is met 'n eksperiment om 'n chemiese reaksie te bewerkstellig, of 'n geoloog om die ontwikkeling van 'n bergreeks vas te stel, of 'n historikus die redes vir 'n bepaalde gebeure nagaan, of 'n wysgeer 'n bepaalde denkrigting ondersoek, of 'n teoloog met eksegese van 'n perikoop besig is, of 'n ingenieur die toepassing van 'n natuurwet in sy gebied ondersoek - weet al hierdie wetenskap- likes een ding seker, ondanks die uiteenlopende verskille in die aard van hulle onderskeie wetenskappe, en dit is dat hul besig is om vk^etenskap te beoefen en nie iets anders nie. Hulle is nie besig met sport, kuns of handel dryf nie, maar wetenskap. Almal is toegewyd besig met die oplos van probleme. Almal het wette en besef dat die kriteria van wetenskaplikheid vir hulle geld. Hoe is dit moontlik? Waar kom dit vandaan? Op die chronologiese vraag sal 'n antwoord waarskynlik nie moont lik wees nie. Logies gesproke, blyk daar egter iets merkwaardig, naam- lik dat mense onafhanklik van mekaar, sedert eeue gelede, gehoor gee aan die kriteria van wetenskaplikheid. Wysgerig gesproke, sou mens kon sê dat in soverre daar sprake is van menswees, daar 'n wekroep vir hom geld, en dit is om op 'n bepaalde en wel teoretiese manier met sy wêreld om te gaan. Sonder dat hy eksplisiet besin het oor die wekroep van teoretiese waardes, wat objektief daar is, het hy gehoor gegee aan 397 hierdie wekroep. Die uitkoms hiervan is wat ons noem wetenskap. Ons tref wetenskap aan in klassieke Griekeland, die Hellenistiese tyd- perk, Middeleeue, Renaissance, moderne en hedendaagse tydvakke. 'n Mens sou kon vra of ons by die Grieke van wetenskap kan praat. Wat nog te sê wat die Middeleeue betref. Twee sake moet mens toegee. Eerstens is die Middeleeue in vergelyking met die hedendaagse wetenskap darem heel anders: Ons hoor van geen wetenskaplike deur- brake, bevindinge ensovoorts nie. Tweedens is die Middeleeuse wetenskap baie eensydig en hoofsaaklik tot die teologie-wysbegeerte beperk. Tog openbaar die Middeleeuers 'n baie goeie kritiese inge- steldheid - hy probeer streng logies redeneer. Abelardus (1079-1142) stel selfs eksplisiet geformuleerde vereistes waaraan kennis behoort te voldoen. Kom 'n mens by die Grieke dan vind ons dat Aristoteles die grondlegger was van metodes en gebiede wat in die hedendaagse wetenskap nie net gebruik en ontwikkel word nie, maar van kardinale belang is. 1. INLEIDEND 'n Mens sou dus kon aflei dat die mens dit as sinvol beleef om gehoor te gee aan die wekroep van teoretiese waardes wat hom aanspreek. Hoé die mens daaraan gehoor gee, verskil van tyd tot tyd, plek tot plek en wetenskaplike tot wetenskaplike. 2. BETEKENIS VAN RADIKALE (WETENSKAPSFILOSOFIESE) REFLEKSIE In enige wetenskap gebeur dit van tyd tot tyd dat nuwe insigte, teoriee, paradigmas, hipoteses, modelle en werkwyses aan die lig tree. Reeds dit vertel ons iets van wat wetenskap is, naamlik 'n dinamiese gebeure. So vind ons 'n groot deurbraak en omwenteling in die geestesweten- skappe met die koms van die fenomenologies-hemeneutiese werk wyses; in die natuurwetenskappe weer die koms van die relatiwiteits- en kwantumteorie; in die teologiese wetenskappe die historiese kritiek en teksimmanente werkwyse. In meeste gevalle, val hierdie verandering en ontwikkeling enersyds nie sommer uit die lug nie en andersyds spruit dit ook nie altyd voort uit 'n blote logiese voortgang van die wetenskap nie (die sg innerUke logika van wetenskaplike vooruitgang). Sodanige veranderings is dik- wels radikaal van aard in 'n bepaalde wetenskap en is die deurwerking van sulke veranderings in 'n wetenskap dikwels ingrypend van aard. Daar kan meer as een rede hiervoor wees. Ons wil egter die volgende twee opmerkings maak. 398 Eerstens val dit op dat veranderinge in 'n wetenskap wat nie opper- vlakkig van aard is nie, dit wil sê min of meer radikaal, gewoonlik tot radikale en dus wysgerige refleksie lei. Wetenskaplikes begin om radi- kale vrae te vra cor die omvang, geldigheid, trefsekerheid en gronde ('radikaal' impliseer dat dit 'n wetenskap tot in sy grond raak) van hul vakke. Nuwe verwikkelinge word wysgerig geïnterpreteer. 'n Goeie voorbeeld by die natuurwetenskappe is die opkoms van die kwantum- teorie en die onsekerheidsprinsipe by die mikro-fisika. Heisenberg, die formuleerder van die onsekerheidsprinsipe, die atoomfisikus, Niels Bohr en CF von Weiszacker het almal intensiewe wetenskapsfilosofiese re fleksie en arbeid verrig. Die gevolg hiervan vir die wetenskapsfilosofie en terugwerkend weer op die wetenskappe, was tydig. Ons dink byvoor- beeld aan Niels Bohr se komplementariteitsprinsipe wat die weten- skaplike wêreldbeeld van perspektiwiteit (wat in die natuur- sowel as geesteswetenskappe vandag aanvaar word) mede help vorm het. Die omwenteling van die Newtonse tot die Einsteinse benadering in die fisika, beïnvloed die fisikus-filosoof Thomas Kuhn se paradigma-filo- sofie. Die nawerking hiervan word vandag nog gevind in die weten skapsfilosofie en talle vakwetenskappe. Die debat wat in wetenskapsfi losofiese kringe hierop gevolg het, gaan nog voort. Tweedens gebeur dit van tyd tot tyd dat die omgekeerde ontwikke- ling plaasvind. Dit beteken dat ingrypende wysgerige refleksie, insigte en omwentehnge lei tot betekenisvolle veranderinge in baie vak wetenskappe. 2. BETEKENIS VAN RADIKALE (WETENSKAPSFILOSOFIESE) REFLEKSIE Ten opsigte van die geesteswetenskappe is dit meer direk en ten opsigte van die natuurwetenskappe meer indirek. So het die filosofie van Ernst Mach vir Einstein in die ontwikkeling van sy denke beinvloed. Oneindig groter as Mach is die betekenis van die wiskundige-filosoof Rene Descartes (1596-1650). Descartes se filosofie rondom die res extensa en res cogitans word uiteindelik die wêreldbeeld waarmee die natuurwetenskappe gewerk het en wat hulle veronderstel het dwarsdeur die modeme tyd en vandag nog in die veld is. Hierdie wêreldbeeld behels die Cartesiaanse dualisme (Cartesiaans van Des cartes). Die wysgerige besinning van Heisenberg en Bohr het hierdie wêreldbeeld mede help verander. In die geesteswetenskappe het Husserl, Heidegger en Scheler 'n ingrypende wending in die filosofie gebring, bekend as die fenomeno- logie. Husserl het geen spesifieke vakwetenskaphke tegnieke uitge- werk nie, maar 'n basiese wysgerige benadering en werkwyse daarge- stel. Dit het deurgesuur na talle geesteswetenskappe soos die geskiede- nis, psigologie, sosiologie en opvoedkunde. Hierdie wetenskappe het 399 elkeen op sy eie manier en vanuit sy eie perspektief 'n vakwetenskap- like metode daarvan gemaak en ook aansluitende tegnieke ontwikkel. 'n Mens sou dus hierdie radikale refleksie as een van die redes noem vir omwentelinge en veranderinge in 'n vakwetenskap. elkeen op sy eie manier en vanuit sy eie perspektief 'n vakwetenskap- like metode daarvan gemaak en ook aansluitende tegnieke ontwikkel. 'n Mens sou dus hierdie radikale refleksie as een van die redes noem vir omwentelinge en veranderinge in 'n vakwetenskap. Hierdie veranderinge laat ons die vraag vra: Kom die wetenskap dan nie by die waarheid uit nie? Het dit nie gearriveer nie? Kan dit arri- veer? Dit roep die belangrike kwessie van die waarheid op. Die vraag, 'wat is wetenskap?', impliseer juis die waarheidsvraag. Die 'wete' van wetenskap dui op kennis. (Vgl die wissen in die Duitse Wissenschaft.) Praat jy van kennis, praat jy van waarheid. Die wetenskap wil die waarheid ken. 3. WAARHEID EN OBJEKTIWITEIT BY DIE WETENSKAP: EEN WAARHEID OF VELE? Ons sal dikwels kry dat mense buite die wetenskap tot 'n ware insig kom. Hulle praat dan die waarheid. Menigmaal het 'n boer al gesê dat koolstof goed is vir beeste wat siek is as gevolg van tulpe wat hul geëet het. Dit blyk korrek te wees. Die boer het dit nie wetenskaplik vasgestel nie, maar hy praat die waarheid. Ons vind dat sekere Griekse gedigte menslike gevoelens en emosies baie waar beskryf, asook wat die ge- bruik van slawe te Mycenae betref. Ook dit is nie 'n geesteswetenskap- like insig nie. Dis nogtans waar. Hoekom sou waarheid dan juis so prominent figureer by die vraag na wat die wetenskap is? Anders as by die boer of die digter gaan dit in die wetenskap nie net by uitnemendheid om die waarheid nie, maar dit gaan om 'n meto- diese oopdek of soeke na waarheid. Anders as die digter of boer wat geen sodanige rekenskap gee of hoef te gee nie, want dit gaan vir hulle om 'n heel ander omgang met hul wêreld, moet die wetenskaplike kan rekenskap gee van sy meta hodos, van die weg waarlangs hy geloop het om by hierdie insig te kom. Hodos dui op die pad of weg. Meta sê dat om by 'n bepaalde doel uit te kom, jy 'n geskikte en nie onvanpaste pad moet loop nie. Die wetenskap is 'n gesistimatiseerde metodiese manier om by die waarheid uit te kom. Die waarheid? Sou ons van die waarheid as een waarheid kan praat? Hike wetenskap ondersoek 'n bepaalde deel van die werklikheid, elkeen het te doen met 'n synde van die syn. Dis wat die begrip 'vak' by vakwetenskap wil sê, naamlik dat elke vak wetenskap met 'n vakkie of deeltjie van die wêreld te doen het. Ons het 400 met een en dieselfde wêreld te doen. Impliseer dit nie ook sy korrelaat, naamlik een waarheid nie? In die Cartesiaans-georiënteerde natuurwetenskaplike denke, veral in sy sterkste uitloper, naamlik die positivisme, wat sedert die dertiger- jare 'n opbloei beleef het, geld dat die werklikheid een groot synstotali- teit is. Laasgenoemde is 'n op-sigself-staande-wêreld. In terme van die Cartesiaanse dualisme staan daar teenoor hierdie objektivistiese wê- reld 'n suiwere bewussyn (van die wetenskaplike) wat hierdie wêreld op 'n objektiev^e wyse ken soos wat hy in homself is. 3. WAARHEID EN OBJEKTIWITEIT BY DIE WETENSKAP: EEN WAARHEID OF VELE? (Kuhn en Feyerabend is albei natuurv^etenskaplike filosow^e.) Alleen radikale deurdenking, soos wat onder andere in genoemde reak- sie plaasgevind het, sou 'n beter kognitiev^e model tot gevolg kon hê. Naas die bogenoemde omwenteling in natuurwetenskaplike kringe in die huidige periode, vind daar ook in dieselfde tydvak 'n ander radi kale deurdenking plaas en wel by die wysbegeerte. Wat merkwaardig is, is dat hierdie tv^ee omwentelinge heeltemal onafhanklik van mekaar plaasgevind het, maar dat die uitkoms daarvan 'n kognitiewe model vir die wetenskap v^as, asook 'n nuwe v^etenskaplike wêreldbeeld, v^at groot ooreenkoms met mekaar vertoon. Ten opsigte van die natuurwetenskappe is dit eintlik tv^^ee omwrentelinge: Eerstens is dit die revolusionêre verandering en refleksie van die kwantum-teorie, onsekerheidsprinsipe en relatiwiteitsteorie. Tweedens behels Popper, Kuhn, Lakatos, Feyerabend en Toulmin se anti-induktivsristiese en anti- positiw^istiese konstruksies 'n omv^enteling in natuurwetenskaplike denke. By die wysbegeerte is dit die fenomenologies-hermeneutiese omwenteling. waarheidsvisie gehad het, het die pendulum na die ander uiterste beweeg, naamlik dié van selfs radikale skeptisisme. Ons dink aan die 'anargie'-model van Feyerabend in die hedendaagse tyd, asook die minder radikaal-skeptiese, maar wel relativ^^istiese denke by Thomas Kuhn. (Kuhn en Feyerabend is albei natuurv^etenskaplike filosow^e.) Alleen radikale deurdenking, soos wat onder andere in genoemde reak- sie plaasgevind het, sou 'n beter kognitiev^e model tot gevolg kon hê. Naas die bogenoemde omwenteling in natuurwetenskaplike kringe in die huidige periode, vind daar ook in dieselfde tydvak 'n ander radi kale deurdenking plaas en wel by die wysbegeerte. Wat merkwaardig is, is dat hierdie tv^ee omwentelinge heeltemal onafhanklik van mekaar plaasgevind het, maar dat die uitkoms daarvan 'n kognitiewe model vir die wetenskap v^as, asook 'n nuwe v^etenskaplike wêreldbeeld, v^at groot ooreenkoms met mekaar vertoon. Ten opsigte van die natuurwetenskappe is dit eintlik tv^^ee omwrentelinge: Eerstens is dit die revolusionêre verandering en refleksie van die kwantum-teorie, onsekerheidsprinsipe en relatiwiteitsteorie. Tweedens behels Popper, Kuhn, Lakatos, Feyerabend en Toulmin se anti-induktivsristiese en anti- positiw^istiese konstruksies 'n omv^enteling in natuurwetenskaplike denke. By die wysbegeerte is dit die fenomenologies-hermeneutiese omwenteling. Die nuwe kognitiewe model het die oue verbygegaan. Die verbygaan is egter logics en nie chronologies nie, aangesien die ou model nog in die veld is. Die afgelope twee jaar is pogings aangewend tot 'n meer gesofistikeerde uitwerk van die ou model. So kan ons die poging noem van onder andere Solomon. 3. WAARHEID EN OBJEKTIWITEIT BY DIE WETENSKAP: EEN WAARHEID OF VELE? Laasgenoemde het belangrike kenteoretiese implikasies: Dit beteken dat as jy die wêreld kan ken soos wat hy in sigself is, dan besit hy (die wêreld) 'n rasionaliteit wat volledig deursigtig is en so geken kan word; dit bete ken dat kennis daarvan neerkom op 'n weerspieëling van so 'n volkome verklaarbare wêreld. As dit 'n weerspieëling behels, beteken dit weer dat jou kennis ook die waarheid oor die wêreld is - al wat verder kan gebeur, is dat daar 'n kontinue, kumulatiewe vermeerdering van ken nis kan wees. Dié kumulatiewe waarheidsvisie is tot vandag die sie- ning van die logiese empirisme en logiese positivisme. Nuwe teorieë omvat volgens hierdie rigtings ouer teorieë. Wat as waarheid bevind word, is die waarheid, omdat dit induktivisties-empiristies verifieer- baar en gerugsteun is. Hoewel daar verskillende wetenskappe is, geld dit dat hulle almal saam één objektiewe sintese vorm, waar mens dan één objektiewe waarheid het. Die kriteria wat objektiwiteit bepaal, het die empirisme en kwantitatiewe as inhoud. Wat nie empiristies-kwan- titatief is nie, is nie objektief nie. In werklikheid kom dit neer op een enkele wetenskap wat met één enkele metode op 'n volkome rasionele manier te ken is: Één objektivistiese wêreld wat ooplê en in één enkele wetenskap objektief te ken is. Dit impliseer 'n metodemonisme en wel dié van die fisika en chemie of liewers 'n bepaalde (positivistiese) benadering daarvan. Ook die geesteswetenskappe word 'n sosio-fisika. As ons praat van 'n suiwer bewussyn wat 'n objektivistiese wêreld kan weerspieël, dan volg dit dat die subjek- en objek-pool geskei is, elkeen onafhanklik op sigself (kenteoretiese dualisme). Dit volg ook dat die mens, met ander woorde subjektiwiteit, geelimineer is uit die wêreld, want die wêreld is mos 'objektief' daar. Die menslike wetenskap as subjek is min of meer 'n passiewe toeskouer van die wêreld. Hoogstens registreer en akkumuleer hy feite. Hierdie kognitiewe model van die wetenskap het die wetenskap grootliks aan bande gelê, verarm en tot reaksie gelei in die vorm van skeptisisme. Waar die eensydige kognitiewe model 'n te dogmatistiese 401 waarheidsvisie gehad het, het die pendulum na die ander uiterste beweeg, naamlik dié van selfs radikale skeptisisme. Ons dink aan die 'anargie'-model van Feyerabend in die hedendaagse tyd, asook die minder radikaal-skeptiese, maar wel relativ^^istiese denke by Thomas Kuhn. 3. WAARHEID EN OBJEKTIWITEIT BY DIE WETENSKAP: EEN WAARHEID OF VELE? Een van die belangrikste insigte van die nuwe kognitiewe model is dat wetenskappe nie met een wêreld te doen het nie, maar met vele wêrelde omdat die mens veelsydig gesitueerd is. Die wetenskap word gevoed en sal steeds gevoed word deur 'n oorspronklike situasie, waar die mens nie passief teenoor gegewe reële objekte staan nie, maar in 'n aksie van singewing. Oorspronklik is die menslike subjek dus primer geen passiewe 'innemer' van louter waamemings of gegewens nie - elke waameming geskied vanuit 'n bepaalde standpunt en onder 'n bepaalde gesigspunt of konteks wat te doen het met elke mens se unieke konkrete gesitueerdheid. Maar dit kleur elke waameming. Daar is sprake van perspektiwiteit. Te midde van die perspektiwiteit, is daar tussen subjek en objek 'n dialektiese verhouding waar sowel subjek- as objek-pool mede-betrokke is. In hierdie ontmoeting tussen mens en wêreld, bloei sin op. As dit so is, dan beteken dit heel konkreet dat te 402 midde van 'n gemeenskaplike leefwêreld, daar vele wêrelde is. So is daar vir elke mens sy eie wêreld wat vir horn oorspronklik is. Maar omdat elkeen in sy eie wêreld veelsydig gesitueerd is, is daar ook by elkeen veelheid van sin. Die wetenskap is nou (volgens die nuw^e kognitiewe model) 'n be- sondere vergestalting van bogenoemde. As dit so is, dan is wetenskap- like kennis 'n besondere ontmoeting met jou v^êreld, waarin die gel- digheid van teoretiese v^aardes meespreek en wetenskaplike sin op- bloei. Gevolglik bloei besondere sinvelde op en dit word die veld van wetenskaplike ondersoek. In die lig van wat ons tot dusver gesê het, beteken dit dat die wetenskaplik-teoretiese ontmoeting met die wêreld eweneens 'n veelsydige gestalte gaan openbaar. Anders gesê: Die sin velde wat na vore tree, word getematiseer (tot tema gemaak, dus tot objeksgebied gemaak). Hierdie tematisering geskied dan in baie ver- skillende vorme sodat daar soveel wetenskappe gaan wees as wat daar wyses is waarop die dinge (objekte) hulle in verskillende tematiserings laat konstitueer. In hierdie dialoog of ontmoeting waarby die subjek nie meer passief net feite registreer nie, maar tesame met die objek ak- tief is, word 'n bepaalde vakwetenskaplik-tematiese veld afgebaken. Ons kan sê dit word 'uitgehaal' of geabstraheer uit sy heel oorspronk- like veld. Dit beteken dat 'n bepaalde aspek van 'n saak belig word. 3. WAARHEID EN OBJEKTIWITEIT BY DIE WETENSKAP: EEN WAARHEID OF VELE? Volgens hierdie kenmodel het ons dus nie meer met 'n objektiwistiese wêreld met sy objekte te doen nie, maar 'objektief' lê by sowel objek as subjek; meer presies: in die dialoogrelasie tussen die subjek- en objek- pool. So 'n afgebakende sinveld van 'n bepaalde wetenskap, kan alleen ontsluit word deur 'n wetenskaplike metode, tegnieke, werkwyse, be- gripsapparatuur en taal wat korrelatief tot die objek is. Subjek en objek is nie meer twee op sigself algeheel onafhanklike wêrelde nie. Subjek-pool en objek-pool bly steeds belangrik, want anders kan die kenrelasie oorswaai na 'n subjektivisme. Nou is die wêreld deurtrek met subjektiwiteit en andersom. Kennis van die wê- reld is kennis van 'n menslike wêreld en die kennende subjek is wê- reld-gesitueerd. Sprake van 'n één wêreld is daar nie meer nie. Die korrelaat hiervan is dat die kennis van die verskillende wetenskappe nie 'n homogene eenheid vorm nie. Om te praat van 'n sintese van al hierdie kennis is nie moontlik nie, want daar is nie meer één waarheid nie. In elke wetenskaplike singebied geld waarheid onder voorwaardes wat konstituerend van daardie vakgebied is. Elke wetenskap het sy eie wêreld. Vir byvoorbeeld die eksegeties-teologiese wetenskappe geld die wêreld van die teks. Teologie is geen herhaling van die teks nie 403 (geen blote weerspieëling nie), maar ook hier is daar dialoog en ont- moeting tussen subjek en objek. Hier is die subjek in die laaste in- stansie God. Waarheid is relasionele waarheid. Ook hier is waarhei’d nie buite die mens moontlik nie (kyk Velthuysen 1985). Die nuwe kognitiewe model word verder gekenmerk deur die klem op probleemstelling en probleemformulering. Die hedendaagse wetenskap het 'n probleemgerigte houding. Uiteraard speel die subjek- pool hier 'n meer belangrike rol. HG Gadamer wys in sy opstel 'Practi cal philosophy as a model of the human sciences' daarop dat 'n histo- riese feit in die hedendaagse wetenskap van die geskiedenis nie iets is wat bloot werklik gebeur het nie, maar iets wat werklik gebeur het op so 'n manier dat dit besondere betekenis het vir 'n historiese vraag. In die geskiedeniswetenskap (en so ook ander geesteswetenskappe) gaan dit ook nie om 'n samebondeling van feitlike gegewens nie. Die doel is selfs nie eens die gegewens nie. Dit is slegs middele tot 'n doel, naam- lik die konstruksie van leefwêrelde en menslike handelinge (kyk Dreyer 1974). 3. WAARHEID EN OBJEKTIWITEIT BY DIE WETENSKAP: EEN WAARHEID OF VELE? Hier is wetenskaplike kennis geen hiaatlose kontinui'teit nie - daar is diskontinuiteite, hiate en onsekerhede. Probleemformule ring is aan die orde van die dag. Ons vind verskillende soorte weten skaplike verklarings, ook binne een en dieselfde wetenskap, omdat ons verskillende soorte vrae vra. Verskillende teorieë is 'n gevolg hiervan wat weer tot verdere probleemstelling lei. Dat probleemstelling soos pas genoem, die rol van die subjek beklemtoon en ons derhalwe by die wetenskap nie meer die objektivisme het waar die objek-pool hoof- saaklik alleen praat en die wetenskap objek-bepaald is nie, het ons in die hedendaagse wetenskap die situasie waar ons vind dat uiteenlo- pend verskillende wetenskappe dieselfde objek ondersoek. Dis die selfde objek, maar verskillende vrae word daaroor gevra; verskillende aspekte van die objek word uitgelig. So byvoorbeeld kry ons dat spier- weefsel deur verskillende wetenskappe tot objek in hul onderskeie sinvelde gekonstitueer word. Spierweefsel word onder meer ondersoek deur elektrofisiologie, biochemie en termodinamika. Die groter voor- rang van subjektiwiteit in die wetenskap-kenteoretiese relasie, word ook treffend geillustreer in die atoomfisika: Die ontdekkings van Ru therford en Thompson was nie die uitkoms van eksperimenteel-empi- riese toetsing ('objektiewe'-data) nie, maar vanweë subjektief-teore- tiese konstruksies en kategorieë. Einstein se relatiwiteitsteorie vervang dié van Newton ook nie as gevolg van die 'objektief-waargenome feite' nie, maar is 'n primêr teoretiese konstruksie. Dit staan gevolglik be- kend as 'n 'hoë-vlak'-teorie Dis probleme wat die mens beweeg om 404 aandag aan sake in 'n area te gee. Navorsing ontstaan as iets problema- ties oor sake in 'n area ontstaan. Langs heel ander weë as die fenomenologie het die natuurweten- skappe die afgelope dekades tot baie soortgelyke resultate met betrek- king tot die nuwe kognitiewe model gekom. In die mikrofisika is tot die insig gekom dat partikels nie meer as objektief-vasstaande objekte geken kan word nie (die kwantum-teorie en onsekerheidsprinsipe). Werner von Heisenberg en ook Niels Bohr sê dat die wetenskaplike subjek nie meer 'n passiewe registreerder van objektivistiese feite is nie, maar 'n medespeler. Die wetenskaplike ontdek in die wêreld wat hy ondersoek niemand anders as homself nie! Dis nie meer louter weergawe of weerspieeling nie, maar-'n-deur-die-mens-ondervraagde- natuur. By Popper verkry teorie voorrang bo die empiriese: Daar is geen louter onafhanklike empiriese feite nie, maar feite is altyd en alreeds geinterpreteerde (deur teorie as 'n subjektief-menslike skep- ping) feite. Daar is geen bruta facta in die natuurwetenskappe nie. 3. WAARHEID EN OBJEKTIWITEIT BY DIE WETENSKAP: EEN WAARHEID OF VELE? Teorie en empirie is wisselwerkend tot mekaar verbind en is nooit los te maak nie. Die positivistiese verifikasie-kriterium asook indukti- visme word afgewys. 4. PERSPEKTIWITEIT VAN WAARHEID EN HERMENEUTIESE DIMENSIE Ons het reeds gesien dat 'n bepaalde vakwetenskap self 'n perspektief op die werklikheid met sy eie sinveld het. As al hierdie dinge nou so is, dan is dit logies dat binne een en dieselfde wetenskap die veelheid van menslike konkrete gesitueerdheid ook tot uiting gaan kom. Daar is nie net 'n rykdom van verskeie wetenskappe nie, maar binne dieselfde wetenskap eweneens 'n verskeidenheid van perspektiewe op dieselfde saak. Die begrip perspektief (van Latyns perspicere = om deur te sien, aandagtig te kyk, ondersoek, verken) sê dat geen mens bloot waameem nie, maar op 'n besondere manier, by 'n besondere situasie vir 'n besondere doel. Niels Bohr se komplementariteitsgedagte lui dat geen enkele wetenskaplike beeld van die wêreld die werklikheid in al sy fasette kan omvat nie. 'n Pluraliteit van beelde of perspektiewe is onvermydelik en selfs nodig. In feite kry ons binne die natuur-, geestes- en teologiese wetenskappe verskillende perspektiewe in die vorm van paradigmas, teorieë en ander kontekste. In die natuurwetenskappe is daar onder andere die Newtonse, Einsteinse en kwantum-paradigmas. In die 405 geesteswetenskappe is daar die Hempeliaanse, fenomenologiese en funksionalistiese perspektiewe; in die teologies-eksegetiese weten- skappe die historiese kritiek en teksimmanente teoriee. Hierdie para- digmas of teorieë ('n teorie is 'n raamwerk van verklaring of interpreta- sie en nie iets voorlopigs soos dikwels populêr gemeen word nie) is kontekste of perspektiewe. Ons vind dan dat sake binne die weten- skaplike dialoogsituasie vanuit 'n perspektief verskillend gesien kan word. Hier kom 'n hermeneutiese dimensie ter sprake. Gadamer stel tereg dat 'n feit alleen 'n feit is met betrekking tot betekenisvolheid: Dis nie die feit nie, maar die konteks wat die betekenis van 'n feit definieer. Feit het betekenis vir 'n bepaalde wetenskaplike vraag wat weer moontlik word vanweë 'n bepaalde teorie. Hierdie hermeneutiese dimensie is nie net beperk tot die geesteswetenskappe nie. Dis nie so dat natuurwetenskappe te doen het met eksperimentele feite, terwyl die geesteswetenskappe te doen het met geinterpreteerde feite nie. Die hermeneutiese dimensie geld ook in die hedendaagse natuurwetenskappe: Hier is dit eweneens die konteks wat iets betekenisvol maak. Dis nie die blote vermeerdering en uit- breiding van eksperimentele data wat vooruitgang konstitueer nie, maar juis die hermeneutiese dimensie! Dis die probleem, die vraag, wat 'feite' konsitueer (kyk Dreyer 1974). So byvoorbeeld gee die Ptolemaise en Newtonse teorieë dikwels aanleiding tot verskillende indelings van dieselfde objekte. 4. PERSPEKTIWITEIT VAN WAARHEID EN HERMENEUTIESE DIMENSIE Vanuit die een teorie se dimensie is sekere sake relevant en soms van kardinale belang, terwyl dit in die ander teorie nie juis van belang is nie en soms heeltemal irrelevant. In die Newtonse meganiese teorie is ruimte en tyd twee verskillende kriteria, maar nie in die relatiwiteitsteorie nie. 'n Betreklik onlangse voorbeeld illustreer hoedat twee verskillende teorieë in dieselfde wetenskap aanleiding gee tot verskillende probleemstel- lings en dan weer tot die verskynsel dat daar glad nie 'n probleem ervaar word nie. By die wetenskap van die astronomie het Australiese sterrekundiges waarnemings gemaak van die verafgeleë ster Quasar 3 C 273. Radio-teleskope het aangedui dat 'n bondel van materie wegbe- weeg het vanaf die hoofliggaam van die Quasar. Waarnemings is ge maak tydens 'n periode van drie jaar. Dit het geblyk dat die bondel omtrent 25 ligjare in die periode beweeg het, wat beteken dat die materie se snelheid die snelheid van lig aansienlik moes oorskry het. Volgens die ouer Newtonse teorie se interpretasie van bogenoemde waarnemings, is daar geen probleem hiermee rfie. Vanuit die perspek tief van die jongere relatiwiteitsteorie is hierdie waarneming 'n enorme 406 probleem - tewens die waamemings kan nie wees nie. Dis nie ver- onderstel om voor te kom nie, want volgens die relatiwiteitsteorie kan niks vinniger beweeg as die snelheid van lig nie (kyk Brown 1977). Die belangrikheid van die hermeneutiese dimensie blyk ook daarin dat die oplossing van die meningsverskil tussen die twee teorieë, nie geleë is in 'n verdere noukeurige ondersoek van die data of feite nie. Bruta facta sou nie die probleem oplos nie. In die teologies-eksegetiese wetenskappe vind ons dat daar gekom is tot die insig dat daar in die evangelies teenstrydige vertellinge is ten opsigte van die detail oor ruimte of tyd met betrekking tot 'n bepaalde gebeure. Lukas en Matteus byvoorbeeld se vertel van dieselfde aange- leentheid weerspreek mekaar dikwels. Volgens 'n ouer teologiese teorie is die teenstrydighede 'n probleem. Vir jonger teologiese teorieë is dieselfde saak nie veel van 'n probleem nie. Weer eens is dit die hermeneutiese dimensies van die paradigma - perspektief wat pro- bleemstelling bepaal en nie bruta facta nie. Elke perspektief het sy eie veronderstellings, basiese kategoriee en interpretasies. Die historikus-filosoof RG Collingwood het reeds in die laat dertiger- jare gevra by watter geleenthede, en deur watter gebeure word een stel fundamentele konsepte of konstellasies van absolute veronderstellings deur 'n ander vervang? BROWN, HI 1977. A functional analysis of scientific theories. Zeitschrift fur Allgemeine Wissenschaftstheorie 8. 4. PERSPEKTIWITEIT VAN WAARHEID EN HERMENEUTIESE DIMENSIE Hierop antwoord die fisikus-filosoof TS Kuhn in 1962 met sy The structure of scientific revolutions (kyk Toulmin 1972). Kuhn meen dat verskillende paradigmas mekaar opvolg in die tyd, dat die breuk en verskil tussen twee paradigmas so fundamenteel diskon- tinu is, dat kommunikasie tussen die twee buite die kwessie is, omdat elkeen in verskillende wêrelde leef, byvoorbeeld die Newtonse en Einsteinse teorieë. Toulmin, weer, meen dat die diskontinuiteit nie so revolusionêr-fundamenteel is nie. Veel eerder lyk dit of ons die bestaan van 'n veelheid van paradig mas, teoriee, perspektiewe binne dieselfde wetenskap moet sien as voortspruitend uit wat ons vantevore oor tematisering, dialoog en gesi- tueerdheid gesê het. Dit is omdat ons mense is. Aan die een kant het die positivistiese wêreldbeeld van één wêreld, één waarheid, één wetenskap prinsipieel en in feite verbygegaan. Aan die ander kant weer beteken dit egter nie dat verskillende perspektiewe en verskil lende wetenskappe in 'n monadiese eensaamheidswêreld leef nie. We- tenskaplike gesprek, kritiek, kruisbestuiwing en korreksie is nie net wenslik nie, maar selfs noodsaaklik omdat geen perspektief gearriveer het nie. Geen mens, geen wetenskaplike gaan die waarheid finaal inpalm nie. Perspektiewe is aanvullend tot mekaar. Dit beteken nie dat 407 'n sintese as mikpunt gestel moet word nie, want dan val ons weer terug op die positivistiese waarheidsideaal. Martin (1965) het gelyk wanneer hy sê dat daar in 'n wetenskap kontradiksie is, maar nie alles is en kan kontradiksies wees nie. Ons kan dit so formuleer: Algehele eenstemmigheid in alles is sinneloos; algehele verskil in alles is ewe sinneloos. 'n sintese as mikpunt gestel moet word nie, want dan val ons weer terug op die positivistiese waarheidsideaal. Martin (1965) het gelyk wanneer hy sê dat daar in 'n wetenskap kontradiksie is, maar nie alles is en kan kontradiksies wees nie. Ons kan dit so formuleer: Algehele eenstemmigheid in alles is sinneloos; algehele verskil in alles is ewe sinneloos. Juis hierin is die dinamo van die dinamiek van die wetenskap geleë. MARTIN, G 1965. Allgemeine Metaphysik: Ihre Probleme und ihre Methode. Berlin; De Gruyter. TOULMIN S 1972 Human understanding Volume I: General introduction and Part I Oxford: TOULMIN, S 1972. Human understanding, Volume I: General introduction and Part I. Oxford: Clarendon Press. VELTHUYSEN, GC 1985. Die betekenis van die relasionele waarheidsbegrip vir die Skrifbeskouing. HTS 4 1 ,1 0 8 -1 1 3 . BROWN, HI 1977. A functional analysis of scientific theories. Zeitschrift fur Allgemeine Wissenschaftstheorie 8. DREYER, PS 1974. Inleiding tot die filosofie van die geskiedenis. Kaapstad: HAUM. MARTIN, G 1965. Allgemeine Metaphysik: Ihre Probleme und ihre Methode. Berlin; De Gruyter. TOULMIN, S 1972. Human understanding, Volume I: General introduction and Part I. Oxford: Clarendon Press. VELTHUYSEN, GC 1985. Die betekenis van die relasionele waarheidsbegrip vir die Skrifbeskouing. HTS 4 1 ,1 0 8 -1 1 3 . f R, PS 1974. Inleiding tot die filosofie van die geskiedenis. Kaapstad: HAUM. Gruyter. TOULMIN, S 1972. Human understanding, Volume I: General introduction and Part I. Oxford: Clarendon Press. VELTHUYSEN, GC 1985. Die betekenis van die relasionele waarheidsbegrip vir die Literatuurverwysings 408
https://openalex.org/W4393443495	http://jtein.ppj.unp.ac.id/index.php/JTEIN/article/download/446/203	Indonesian	null	Rancang Bangun Robot Humanoid Penari Untuk Berjalan Pada Lantai Berundak	Jurnal Teknik Elektro Indonesia	2,023	cc-by	4,588	Abstract The development of the world of robotics in Indonesia has progressed rapidly. One of the robot developments is the humanoid robot, humanoid robot is a form of technology in the world of robotics where this robot has the ability to be able to imitate several human activities, for example, namely squatting, standing and walking. One example of a humanoid robot is a dancing robot, which is usually held in humanoid robot competitions in Indonesia, namely the Indonesian Dance Robot Contest (KRSTI). The type of servo motor used in humanoid robots is the Dynamixel MX- 28T servo. In this research a humanoid robot design was carried out using the OpenCM9.04 and OpenCM 485 EXP controllers, as well as using robotic software. The test uses variations of MotionPagePlay, namely 1800, 2200, 2500, 2800, and 3200. The MotionPagePlay used can affect the movement of humanoid robots. The results showed that with a stepped surface that has a height difference of 4mm the robot can pass well, with MotionPagePlay worth 2500 and 2800. The percentage of success reaches 90%. Meanwhile, for walking in flat areas with MotionPagePlay worth 2200 with an average error percentage of 0.33%. Rancang Bangun Robot Humanoid Penari Untuk Berjalan Pada Lantai Berundak Nikmatul Azizah1, Bustanul Arifin)2, Eka Nuryanto Budisusila3 123Program Studi Teknik Elektro, Fakultas Teknologi Industri, Universitas Islam Sultan Agung, Indonesia )Corresponding author, email: bustanul@unissula.ac.id Abstrak Perkembangan dunia robotika di Indonesia telah maju dengan pesat. Salah satu perkembangan robot yaitu robot humanoid, robot humanoid merupakan salah satu bentuk teknologi dalam dunia robotika yang dimana robot ini memiliki kemampuan untuk dapat menirukan beberapa kegiatan manusia contohnya yaitu jongkok, berdiri, dan berjalan. Salah satu contoh robot humanoid adalah robot penari, yang biasanya di ajang perlombaan robot humanoid yang ada di Indonesia adalah Kontes Robot Seni Tari Indonesia (KRSTI). Jenis motor servo yang digunakan pada robot humanoid yaitu servo Dynamixel MX-28T. Pada penelitian ini dilakukan sebuah perancangan robot humanoid dengan menggunakan kontroler OpenCM9.04 dan OpenCM 485 EXP, serta menggunakan sofware robotis. Pengujian menggunakan variasi MotionPagePlay yaitu 1800, 2200, 2500, 2800, dan 3200. MotionPagePlay yang digunakan dapat mempengaruhi perpindahan gerak robot humanoid. Hasil penelitian menunjukkan bahwa dengan permukaan berundak yang mempunyai perbedaan ketinggian 4mm dapat dilalui oleh robot dengan baik, dengan MotionPagePlay senilai 2500 dan 2800. Persentase keberhasilannya mencapai 90%. Sedangkan untuk berjalan di area datar dengan MotionPagePlay senilai 2200 dengan rata-rata persentase error 0,33%. Info. Artikel: No. 446 Received. July, 21, 2023 Revised. August, 03, 2023 Accepted. August, 08, 2023 Page. 555 – 565 Kata kunci: ✔ Robot Humanoid ✔ KRSTI ✔ Dynamixel MX 28T ✔ OpenCM ✔ MotionPagePlay PENDAHULUAN Perkembangan dunia robotika di Indonesia sudah maju sangat pesat. Dengan terbuktinya banyak kontes-kontes robot yang telah diselenggarakan dan juga jumlah pesertanyapun semakin meningkat dari tahun ke tahun. Salah satu jenis robot yang banyak dilakukan penelitian yaitu roobot humanoid. Robot humanoid merupakan robot yang meyerupai manusia yang dimana robot ini dapat digunakan dalam bidang, kesenian, kesehatan, pendidikan dan industri[1]. Berdasarkan panduan umum Kontes Robot Indonesia 2023 dijelaskan bahwa Kontes Robot Indonesia (KRI) adalah kegiatan perlombaan yang diselenggarakan oleh Balai Pengembangan Talenta Indonesia (BPTI) Pusat Prestasi Nasional (Puspresnas) Kementrian, Pendidikan, Kebudayaan, Riset, dan Teknologi (Kemendikbudristek) Republik Indonesia[2], [3]. 555 JTEIN: Jurnal Teknik Elektro Indonesia Vol. 4, No. 2, 2023 JTEIN: Jurnal Teknik Elektro Indonesia Vol. 4, No. 2, 2023 Nikmatul Azizah, Bustanul Arifin, Eka Nuryanto Budisusila Di Indonesia terdapat ajang tahunan tentang dunia robotika yaitu KRI (Kontes Robot Indonesia) yang diikuti oleh perguruan tinggi seluruh Indonesia baik negeri maupun suwasta. Dalam Kontes Robot Indonesia terdapat berbagai divisi, salah satunya yaitu Kontes Robot Seni Tari Indonesia (KRSTI). Dengan adanya kontes tari nusantara merupakan salah satu upaya dalam pelestarian warisan budaya[4] yang telah memanfaatkan perkembangan teknologi. Kontes Robot Seni Tari Indonesia (KRSTI) merupakan kompetisi perancangan, pembuatan, dan pemrograman robot humanoid yang disertai dengan unsur seni dan budaya bangsa Indonesia khususnya seni tari di tanah air. Untuk pelaksanaan KRSTI tahun 2023 mengambil tema “Robot Penari Denok Semarangan/Robot Penari Gambang Semarang”[3]. Tari denok merupakan tarian tradisional yang berasal dari Kota Semarang ibu kota Jawa Tengah yang sudah terpengaruh oleh gerak gaya Surakarta[5]. Robot humanoid yang dipertandingkan pada KRSTI 2023 mampu melakukan gerakan-gerakan dalam Tarian Denok Semarangan. Pada salah satu peraturan yang dibuat dalam lomba KRI di KRSTI ini yaitu robot dapat melewati Zona B dengan papan yang berukuran 1190x800mm dan ketinggian 4mm. Pada Zona B ini memiliki Zona Larangan dengan diameter 300mm dengan tinggi 10mm, dimana Zona Larangan ini tidak boleh dilewati[3]. Penelitian yang akan dilakukan tentang rancang bangun yang terdapat pada robot KRSTI Tim Saroseta. Robot humanoid pada penelitian ini dapat melakukan gerakan berjalan melewati undakan. Sesuai dengan judul dari penelitian ini yaitu “Rancang Bangun Robot Humanoid Penari Untuk Berjalan Pada Lantai Berundak” maka, penelitian juga akan membahas tentang gerak robot berjalan pada lantai yang berundak dengan melewati zona dengan ketinggian 4mm. PENDAHULUAN Pada prinsipnya, robot humanoid yang dirancang hanya robot dapat berjalan[6] namun dalam penelitian ini robot dapat berjalan pada permukaan yang lebih tinggi 4mm dengan menggunakan motor servo Dynamixel MX-28T dengan kontroler OpenCM9.04 dan OpenCM 485 EXP. Dalam penggunaan motor memerlukan sebuah kontrol agar dapat digunakan sesuai dengan fungsinya[7]. METODE PENELITIAN Dalam penelitian ini dilakukan beberapa proses dengan tujuan untuk mempermudah dalam pengimplementasiannya. Proses yang dilakukan dalam penelitian ini meliputi diagram blok, flowchart sistem, perancangan mekanik, perancangan hardware, dan perancangan software yang diuraikan sebagai berikut: Available online: http://jtein.ppj.unp.ac.id \| 556 Flowchart Sistem Flowchart menjelaskan tentang alur kerja dari keseluruhan sistem yang akan dirancang, berikut ini merupakan penjelasan flowchart dari Perancangan Sistem Gerak Berjalan Robot Humanoid Penari pada Jalan yang Berundak. Flowchart sistem terlihat pada Gambar 2. Mulai Perancangan mekanik robot Robot berjalan dan bergerak sesuai harapan Analisa dan perbaikan Pengujian robot berhasil? Selesai Ya Tidak Pembuatan Program pada Robotis Gambar 1. Flowchart perancangan sistem gerak dan berjalan robot humanoid penari Analisa dan perbaikan Tidak Pengujian robot berhasil? Ya Selesai Gambar 1. Flowchart perancangan sistem gerak dan berjalan robot humanoid penari Available online: http://jtein.ppj.unp.ac.id \| 556 Available online: http://jtein.ppj.unp.ac.id \| 556 JTEIN: Jurnal Teknik Elektro Indonesia e-ISSN 2723-0589 Rancang Bangun Robot Humanoid Penari Untuk Berjalan Pada Lantai Berundak \| 557 Available online: http://jtein.ppj.unp.ac.id Perancangan Mekanik 2 O CM9 04 2. OpenCM9.04 OpenCM 9.04 digunakan untuk kontroler pada motor servo[9]. OpenCM 9.04 yang digunakan yaitu dengan type C. Pada openCM 9.04 type C hanya memiliki sambungan Dynamixel TTL seri XL yang berjumlahkan 4[10] 3. OpenCM 485 OpenCM 485 digunakan sebagai board ekspansion sebagai pengontrol motor servo Dynamixel. Dibutuhkan kontroler OpenCM 9.04 sebagai pendukung Dynamixel menggunakan TTL dan RS- 485[11], [12]. OpenCM 485 EXP digunakan, untuk menerima data motor dari koneksi OpenCM dan menghubungkannya ke Sumber Daya listrik[13]. 4. Servo Dynamixel MX 28T Servo Dynamixel MX series merupakan konsep baru dari Dynamixel dengan fungsi lanjutan, seperti kontrol presisi, kontrol PID, kontrol posisi 360 derajat dan komunikasi kecepatan tinggi. Kontrol posisi pada MX-series ini sebesar 360 derajat tanpa zona mati.[14]. Motor servo tersebut merupakan jenis motor servo yang presusu dan memiliki susunan roda gigi open-circuit dari kontroler yang terdapat dalam satu paket. Circuit dari kontroler merupakan otak dari setiap motor servo[15]. P S f g f Perancangan software atau perangkay lunak pada tugas akhir ini yaitu dengan menggunakan ROBOTIS OpenCM. ROBOTIS OpenCM adalah perangkat lunak pengembangan dan alat download untuk board OpenCM9.04[16]. Program yang telah dikembangkan pada ROBOTIS OpenCM dikompilasi dan diunggah ke OpenCM9.04 untuk mengontrol input maupun output dari keseluruhan sistem gguna untuk menjalankan robot humnaoid untuk dapat berjalan pada permukaan yang berundak dengan ketinggian 4mm. HASIL DAN PEMBAHASAN Setelah dilakukannya beberapa tahapan seperti perancangan mekanik, hardware, dan software. Maka terbentuklah bentuk fisik robot humanoid dan hasil dari implementasi perancangan pergerakan kaki robot. Analisa data menggunakan nilai rata-rata dari keberhasilan robot humanoid untuk dapat mengetahui jumlah persentase keberhasil pada setiap pengujian dengan MotionPagePlay 1800, 2200, 2500, 2800, dan 3200. Perancangan Mekanik Perancangan mekanik pada robot humanoid penari ini didesain seperti dengan manusia yang pada umumnya yang dapat melakukan gerakan tari. Robot humanoid penari menggunakan dua jenis motor servo. Pada kerangka tubuh, tangan dan juga kepala menggunakan motor servo Dynamixel XL- 320 dengan jumlah 13 buah motor servo. Pada bagian kedua kaki dan bagian perut penggunakan jenis servo Dynamixel MX-28T dengan jumlah 14 buah motor servo sedangkan untuk kerangkanya sendiri menggunakan alumunium. Desain pada penelitian ini dapat dilihat pada Gambar 2. Bagian yang dibahas 24 23 22 20 21 3 5 7 9 4 6 8 10 26 25 13 14 15 19 21 17 23 18 20 24 16 22 11 12 = Motor Servo Dynamixel MX-28T = Motor Servo Dynamixel XL-320 Gambar 2 Desai Perancangan Sistem Mekanik Robot Humanoid Bagian yang dibahas 19 = Motor Servo Dynamixel XL-320 Gambar 2 Desai Perancangan Sistem Mekanik Robot Humanoid Perancangan Hardware g Perancangan hardware bermanfaat unruk menentukan perangkat yang akan digunakan, dengan perancangan hardware dapat membantu dalam menentukan mekanik yang diperlukan. Berikut ini merupakan diagram blok dari perancangan hardware pada bagian kaki robot humanoid. = Jalur power = Jalur data = Jalur power dan data = Jalur power = Jalur data = Jalur power dan data Dynamixel servo ID 26 Dynamixel servo ID 25 Dynamixel servo ID 14 Dynamixel servo ID 13 Dynamixel servo ID 10 Dynamixel servo ID 16 Dynamixel servo ID 21 Dynamixel servo ID 24 Dynamixel servo ID 19 Dynamixel servo ID 23 Dynamixel servo ID 22 Dynamixel servo ID 20 Dynamixel servo ID 15 Dynamixel servo ID 17 OpenCM 9.04 11,1V 11,1V 11,1V 11,1V 11,1V Ports = Jalur power = Jalur data = Jalur power dan data Data Catu Daya 11.1V Gambar 3 Diagram blok sistem robot humanoid pada bagian kaki Gambar 3 Diagram blok sistem robot humanoid pada bagian kaki JTEIN: Jurnal Teknik Elektro Indonesia Vol. 4, No. 2, 2023 Nikmatul Azizah, Bustanul Arifin, Eka Nuryanto Budisusila Berdasarkan diagram blok pada Gambar 1, terdapat beberapa komponen yang berfungsi sebagai berikut: Berdasarkan diagram blok pada Gambar 1, terdapat beberapa komponen yang berfungsi sebagai berikut: 1. Catu Daya 11,1V Catu daya dengan tegangan 11,1V digunakan untuk mencatu motor servo[8] Dynamixel 28T. Catu daya yang digunakan yaitu baterai Lipo 3s 25C 2200mAh. 1. Catu Daya 11,1V Catu daya dengan tegangan 11,1V digunakan untuk mencatu motor servo[8] Dynamixel 28T. Catu daya yang digunakan yaitu baterai Lipo 3s 25C 2200mAh. Hasil Perancangan Software Pada hasil perancangan software akan dilakuka langkah variasi gerakan jongkok, stay(berdiri), an berjalan sebagai berikut: Pada hasil perancangan software akan dilakuka langkah variasi gerakan jongkok, stay(berdiri), dan berjalan sebagai berikut: JTEIN: Jurnal Teknik Elektro Indonesia Vol. 4, No. 2, 2023 Rancang Bangun Robot Humanoid Penari Untuk Berjalan Pada Lantai Berundak \| 559 Available online: http://jtein.ppj.unp.ac.id 1. Mekanisme Jongkok dan Stay J g y Terlihat pada Gambar 4 (a) bahwa robot humanoid penari seperti sedang dalam posisi jongkok. Pada saat robot humanoid diberi hambatan dengan angin maupun sentuhan pada robot humanoid, robot tetap dapat berjongkok dengan baik. Begitupun pada saat percobaan pada permukaan dengan kemiringan 2o, 4o, 6o, 8o dan 10o robot humanoid masih dapat berjongkok dengan baik, dapat dilihat pada Gambar 5. g p p Pada saat pengujian mekanisme stay Gambar 4 (b) dengan rentan waktu robot berdiri selama 1 menit 54 detik robot masih dapat berdiri dengan baik. Namun, pada tumpuan lutut robot humanoid penari sedikit mengalami perubahan suhu. Pada saat diberi hambatan angin dan sentuhan sedikit robot humanoid penari masih dapat berdiri dengan baik. Available online: http://jtein.ppj.unp.ac.id \| 558 JTEIN: Jurnal Teknik Elektro Indonesia JTEIN: Jurnal Teknik Elektro Indonesia e-ISSN 2723-0589 (a) (b) Gambar 4 Hasil perancangan software (a) jongkok dan (b) stay (a) (b) (c) (d) (e) Gambar 5 Hasil percobaan mekanisme jongkok dengan kemiringan (a) 20, (b) 40, (c) 60, (d) 80, dan (e) 80 (a) (b) Gambar 4 Hasil perancangan software (a) jongkok dan (b) stay (a) (b) (b) (a) Gambar 4 Hasil perancangan software (a) jongkok dan (b) stay (a) (b) (c) (d) (e) Gambar 5 Hasil percobaan mekanisme jongkok dengan kemiringan (a) 20, (b) 40, (c) 60, (d) 80, dan (e) 80 (e) (d) (a) (b) (c) (a) (e) (b) (c) (d) Gambar 5 Hasil percobaan mekanisme jongkok dengan kemiringan (a) 20, (b) 40, (c) 60, (d) 8 (e) 80 2. Mekanisme Berjalan Gerakan berjalan pada robot humanoid penari ini langkahnya hampir menyerupai berjalannya manusia, yaitu gerakan pergantian langkah kaki kanan dan juga kaki kiri yang dilakukan secara bergiliran. Dapat di lihat pada Gambar 6 terdapat 9 step. Step pertama yaitu robot miring ke kiri, pada menit ke 1 servo tidak dapat menahan beban. Step kedua yaitu miring ke kiri kaki kanan melangkah, pada detik ke 23 servo tidak dapat menahan beban. Step ketiga yaitu kaki kanan robot menyentuh tanah dengan posisi robot masih miring, pada detik ke 17 servo tidak dapat menahan beban. Step keempat yaitu posisi badan robot stabil di tengah, pada detik ke 17 servo tidak dapat menahan beban. Step kelima yaitu robot miring ke kanan, robot dapat bertahan selama 50 detik. Step keenam yaitu miring ke kanan kaki kiri melangkah, pada detik ke 35 servo tidak dapat menahan beban. Step ketujuh yaitu kaki kiri robot menyentuh tanah dengan posisi robot masih miring, robot dapt bertahan selama 1 menit. Step kedelapan yaitu posisi robot stabil ditengah, robot dapat bertahan selama 1 menit. Step kesembilan merupakan step pengganti untuk step pertama jika dilakukan pengulangan berjalan, robot dapat bertahan selama 31 detik. Pada langkah kaki kanan berjarak 8,8 cm, sedangkan langkah kaki kiri berjarak 15,4 cm. JTEIN: Jurnal Teknik Elektro Indonesia Vol. 4, No. 2, 2023 Nikmatul Azizah, Bustanul Arifin, Eka Nuryanto Budisusila Gerakan Berjalan Gerakan Berjalan Gambar 6 Step mekanisme berjalan 1 2 3 4 5 6 7 8 9 3 2 5 4 1 3 6 7 9 7 8 Gambar 6 Step mekanisme berjalan Available online: http://jtein.ppj.unp.ac.id \| 560 Rancang Bangun Robot Humanoid Penari Untuk Berjalan Pada Lantai Berundak \| 561 Available online: http://jtein.ppj.unp.ac.id Hasil Pengujian Jalan pada Permukaan Bidang Hasil Pengujian Jalan pada Permukaan Bidang g j J p g Pengujian robot berjalan pada permukaan yang bidang dilakukan dengan meletakkan robot di atas lapangan yang telah disediakan dengan panjang 71 cm serta jarak selebar 26 cm. Pengujian delay atau MotionPagePlay yang akan dilakukan pengujian sebanyak 10 kali mulai dari MotionPagePlay 1800, 2200, 2500, 2800 dan 3200. Pengaturan MotionPagePlay yang digunakan yaitu untuk mengatur kecepatan gerak atau perpindahan gerak robot humanoid dari gerakan satu kegerakan yang satunya. Dengan pengaturan pada percepatan geraknya yaitu mempengaruhi gerakan robot. Dapat dilihat dari Tabel 1 merupakan rata-rata dari hasil keselurusan pengujian pada permukaan bidang dengan menggunakan variasi MotionPagePlay. Tabel 1 Hasil pengujian berjalan pada permukaan bidang secara keseluruhan MotionPagePlay Rata-rata nilai sudut Rata-rata persentase error Jumlah keberhasilan (dari 10 percobaan) 1800 1,08o 0,60% 4 2200 0,59o 0,33% 7 2500 0,86o 0,48% 6 2800 1,44o 0,80% 5 3200 0,77o 0,43% 6 Tabel 1 Hasil pengujian berjalan pada permukaan bidang secara keseluruhan Dari hasil pengujian dengan menggunakan MotionPagePlay yang berbeda-beda akan memberikan hasil yang berbeda pula. Pada saat percobaan dengan menggunakan MotionPagePlay 1800 menunjukkan bahwa dengan perpindahan pergerakan 1,8 detik setiap pergerakannya akan menghasilkan robot humanoid kurang seimbang dalam berjalannya, sehingga mengakibatkan robot menyerong pada satu sisi. Percobaan dengan menggunakan MotionPagePlay 2200 memiliki hasil yang baik jika dibandingkan dengan MotionPagePlay yang lainnya. Pada pengujian tersebut robot dapat berjalan dengan lurus walaupun terdapat percobaan yang dimana robot melewati batas. Adapun hasil percobaan secara lengkap dengan MotionPagePlay 2200 dapat dilihat pada Tabel 2. Pada percobaan dengan MotionPagePlay 2500 dan 2800 menghasilkan bahwa perpindahan pergerakan menghasilkan Available online: http://jtein.ppj.unp.ac.id \| 560 JTEIN: Jurnal Teknik Elektro Indonesia e-ISSN 2723-0589 pergeseran pada tiap langkahnya. Kemudian, pada hasil percobaan dengan MotionPagePlay 3200 menghasilkan rata-rata persentase error 0,43% yang dimana untuk pergeseran yang terjadi pada saat robot berjalan tidak lebih dari 1 cm. Rata-rata dari hasil percobaan tersebut jika robot menyerong, robot akan menyerong ke kiri. Pengujian pada permukaan bidang dengan MotionPagePlay yang telah diujikan menghasilkan bahwa dengan menggunakan yang terlalu cepat maupun lambat dapat mempengaruhi jalannya robot humanoid. Pada pengujian ini posisi awal roboot juga mempengaruhi hasil akhir dari robot. Apabila robot bergeser 1 milimeter dapat mengakibatkan jalan robot sedikit menyerong. Gambar 7 merupakan hasil akhir pada saat robot berhenti yaitu yang dimana kedua kaki robot masih berada dalam garis jarak yang telah ditentukan. dalam garis jarak yang telah ditentukan. Rancang Bangun Robot Humanoid Penari Untuk Berjalan Pada Lantai Berundak \| 561 Available online: http://jtein ppj unp ac id Hasil Pengujian Jalan pada Permukaan Bidang Tabel 2 Hasil pengujian berjalan pada permukaan bidang dengan MotionPagePlay 2200 Percobaan Jarak menyimpang (cm) Sudut Persentase error 1 0,5 0,40o 0,22% 2 0,5 0,40o 0,22% 3 0,5 0,40o 0,22% 4 1,5 1,21o 0,67% 5 0,5 0,40o 0,22% 6 0,5 0,40o 0,22% 7 0,7 0,56o 0,31% 8 0,5 0,40o 0,22% 9 0,5 0,40o 0,22% 10 1,6 1,29o 0,72% Rata-rata 0,33% Jarak menyimpang Jarak tempuh START FINISH Gambar 7 Ilustrasi pengukuran menyimpang Pada Tabel 2 terdapat perhitungan sudut[17] yang dihasilkan dari persamaan (1): Tabel 2 Hasil pengujian berjalan pada permukaan bidang dengan MotionPagePlay 2200 Percobaan Jarak menyimpang (cm) Sudut Persentase error 1 0,5 0,40o 0,22% 2 0,5 0,40o 0,22% 3 0,5 0,40o 0,22% 4 1,5 1,21o 0,67% 5 0,5 0,40o 0,22% 6 0,5 0,40o 0,22% 7 0,7 0,56o 0,31% 8 0,5 0,40o 0,22% 9 0,5 0,40o 0,22% 10 1,6 1,29o 0,72% Rata-rata 0,33% Tabel 2 Hasil pengujian berjalan pada permukaan bidang dengan MotionPagePlay Percobaan Jarak menyimpang (cm) Sudut Persenta Jarak menyimpang Jarak tempuh START FINISH Gambar 7 Ilustrasi pengukuran menyimpang START Gambar 7 Ilustrasi pengukuran menyimpang Pada Tabel 2 terdapat perhitungan sudut[17] yang dihasilkan dari persamaan (1): 𝜃= 𝑡𝑎𝑛−1 ( 𝐽𝑎𝑟𝑎𝑘 𝑚𝑒𝑛𝑦𝑖𝑚𝑝𝑎𝑛𝑔 𝐽𝑎𝑟𝑎𝑘 𝑡𝑒𝑚𝑝𝑢ℎ ) (1) 𝜃= 𝑡𝑎𝑛−1 ( 0,5 71) 𝜃= 𝑡𝑎𝑛−1 ( 𝐽𝑎𝑟𝑎𝑘 𝑚𝑒𝑛𝑦𝑖𝑚𝑝𝑎𝑛𝑔 𝐽𝑎𝑟𝑎𝑘 𝑡𝑒𝑚𝑝𝑢ℎ ) 𝜃= 𝑡𝑎𝑛−1 ( 0,5 71) (1) 𝜃= 0,40° Setelah mendapatkan hasil dari sudut maka dapat dilakukan perhitungan persentase error dengan menggunakan rumus persamaan (2) berikut: JTEIN: Jurnal Teknik Elektro Indonesia Vol. 4, No. 2, 2023 Nikmatul Azizah, Bustanul Arifin, Eka Nuryanto Budisusila 𝑃𝑒𝑟𝑠𝑒𝑛𝑡𝑎𝑠𝑒 𝑒𝑟𝑟𝑜𝑟= 𝐻𝑎𝑠𝑖𝑙 𝑠𝑢𝑑𝑢𝑡 180° 𝑥100% (2) 𝑃𝑒𝑟𝑠𝑒𝑛𝑡𝑎𝑠𝑒 𝑒𝑟𝑟𝑜𝑟= 0,40° 180° 𝑥100% 𝑃𝑒𝑟𝑠𝑒𝑛𝑡𝑎𝑠𝑒 𝑒𝑟𝑟𝑜𝑟= 0,22% 𝑃𝑒𝑟𝑠𝑒𝑛𝑡𝑎𝑠𝑒 𝑒𝑟𝑟𝑜𝑟= 𝐻𝑎𝑠𝑖𝑙 𝑠𝑢𝑑𝑢𝑡 180° 𝑥100% (2) 𝑃𝑒𝑟𝑠𝑒𝑛𝑡𝑎𝑠𝑒 𝑒𝑟𝑟𝑜𝑟= 0,40° 180° 𝑥100% 𝑃𝑒𝑟𝑠𝑒𝑛𝑡𝑎𝑠𝑒 𝑒𝑟𝑟𝑜𝑟= 0,22% (2) 𝑃𝑒𝑟𝑠𝑒𝑛𝑡𝑎𝑠𝑒 𝑒𝑟𝑟𝑜𝑟= 0,22% Dari 10 hasil persentase error maka didapatkan rata-rata persentase error yaitu dengan menggunakan persamaan (3) berikut: 𝑅𝑎𝑡𝑎−𝑟𝑎𝑡𝑎 𝑝𝑒𝑟𝑠𝑒𝑛𝑡𝑎𝑠𝑒 𝑒𝑟𝑟𝑜𝑟= 𝐽𝑢𝑚𝑙𝑎ℎ 𝑝𝑒𝑟𝑠𝑒𝑛𝑡𝑎𝑠𝑒 𝑒𝑟𝑟𝑜𝑟 𝐽𝑢𝑚𝑙𝑎ℎ 𝑝𝑒𝑟𝑐𝑜𝑏𝑎𝑎𝑛 . 𝑅𝑎𝑡𝑎−𝑟𝑎𝑡𝑎 𝑝𝑒𝑟𝑠𝑒𝑛𝑡𝑎𝑠𝑒 𝑒𝑟𝑟𝑜𝑟= 𝐽𝑢𝑚𝑙𝑎ℎ 𝑝𝑒𝑟𝑠𝑒𝑛𝑡𝑎𝑠𝑒 𝑒𝑟𝑟𝑜𝑟 𝐽𝑢𝑚𝑙𝑎ℎ 𝑝𝑒𝑟𝑐𝑜𝑏𝑎𝑎𝑛 . (3) 𝑅𝑎𝑡𝑎−𝑟𝑎𝑡𝑎 𝑝𝑒𝑟𝑠𝑒𝑛𝑡𝑎𝑠𝑒 𝑒𝑟𝑟𝑜𝑟= 3,3 10. 𝑅𝑎𝑡𝑎−𝑟𝑎𝑡𝑎 𝑝𝑒𝑟𝑠𝑒𝑛𝑡𝑎𝑠𝑒 𝑒𝑟𝑟𝑜𝑟= 0,33%. Nilai error pada pengujian permukaan bidang ini dipengaruhi oleh percepatan perpindahan pada pergantian gerak robot humanoid. Sehingga jalan robot humanoid menjadi kurang seimbang pada saat berjalan, sehingga mengakibatkan pergeseran. Selain itu, terdapat faktor lain seperti halnya kondisi suhu pada servo yang meningkat, serta tegangan baterai yang kurang dari 12,2V. Gambar 8 Hasil percobaan pada permukaan bidang dengan MotionPagePlay 2200 Gambar 8 Hasil percobaan pada permukaan bidang dengan MotionPagePlay 2200 Available online: http://jtein.ppj.unp.ac.id \| 562 Hasil Pengujian Jalan pada Permukaan Berundak Pengujian robot berjalan pada permukaan yang terdapat undakan dengan ketinggian 4mm dan terdapat zona larangan di tengah dengan diameter 300mm[3] dilakukan dengan meletakkan robot di atas lapangan yang telah disediakan. Pada pengujian permukaan berundak yaitu dengan menggunakan variasi MotionPagePlay 1800, 2200, 2500, dan 3200. Terlihat pada Tabel 3 merupakan rata-rata dari hasil keselurusan pengujian pada permukaan berundak dengan menggunakan variasi MotionPagePlay. Persentase keberhasilan didapatkan dari persamaan (4); 𝑃𝑒𝑟𝑠𝑒𝑛𝑡𝑎𝑠𝑒 𝑘𝑒𝑏𝑒𝑟ℎ𝑎𝑠𝑖𝑙𝑎𝑛= 𝐽𝑢𝑚𝑙𝑎ℎ 𝑘𝑒𝑏𝑒𝑟ℎ𝑎𝑠𝑖𝑙𝑎𝑛 𝐽𝑢𝑚𝑙𝑎ℎ 𝑝𝑒𝑟𝑐𝑜𝑏𝑎𝑎𝑛𝑥100%. (4) 𝑃𝑒𝑟𝑠𝑒𝑛𝑡𝑎𝑠𝑒 𝑘𝑒𝑏𝑒𝑟ℎ𝑎𝑠𝑖𝑙𝑎𝑛= 3 10 𝑥100%. 𝑃𝑒𝑟𝑠𝑒𝑛𝑡𝑎𝑠𝑒 𝑘𝑒𝑏𝑒𝑟ℎ𝑎𝑠𝑖𝑙𝑎𝑛= 30%. 𝑃𝑒𝑟𝑠𝑒𝑛𝑡𝑎𝑠𝑒 𝑘𝑒𝑏𝑒𝑟ℎ𝑎𝑠𝑖𝑙𝑎𝑛= 30%. Available online: http://jtein.ppj.unp.ac.id \| 562 JTEIN: Jurnal Teknik Elektro Indonesia e-ISSN 2723-0589 Tabel 3 Hasil pengujian berjalan pada permukaan berundak secara keseluruhan MotionPagePlay Jumlah Keberhasilan (dari 10 percobaan) Persentase keberhasilan 1800 3 30% 2200 6 60% 2500 9 90% 2800 9 90% 3200 1 10% Tabel 3 Hasil pengujian berjalan pada permukaan berundak secara keseluruhan Tabel 3 merupakan ringkasan hasil pengujian yang telah dilakukan pengujian pada robot humanoid penari dapat disimpulkan bahwa MotionPagePlay pada pengujian ini berpengaruh. Pengujian pada permukaan berundak dapat dilihat pada Gambar 9 yang terdapat perbedaan tinggi 4mm pada permukaan lapangan. Pada saat pengujian dengan menggunakan MotionPagePlay 1800 pada robot humanoid penari tingkat keberhasilan pada percobaan ini adalah 30%. Pada percobaan ini robot humanoid dapat berjalan dengan perpindahan gerak yang cepat dan jalan lurus tetapi robot humanoid penari ini pada saat menaiki undakan robot humanoid hampir jatuh hanya kurang seimbang dan pada saat menuruni undakan robot terjatuh. Pada saat pengujian dengan MotionPagePlay 2200 pada robot humanoid penari tingkat keberhasilannya pada percobaan ini adalah 60%. Pada saat pengujian robot masih kurang stabil dalam menaiki undakan maupun turunan. Pada saat pengujian dengan MotionPagePlay 2500 dan 2800 pada robot humanoid penari tingkat keberhasilannya adalah 90% dapat dilihat pada Tabel 4. Hasil pengujian dengan variasi MotionPagePlay ini adalah hampir semua percobaan robot dapat berjalan melewati undakan dan juga turunan dengan baik dan tanpa mengenai zona larangan. Pada pengujian terakhir yaitu dengan menggunakan variasi MotionPagePlay 3200 pada robot humanoid penari tingkat keberhasilannya adalah 10%. Hasil pengujian ini robot berjalan dengan lambat dan pada saat berjalan ke jalan berundak robot sedikit kurang stabil mengakibatkan robot menyerong dan berjalan mengenai zona larangan, sehingga harus dilakukan pengangkatan robot untuk meletakkan posisi robot agar lurus kembali. Sedangkan, pada saat turun dari permukaan yang berundak robot humanoid penari dapat berjalan dengan baik. Sama halnya dengan permukaan bidang, pada permukaan berundak ini posisi awal robot juga mempengaruhi hasil akhir dari robot. Hasil Pengujian Jalan pada Permukaan Berundak Selain itu, perubahan suhu pada motor servo dan tegangan pada baterai juga mempengaruhi kinerja dari robot humanoid. Apabila robot bergeser 1 milimeter dapat mengakibatkan jalan robot sedikit menyerong. Dalam hasil pengujian yang telah digunakan yaitu dengan MotionPagePlay 2500 dan 2800 yang menunjukkan jumlah keberhasilan sebanyak 9 dari 10 percobaan. Gambar 9 Hasil percobaan pada permukaan berundak dengan MotionPagePlay Gambar 9 Hasil percobaan pada permukaan berundak dengan MotionPagePlay Rancang Bangun Robot Humanoid Penari Untuk Berjalan Pada Lantai Berundak \| 563 Available online: http://jtein.ppj.unp.ac.id JTEIN: Jurnal Teknik Elektro Indonesia Vol. 4, No. 2, 2023 Nikmatul Azizah, Bustanul Arifin, Eka Nuryanto Budisusila Tabel 4 Hasil pengujian berjalan pada permukaan bidang dengan MotionPagePlay 2500 Percobaan Hasil pengujian Keterangan 1 Berhasil Robot berjalan dengan baik pada saat naik undakan dan pada saat turun undakan, tetapi jalan agak menyerong ke kiri dan hampir mengenai zona larangan 2 Berhasil Robot berjalan dengan baik pada saat naik undakan dan pada saat turun undakan, tetapi jalan agak menyerong ke kiri dan hampir mengenai zona larangan 3 Berhasil Robot berjalan dengan baik pada saat naik undakan dan pada saat turun undakan, tetapi jalan agak menyerong ke kiri dan hampir mengenai zona larangan 4 Tidak berhasil Robot berjalan dengan baik pada saat naik undakan dan pada saat turun undakan, tetapi menyentuh zona larangan 5 Berhasil Robot berjalan dengan baik pada saat naik undakan dan pada saat turun undakan, tetapi jalan agak menyerong ke kiri 6 Berhasil Robot berjalan dengan baik pada saat naik undakan dan pada saat turun undakan, tetapi jalan agak menyerong ke kiri 7 Berhasil Robot berjalan dengan baik pada saat naik undakan dan pada saat turun undakan, tetapi jalan agak menyerong ke kiri dan hampir menyentuh zona larangan 8 Berhasil Robot berjalan dengan baik pada saat naik undakan dan pada saat turun undakan, tetapi jalan agak menyerong ke kiri dan pada saat jalan sedikit tidak stabil 9 Berhasil Robot berjalan dengan baik pada saat naik undakan dan pada saat turun undakan, tetapi jalan agak menyerong ke kiri 10 Berhasil Robot berjalan dengan baik pada saat naik undakan dan pada saat turun undakan, tetapi jalan agak menyerong ke kiri DAFTAR PUSTAKA [1] M. A. Fahd, D. Purwanto, and M. H. Fatoni, “Rancang Bangun Robot Penari Humanoid dengan 25 DoF untuk Melakukan Gerakan Tari Remo,” J. Tek. ITS, vol. 7, no. 2, pp. 2337–3539, 2018. [2] Kementrian Pendidikan Kebudayaan Riset dan Teknologi, “KRI 2023 - Kontes Robot Indonesia,” Panitia Pusat Kontes Robot Indonesia, 2023. https://kontesrobotindonesia.id/kri-2023.html (accessed Apr. 20, 2023). [3] B. Kusumoputro et al., Buku Pedoman Kontes Robot Indonesia ( Kri ) Tahun 2023. Jakarta: Balai Pengembangan Talenta Indonesia Pusat Prestasi Nasional Kementerian Pendidikan, Kebudayaan, Riset dan Teknologi, 2023. g [4] F. Hermawanto and I. Karim, “Rancang Bangun Robot Penari Tidi,” Energy-Jurnal Ilm. Ilmu-Ilmu Tek., vol. 8, no. 2, pp. 35–41, 2018. . W. K. Andriani, “Minat Remaja Kelurahan Mugassari Kota Semarang Terhadap Tari Denok,” 2015. [6] S. Soim, B. Joni, and F. D. Junaidi, “Perancangan Robot Humanoid Berbasis Mikrokontroler ATmega 32,” Pros. Semnastek, pp. 1–6, 2015. [7] B. Arifin, A. A. Nugroho, B. Suprapto, S. A. D. Prasetyowati, and Z. Nawawi, “Review of Method for System Identification on Motors,” in 2021 8th International Conference on Electrical Engineering, Computer Science and Informatics (EECSI), 2021, pp. 257–262. [8] L. A. Sutawati, I. N. S. Kumara, and W. Widiadha, “Pengembangan Three Degree of Freedom Hexapod sebagai Robot Pemadam Api dengan Sensor UVTron Hamamatsu,” Maj. Ilm. Teknol. Elektro, vol. 17, no. 3, pp. 417–426, 2018. pp [9] H. D. Saputro, R. Maulana, and M. H. H. Ichsan, “Implementasi Real Time pada Pergerakan Robot Quadruped mengggunakan Multisensor dan RTOS,” J. Pengemb. Teknol. Inf. dan Ilmu Komput., vol. 2, no. 12, pp. 6868– 6875, 2018. , “OpenCM 9.04.” https://emanual.robotis.com/docs/en/parts/controller/opencm904/ (accessed 2023). [10] Robotis, “OpenCM 9.04.” https://emanual.robotis.com/docs/en/parts/controller/opencm9 Jul. 19, 2023). J ) [11] Robotis, “OpenCM 485 EXP.” https://emanual.robotis.com/docs/en/parts/controller/opencm485exp/ (accessed Sep. 26, 2022). ( p ) [12] Robotis, “OpenCM 485 Expansion Board - Robotis.” https://www.robotis.us/opencm-485-expansion- boar/ (accessed Sep. 26, 2022). [13] A. N. de Paula et al., “Edrom Humanoid Kid Size 2019,” 2019. [13] A. N. de Paula et al., “Edrom Humanoid Kid Size 2019,” 2019. [13] A. N. de Paula et al., “Edrom Humanoid Kid Size 2019,” 2019. [14] Robotis, “Dynamixel MX-28T - Robotis,” 2022. https://www.robotis.us/dynamixel-mx-28t/ (accessed Sep. 26, 2022). [14] Robotis, “Dynamixel MX-28T - Robotis,” 2022. https://www.robotis.us/dynamixel-mx-28t/ 26, 2022). [15] U. W. Putri and T. Thamrin, “Perancangan Pergerakan Kaki Robot Humanoid Menggunakan Servo Dynamixel Berbasis OpenCM 9.04,” Voteteknika (Vocational Tek. Elektron. dan Inform., vol. 7, no. 3, pp. 76– 84, 2019. KESIMPULAN Hasil penelitian menunjukkan bahwa penggunaan MotionPagePlay yang direkomendasikan adalah 2200 sampai dengan 2500. Jika MotionPagePlay diatur dengan lebih lambat maupun cepat maka akan mempengaruhi gerakan robot. Pada saat pengujian jalan pada permukaan bidang menunjukkan bahwa dengan menggunkan MotionPagePlay 2200 dapat berjalan dengan lurus menghasilkan keberhasilan sebanyak 7 dari 10 percobaan dengan persentase keberhasilan 70% serta rata-rata persentase error sebesar 0,33%. Sedangkan pada saat pengujian jalan pada permukaan berundak dengan MotionPagePlay 2500 sampai dengan 2800 menghasilkan keberhasilan sebanyak 9 dari 10 percobaan dengan persentase keberhasilan 90%. Kondisi baterai dan juga perubahan suhu pada servo mempengaruhi kinerja terhadap gerakan pada robot humnaoid. Kondisi baterai yang optimal untuk menggerakkan servo yaitu 12,6V sampai dengan 12,2V. Di bawah kondisi baterai tersebut maka kinerja dari servo akan menurun. Selama pengujian robot sebanyak 4 kali percobaan motor servo MX-28T mengalami peningkatan suhu pada servo ssehingga dapat mempengaruhi kinerja gerakan pada robot humanoid. Untuk penelitian lebih lanjut robot humanoid penari pada jalan berundak dapat menambahkan menambahkan sensor Gyroskop dan accelerometer yang digunakan untuk menunjang dalam pengenalan gerakan robot, menjaga keseimbangan serta mengontrol postur tubuh. Robot humanoid memerlukan pemeriksaan secara berkala, terutama pada bagian dengan beban tumpuan yang berat. Pemeriksaan visual meliputi kerusakan dan keausan pada servo, uji gerak robot untuk memastikan bahwa servo bergerak dengan lancar, pemeriksaan suhu untuk memastikan bahwa servo telah bekerja lebih baik, pemeriksaan koneksi antar servo untuk memastikan bahwa antar servo telah terhubung dengan baik dan pemeriksaan catu daya yang cukup. Available online: http://jtein.ppj.unp.ac.id \| 564 JTEIN: Jurnal Teknik Elektro Indonesia e-ISSN 2723-0589 Rancang Bangun Robot Humanoid Penari Untuk Berjalan Pada Lantai Berundak \| 565 Available online: http://jtein.ppj.unp.ac.id DAFTAR PUSTAKA [16] Robotis, “OpenCM IDE.” https://emanual.robotis.com/docs/en/software/opencm_ide/getting_started/# (accessed Jul. 19, 2023). [17] A. A. Syakur, R. Maulana, and E. Setiawan, “Perancangan Dan Implementasi Sistem Pola Berjalan Pada Robot Hexapod Menggunakan Metode Inverse Kinematic,” J. Pengemb. Teknol. Inf. dan Ilmu Komput., vol. 4, no. 6, pp. 1875–1881, 2020. Rancang Bangun Robot Humanoid Penari Untuk Berjalan Pada Lantai Berundak \| 565 Available online: http://jtein.ppj.unp.ac.id Rancang Bangun Robot Humanoid Penari Untuk Berjalan Pada Lantai Berundak \| 565 Available online: http://jtein.ppj.unp.ac.id
https://openalex.org/W3135008695	https://jnanobiotechnology.biomedcentral.com/track/pdf/10.1186/s12951-021-00893-6	English	null	A cyclic nano-reactor achieving enhanced photodynamic tumor therapy by reversing multiple resistances	Journal of nanobiotechnology	2,021	cc-by	9,655	RESEARCH Open Access © The Author(s) 2021. 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://creativeco mmons.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. Liu et al. J Nanobiotechnol (2021) 19:149 https://doi.org/10.1186/s12951-021-00893-6 Liu et al. J Nanobiotechnol (2021) 19:149 https://doi.org/10.1186/s12951-021-00893-6 Journal of Nanobiotechnology Abstract Background: Photodynamic therapy (PDT) is a clinically implemented modality to combat malignant tumor, while its efficacy is largely limited by several resistance factors from tumor microenvironment (TME), such as hypoxia, anti- oxidant systems, and ATP-dependent tumor adaptive resistances. The aim of this work is to construct a multifunc- tional nanoplatform to remodel multiple resistant TME for enhanced PDT. Results: Here, a targeting nano-reactor was facilely constructed to reverse the multiple resistances of PDT by incor- porating glucose oxidase (GOx) and chlorin e6 (Ce6) into poly (D, L-lactic-co-glycolic acid) (PLGA)/ metal–organic framework (MOF) core–shell nanoassembly, with surface deposition of hyaluronic acid (HA) stabilized MnO2. The nano-reactor could selectively target tumor cells by virtue of surface HA modification, and once internalization, a few reactions were initiated to modulate TME. Glucose was consumed by GOx to inhibit ATP generation, and the produced H2O2 was catalyzed by MnO2 to generate O2 for tumor hypoxia alleviation and photodynamic sensitization, and glutathione (GSH) was also effectively depleted by MnO2 to suppress the tumor antioxidant defense. Conse- quently, the nano-reactor achieved robust PDT with amplified tumor therapy via intravenous injection. Conclusions: This nano-reactor offers a multifunctional nanoplatform to sensitize TME-limited tumor treatment means via reversing multiple resistances. Keywords: Nanomedicine, Targeting, Tumor hypoxia, Drug resistance, Starvation therapy, GSH depletion, Oxygenation, Metal–organic-frameworks Peng Liu1, Yanbin Zhou1, Xinyi Shi1, Yu Yuan1, Ying Peng1, Surong Hua3, Qiange Luo1, Jinsong Ding1, Yong Li2* and Wenhu Zhou1* Peng Liu1, Yanbin Zhou1, Xinyi Shi1, Yu Yuan1, Ying Peng1, Surong Hua3, Qiange Luo1, Jinsong Ding1, Yong Li2* and Wenhu Zhou1* Peng Liu1, Yanbin Zhou1, Xinyi Shi1, Yu Yuan1, Ying Peng1, Surong Hua3, Qiange Luo1, Jinsong Ding1, Yong Li2* and Wenhu Zhou1* Background are activated by light to convert oxygen into toxic reactive oxygen species (ROS), which bind and dis-functionalize some bio-macromolecules in tumor cells, such as DNA and lipid, resulting in cell apoptosis or necrosis, immune responses and microvascular damage [3–7]. Such process is highly efficient to kill cancer at cellular level, and some PSs, such as verteporfin, 5-ALA and temoporfin, have been demonstrated as promising candidate for clinical translation [8, 9]. However, while PDT has been accepted for several types of cancer in clinic [10], its widespread implementation is still hindered by various biological limitations. Aside from the inherent barrier of light pen- etration that can be partially addressed by deep PDT Photodynamic therapy (PDT) has gain tremendous fun- damental and translational attention for tumor therapy, owing to its advantages of low systemic toxicity, non- invasive, spatial and temporal controllable activation [1, 2]. During the process of PDT, the photosensitizers (PSs) Liu et al. J Nanobiotechnol (2021) 19:149 Page 2 of 13 techniques, tumor resistances are the main mechanism to weaken the efficacy of PDT [11]. strategy to alleviate hypoxia, which can be achieved by either oxygen delivery (using hemoglobin or perfluoro- carbons) or endogenous oxygen generation (through catalytic converting tumor abundant H2O2 into O2) [24–26]. Oxygen generation can be further boosted by cascade equipping the nano-system with glucose oxidase (GOx) to supplement H2O2 substrate [27]. To augment PDT efficacy, several nano-vehicles were also designed to suppress the tumor antioxidant defense and restore the ROS damage effect of PDT via antioxidants depletion [28, 29]. Moreover, inhibition of ATP was reported to sensi- tize PDT by inhibiting drug efflux and aggravating PDT- induced DNA damage [30, 31]. Ideally, simultaneous remodeling various TME to alleviate multiple biological resistances is preferred for optimized PDT, considering the cunning nature of cancer. Unfortunately, the devel- opment of nanoparticles with multifunctionalities often requires sophisticated materials design and complicated preparation procedure, thus imposing the difficult of cost-effective, reproducible, and scalable production. fi Tumor cells can resist PDT by different pathways due to the complexity of tumor microenvironment (TME), in which the most well-known feature is hypoxia. Hypoxic cells are ~ threefold more resistant to ROS damage than aerobic cells [12], and more importantly, hypoxic envi- ronment could directly decrease the PDT efficacy by blocking the oxygen supply [13, 14]. What’s more, the PDT process would further aggravate tumor hypoxia through oxygen consumption and vascular damage, which activates hypoxia inducible factor-1α (HIF-1α) survival pathway, thus causing PDT resistance [15, 16]. At the same time, cancer cells are equipped with anti- oxidant defense systems, in which the most abundant one is glutathione (GSH), to scavenge the ROS and thus counteract ROS-mediated injury [17–19]. In addition, tumor cells could generate adaptive resistance toward PDT through upregulation of drug efflux proteins, heat shock proteins (HSPs), and DNA repair proteins [20–22]. It has been reported that numerous drug efflux proteins, such as P-glycoprotein (P-gp), and ATP-binding cassette super-family G member 2 (ABCG2), have been impli- cated in PDT resistance via pumping out PSs before their action [23]. f To tackle this, we designed and facilely prepared a core–shell nano-reactor for enhanced PDT via simulta- neous oxygenation, antioxidant suppression, and ATP depletion (Scheme 1). Materials and methods Materials Chlorin e6 (Ce6) and Nile red (NR) were obtained from Frontier Scientific, Inc. (Utah, USA). PLGA (Mw: 20 kDa) was purchased from Daigang Biomaterial Co., Ltd. (Jinan, China). Hyaluronic acid (HA, 7 kDa) was purchased from Lifecore Biomedical Company (MN, USA). Poly (allylamine hydrochloride) (PAH), tannic acid (TA), glu- cose oxidase (GOx), singlet oxygen sensor green (SOSG), and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazo- lium bromide (MTT) were provided by Sigma-Aldrich (Saint Louis, MO, USA). Live & dead viability/cytotox- icity assay kit was obtained by Invitrogen (NY, USA). Ellman’s reagent and 4′, 6-diamidino-2-phenylindole (DAPI) were provided by Solarbio Biotech, Co., Ltd. (Bei- DL = amount of loaded drug in the PTFCG@MH / weight of PTFCG@MH (µg/mg). jing, China). Ferric chloride hexahydrate (FeCl3·6H2O), N-acetylcysteine (NAC) and 2′ 7’-dichlorofluorescin diacetate (DCFDA) were obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). DMEM medium, fetal bovine serum, streptomycin/penicillin and Trypsin–EDTA were provided by GIBCO (NY, USA). The 3,5-dinitrosalicylic acid (DNS) reagent was pur- chased from Coolaber Biotech, Co., Ltd. (Beijing, China). ROS-ID hypoxia detection kit was provided by Enzo Life Sciences Inc. (NY, USA). jing, China). Ferric chloride hexahydrate (FeCl3·6H2O), N-acetylcysteine (NAC) and 2′ 7’-dichlorofluorescin diacetate (DCFDA) were obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). DMEM medium, fetal bovine serum, streptomycin/penicillin and Trypsin–EDTA were provided by GIBCO (NY, USA). The 3,5-dinitrosalicylic acid (DNS) reagent was pur- chased from Coolaber Biotech, Co., Ltd. (Beijing, China). ROS-ID hypoxia detection kit was provided by Enzo Life Sciences Inc. (NY, USA). The PTFCG@MH solution ([Ce6] = 200 μg/mL) was dispersed in 5 mL of various dissolution media (10 mM PBS buffer, 10 mM PBS buffer containing 10 mM GSH). The mixtures were shaken gently in a shaking incubator at 37 °C. At predetermined time intervals (0, 1, 2, 4, 8, 12 h), 0.5 mL of samples were withdrawn and centrifuged (16,000 rpm, 25 min). The supernatant was collected, and the amount of Ce6 was quantified by UV absorbance quantification (λ = 640 nm). Tannic acid (TA), ferric iron (Fe3+) and poly (D, L-lactic-co-glycolic acid) (PLGA) were assembled into core–shell structure via hydrophobic, π-π staking, and electrostatic interactions [32–34], in which chlorin e6 (Ce6, a widely used PS) and GOx were To reverse PDT resistance, various nano-systems capa- ble of modulating TME have been developed. For exam- ple, replenishment of oxygen is a commonly employed Scheme 1 Schematic illustration the preparation of core-sell nano-reactor, and its targeted delivery in vivo for enhanced PDT against tumor by reversing multiple resistances Scheme 1 Schematic illustration the preparation of core-sell nano-reactor, and its targeted delivery in vivo for enhanced PDT against tumor by reversing multiple resistances Scheme 1 Schematic illustration the preparation of core-sell nano-reactor, and its targeted delivery in vivo for enhanced PDT against tumor by reversing multiple resistances Liu et al. J Nanobiotechnol (2021) 19:149 Page 3 of 13 Page 3 of 13 co-loaded. MnO2 was deposited on particle surface and stabilized by hyaluronic acid (HA) through layer-by-layer electrostatic adsorption. In our system, the MnO2 played dual roles of self-oxygen supply and GSH depletion via its catalase-mimic activity and oxidbillity, respectively. The GOx boosted the oxygenation by in-situ generation of H2O2, and its capability of glucose consumption blocked the energy supply to decrease ATP generation. All these functions collectively remodeled the TME from different aspect to sensitize PDT efficacy, which has been dem- onstrated by solution, in vitro and in vivo experimental results. The nanosystem could passively accumulate into tumor, actively internalize tumor cells via HA-mediated targeting, and impose potent PDT effect via reversing multiple resistances. was stirred at 30 °C for 3 h to evaporate acetone. The PTFCG was separated by centrifugation (16,000 rpm, 25 min) and washed by deionized water. To prepare PTFCG@MH, 50 μL PAH (20 mg/mL) was mixed with 1 mL PTFCG solution and mild stirred for 30 min. Then, 25 μL potassium permanganate (10 mg/mL) was added and stirred for another 1 h. After centrifugation (16,000 rpm for 25 min) and washing with deionized water, the PTFCG@M was obtained. Finally, 500 μL HA (20 mg/mL) was introduced into 1 mL of PTFCG@M solution and stirred for 1 h. Excess HA was removed by centrifugation (16,000 rpm, 25 min), and PTFCG@MH was collected. Characterizations of PTFCG@MHh The particle sizes and ζ potential of PTFCG, PTFCG@M and PTFCG@MH were measured by a Malvern Zeta Sizer Nano series (Malvern, UK) at 25 °C. The morpholo- gies and element content were investigated by TEM-EDS (Titan G2 60–300, FEI, USA). For TEM-EDS analysis, the PTFCG or PTFCG@MH solution was dropped onto carbon film-coated copper grid and observed under TEM-EDS instrument. The UV–vis absorption spec- tra were carried out on UV–visible spectrophotometer (Shimadzu, Japan). The fluorescence spectra were col- lected using FL-2700 spectrofluorometer (HITACHIH). The Ce6 concentration was determined by the UV–vis- ible spectra spectrophotometer at 640 nm. The loading amount of GOx was estimated using bicinchoninic acid (BCA) protein assay (Beyotime) and the protocol was provided by the supplier. The drug loading (DL) of Ce6 and GOx in the PTFCG@MH was calculated as follows: In vitro cytotoxicity studies y y MDA-MB-231 cells were seeded in a 96-well plate (5000 cells/well) for overnight incubation. The cells were treated with PTFCG or PTFCG@MH at different con- centrations for 24 h, followed by laser irradiation for 1 min (100 mW/cm2, 635 nm). After 24 h, MTT solution (1 mg/mL) was added for another 4 h incubation. The medium was replaced with 100 μL of DMSO. The absorb- ance values at 570 nm were recorded to calculate the cell viability. Then, the Live/Dead assay was carried out by calcein AM/propidium iodide double staining. The cells were seeded and treated as described above. After laser irradiation (100 mW/cm2, 635 nm), the fresh medium containing calcein AM (2 μM) and propidium iodide (8 μM) was added for 20 min incubation. Finally, the cells were imaged by fluorescence imaging system (NIKON, Ti-S, Japan). Intracellular O2 consumption and ROS detection 2 p ROS-ID and DCFDA were used to detect the intracellu- lar O2 consumption and ROS generation inside cells. The MDA-MB-231 cells were seeded in 24-well culture plate (105 cells/well) and cultured overnight. After sealing by liquid paraffin, the cells were incubated with PTFCG@ MH, PTF@MH, PTFCG, or free Ce6 ([Ce6] = 1 μg/mL) for 2 h. Then, laser irradiation (635 nm, 100 mW/cm2) was applied for 2 min. With PBS washing for three times, the mixture of ROS-ID (0.5 μM) and DCFDA (10 μM) was added into each group for 30 min further incuba- tion. After washing with pre-cooled PBS, fluorescence inside cells was observed by fluorescence imaging system (NIKON, Ti-S, Japan). Intracellular ATP and GSH detection MDA-MB-231 cells were seeded in 12-well plate at a density of 2 × 105 cells per well for 12 h incubation. Then, various formulations with different concentrations were added for 24 h incubation. After that, the cells were col- lected and lysed using ATP lysis buffer, and ATP con- centration was measured by ATP assay kit (Beyotime). Likewise, the cells were collected and lysed using the Triton-X 100 cell lysis buffer, and GSH concentration was detected by the Ellman’s reagent (Solarbio). The numbers of cells were standardized by measuring total protein concentration using BCA protein assay (Beyotime). PTF@MH or PTFCG@MH ([Ce6] = 1 μg/mL) was added into SOSG solution (2.5 μM), followed by adding H2O2 (10 mM). Then, a continuous laser (100 mW/cm2, 635 nm) was performed at predetermined time points (0, 1, 2, 4, 6, 8, 10 min), and fluorescence intensity was detected immediately by fluorescence spectroscopy (Ex = 490 nm, Em = 525 nm). Cyclic reaction of PTFCG@MHh Ce6, FeCl3 and PLGA were mixed in acetone at a concen- tration of 400 μg/mL, 400 μg/mL and 2 mg/mL, respec- tively. Then, 1 mL of such mixture was added dropwise to 5 mL aqueous solution containing 320 μg/mL TA and 20 μg/mL GOx under constant sonication. The solution The catalytic activity of GOx was determined by measur- ing the glucose consumption and pH change in presence of glucose. For the glucose consumption, the PTF@MH or PTFCG@MH solution ([GOx] = 4 μg/mL) was mixed with glucose (10 mM) in the presence or absence of H2O2 Liu et al. J Nanobiotechnol (2021) 19:149 Page 4 of 13 PBS, digested by Trypsin–EDTA and analyzed using flow cytometry (FACSVerse, BD, USA). (100 μM) at 37 °C, then the glucose concentration was detected by DNS reagent (Coolaber) within 1 h and the protocol was provided by the supplier. For the pH change, the glucose (10 mM) was added into PTFCG or PTFCG@ MH solution ([GOx] = 4 μg/mL) at 37 °C, then the pH value was measured by a pH meter (PHSJ-4F, INESA, China) within 30 min. To monitor the oxygen genera- tion under different acidic conditions, PTFCG@MH ([GOx] = 4 μg/mL) was dispersed in 10 mM PBS buffer (pH 5.0, 6.0, and 7.0) containing 1 mM H2O2. At pre- determined time points, the dissolved O2 was detected using the portable dissolved oxygen meter (JPBJ-609L, INESA, China). Moreover, the PTFCG or PTFCG@MH ([GOx] = 4 μg/mL) was mixed with glucose (10 mM) in the presence or absence of H2O2 (100 μM) and laser irra- diation (ADR-1805 Laser, 100 mW/cm2, 635 nm), then the dissolved O2 was measured within 160 s. Cellular uptakeh The NR-loaded nanoparticles were prepared following the method described above by replacing Ce6 with NR. The cells were seeded in Petri dish (35 mm) at 5 × 104 cells/cm2 and cultured overnight. With or without pre- treatment of free HA (10 mg/mL) for 1 h, the cells were incubated with NR-loaded nanoparticles or free NR ([NR] = 2 μg/mL) for another 2 h. Next, the cells were washed with pre-cooled PBS and fixed with 4% para- formaldehyde. After staining by DAPI (1 μg/mL), the cells were imaged under confocal microscope (LSM780 NLO, Zeiss, Germany). To study the endocytosis mechanism, the cells were seeded in 6-well culture plate (2 × 105 cells/well). After pretreating with chlorpromazine (10 μg/mL), nystatin (15 μg/mL) or colchicine (5 μg/mL) for 1 h, the cells were incubated with NR-loaded nanoparticles ([NR] = 2 μg/ mL) for 2 h. Then, the cells were washed with pre-cooled Cell culture MDA-MB-231 and HUVEC cells were cultured under a humidified atmosphere of 5% CO2 at 37 °C using DMEM and RPMI 1640 complete medium, respectively. The complete medium contains 10% FBS (GIBCO, USA), 1% penicillin/streptomycin (100 U/mL, Solarbio Bioteh). Immunofluorescence analysish The expression of HIF-1α protein was investigated by immunofluorescence assay. Nude mice bearing MDA- MB-231 tumors were sacrificed at 48 h after various treatments mentioned above. Tumor tissues were col- lected for preparing slices of tumor frozen sections. Then, the slices were stained with HIF-1α antibody (mouse pol- yclonal to HIF-1α, Abcam) at 4 °C overnight, followed by adding the secondary antibodies conjugated with FITC for 1 h incubation. After cell nuclei staining with Hoechst 33,258 for 20 min, the tumor sections were imaged by confocal fluorescence microscope (LSM780 NLO, Zeiss, Germany). According to the UV–vis absorbance spectra, PTFCG had characteristic Ce6 peaks at 404 nm and 640 nm, indi- cating the successful Ce6 loading (Fig. 1D). For PTFCG@ MH, a wide absorbance band in the range from 250– 400 nm appeared, which was ascribed to the MnO2. To confirm the MnO2 coating, the energy dispersive X-Ray spectroscopy (EDS) was conducted, and a high Mn ele- ment content (~ 6%) was observed (Fig. 1E). The drug loading (DL) for Ce6 and GOx was measured to be 108.4 μg and 24.6 μg per mg of PTFCG@MH, respec- tively. Interestingly, after encapsulation into nanoparti- cles, the intrinsic fluorescence of Ce6 was significantly quenched, especially for PTFCG@MH (Additional file 1: Figure S4). However, such quenched fluorescence can be Nanoparticles preparation and characterizationh The GOx and Ce6 were co-loaded into nanocompos- ites (PTFCG) using a solvent exchange and evapora- tion method (Scheme 1), in which the ethanol solution (containing Ce6, PLGA and FeCl3) was dropwise added into the aqueous phase (with TA and GOx). During organic evaporation, the TA-Fe metal–organic frame- works (MOFs) were coated on the surface of hydro- phobic PLGA nano-core for particle stabilization, and the resulting PTFCG displayed a dynamic diameter of ~ 175 nm (Fig. 1A, Additional file 1: Figure S1) with ζ potential of − 33.4 mV (Fig. 1C). From the TEM image, the PTFCG displayed a roughly spherical morphology with an obvious core–shell structure (inset in Fig. 1A). To further deposit MnO2 on particle surface, a PAH solu- tion was added, followed by adding KMnO4, allowing for in-situ growth of MnO2 on nanoparticle surface to yield PTFCG@M. Compared with PTFCG, the ζ poten- tial of PTFCG@M was reversed to positive (+ 17.3 mV) (Fig. 1C), which allowed for the subsequent HA coat- ing via electrostatic attraction (termed PTFCG@MH). The successful HA modification was evidenced by the decrease of particle charge to negative (− 21.7 mV) (Fig. 1C), which is beneficial for enhanced colloidal sta- bility. [35] The resulting PTFCG@MH nanoparticles dis- played a dynamic diameter of ~ 205 nm (Fig. 1B), while the size measured by TEM was ~ 160 nm (inset in Fig. 1B, Additional file 1: Figure S2). It is reasonable as DLS meas- urement would distort the particle size being observed due to the particle surface hydration. Importantly, the HA modification not only significantly increased the col- loidal stability (Additional file 1: Figure S3), [36] but also rendered the nanoparticles with tumor targetability (vide infra). For in vivo/ex vivo imaging, the tumor-bearing mice were intravenously injected with free Ce6 or PTFCG@ MH (100 μL, [Ce6] = 2.5 mg/kg), and the fluorescence images were taken by an optical imaging system (IVIS Lumina, PerkinElmer, USA) at 1 h and 24 h post-injec- tion. The mice were sacrificed at 24 h after injection, and the major organs were collected for ex vivo imaging. The images were analyzed using Living Imaging Software (IVIS Lumina LT, PerkinElmer, USA). In vivo antitumor studyh The tumor-bearing mice were randomly divided into 5 groups and injected intravenously with (1) PBS, (2) Ce6 + Laser, (3) PTFCG + Laser, (4) PTFCG@MH, (5) PTFCG@MH + Laser (100 μL, [Ce6] = 2.5 mg/kg) at day 0 and 4. The irradiation groups were exposed to laser (100 mW/cm2, 635 nm) for 5 min at 24 h after injec- tion. The tumor volume was recorded and calculated as follows: V = (length × width2)/2. In addition, the body weights were also obtained. At day 14, all the mice were sacrificed, and the tumors were collected and weighed. For histology analysis, the major organs and tumors were extracted and immersed in 4% formaldehyde, embedded in paraffin, sectioned, stained with H&E and observed by an optical microscope (Leica, German). In vivo/ex vivo fluorescence imaging Healthy female Balb/c mice (aged 4–6 weeks) were pur- chased from the Laboratory Animal Center of Central South University. All experimental procedures were Liu et al. J Nanobiotechnol (2021) 19:149 Liu et al. J Nanobiotechnol (2021) 19:149 Liu et al. J Nanobiotechnol (2021) 19:149 Page 5 of 13 ANOVA analysis of variance was conducted to deter- mine the statistical significance of various groups. P val- ues < 0.05 was regarded as statistically significant. carried out in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals of China, and approved by the Ethics Commit- tee for Research in Animal Subjects at Xiangya School of Pharmaceutical Sciences of Central South University. MDA-MB-231 tumor-bearing mice were obtained by subcutaneously injecting a cells suspension in PBS (106 cells) into the right armpit of mice. When the tumor vol- ume reached ~ 100 mm3, the mice were treated with dif- ferent formulations. carried out in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals of China, and approved by the Ethics Commit- tee for Research in Animal Subjects at Xiangya School of Pharmaceutical Sciences of Central South University. MDA-MB-231 tumor-bearing mice were obtained by subcutaneously injecting a cells suspension in PBS (106 cells) into the right armpit of mice. When the tumor vol- ume reached ~ 100 mm3, the mice were treated with dif- ferent formulations. Statistical analysis Data were presented as mean ± SD. Analysis was per- formed using Graphpad Prism 5 software. One-way Liu et al. J Nanobiotechnol (2021) 19:149 Page 6 of 13 Fig. 1 Dynamic sizes and TEM images of A PTFCG and B PTFCG@MH. C The ζ potential of PTFCG, PTFCG@M and PTFCG@MH. D UV–vis absorbance spectra of Ce6, PTFCG and PTFCG@MH. E Energy dispersive X-Ray spectroscopy (EDS) analysis of PTFCG@MH. F In vitro Ce6 release from PTFCG@MH with or without addition of GSH (10 mM) Fig. 1 Dynamic sizes and TEM images of A PTFCG and B PTFCG@MH. C The ζ potential of PTFCG, PTFCG@M and PTFCG@MH. D UV–vis absorbance spectra of Ce6, PTFCG and PTFCG@MH. E Energy dispersive X-Ray spectroscopy (EDS) analysis of PTFCG@MH. F In vitro Ce6 release from PTFCG@MH with or without addition of GSH (10 mM) strengthened after adding H2O2, which can be ascribed to the decomposition of H2O2 by PTFCG@MH to sup- ply O2. Michaelis–Menten constant (KM) was calculated to quantify the reaction kinetics, and the KM value of PTFCG@MH + H2O2 and PTFCG@MH turned out to be 6.35 and 10.02 mM (Additional file 1: Figure S5), indi- cating the promoted glucose consumption by H2O2. The activity of GOx can also be monitored by the pH decrease due to the generation of gluconic acid byproduct. Upon addition of glucose, both PTFCG and PTFCG@MH showed a gradual pH decrease over time (Fig. 2C). Nota- bly, PTFCG@MH exhibited a relative lower pH decrease rate than that of PTFCG. This is likely due to that the surface deposited MnO2 impeded accessibility of glucose into GOx-loaded nanocore. In addition, the MnO2 could neutralize H+ to slow down pH decrease rate. largely recovered upon addition of GSH, indicating GSH- responsive release of Ce6. To confirm this, Ce6 release behavior from PTFCG@MH was investigated (Fig. 1F). In absence of GSH, the Ce6 showed ~ 20% accumulative release, while a burst drug release was observed after adding GSH, with ~ 60% Ce6 release within 8 h. There- fore, such nanosystem showed a triggered activation of photodynamic activity in response to intracellular stimu- lus, thus minimizing the potential phototoxicity of Ce6 during in vivo delivery. largely recovered upon addition of GSH, indicating GSH- responsive release of Ce6. To confirm this, Ce6 release behavior from PTFCG@MH was investigated (Fig. 1F). Statistical analysis In absence of GSH, the Ce6 showed ~ 20% accumulative release, while a burst drug release was observed after adding GSH, with ~ 60% Ce6 release within 8 h. There- fore, such nanosystem showed a triggered activation of photodynamic activity in response to intracellular stimu- lus, thus minimizing the potential phototoxicity of Ce6 during in vivo delivery. A cyclic reaction nano‑reactor E Kinetics of dissolved oxygen variation for PTFCG@MH and PTFCG under different conditions. F The 1O2 generation kinetics for PTFCG@MH, PTFCG and PTF@MH in the presence or absence of H2O2 or glucose under laser irradiation. The concentration of glucose and H2O2 was 10 mM and 100 μM, respectively measuring the oxygen variation (Fig. 2E). For PTFCG, the dissolved O2 was quickly decreased within 100 s upon addition of glucose. For the PTFCG@MH, by contrast, the O2 consumption rate significantly lessened due to the self-oxygen generation activity of MnO2, demonstrating the cyclic oxygen supply. Moreover, the O2 balance was achieved upon further addition of 100 μM H2O2 (which mimics the tumor microenvironment) [37], and such O2 generation efficiency is strong enough to support the PDT (Fig. 2E, blue trace). strengthen the PDT effect. As a control, the PTFCG group showed lower 1O2 generation due to the lack of oxygen supply. All these results demonstrated the capa- bility of cyclic nano-reactor for boosting PDT effect of Ce6. A cyclic reaction nano‑reactor As illustrated in Fig. 2A, our nanosystem was designed with a few cyclic reactions to enhance the Ce6-based PDT. GOx consumes glucose (Glu) to block the energy (ATP) supply, and the concomitantly produced H2O2 is decomposed by the catalase-mimic MnO2 to generate O2, which in turn boosts the 1O2 production under laser illumination. To demonstrate this concept, each reaction was tested individually. Next, the catalase-mimic activity of MnO2 was tested by monitoring O2 production in presence of H2O2 (Fig. 2D). As a control, the PTFCG@MH alone did not show any O2 generation. However, a rapid increase of the dissolved O2 was observed upon addition of H2O2. We also studied the effect of pH, and a marginal increase of catalytic rate was seen with the decrease of pH from 7.0 to 5.0. Overall, the catalytic efficiency was relatively higher under acidic tumor microenvironment than phys- iological conditions. The catalytic activity of GOx was first measured by monitoring the glucose consumption (Fig. 2B). After 60 min incubation, PTFCG@MH decreased the glu- cose level by 34%, while the nanoparticles without GOx loading (termed PTF@MH) did not show any glucose consumption. Therefore, GOx maintained its catalytic activity after being loaded into nanoparticles. Moreo- ver, the glucose depletion of PTFCG@MH was further After confirming the single catalytic reaction of GOx and MnO2, we next explored the catalytic circulation by Liu et al. J Nanobiotechnol (2021) 19:149 Page 7 of 13 Fig. 2 A Schematic illustration of cyclic reaction of PTFCG@MH. B Glucose consumption kinetics catalyzed by different formulations and conditions. C The kinetics of pH change for PTFCG@MH and PTFCG in absence or presence of glucose. D The kinetics of oxygen generation for PTFCG@MH in absence or presence of H2O2 at different pH values. E Kinetics of dissolved oxygen variation for PTFCG@MH and PTFCG under different conditions. F The 1O2 generation kinetics for PTFCG@MH, PTFCG and PTF@MH in the presence or absence of H2O2 or glucose under laser irradiation. The concentration of glucose and H2O2 was 10 mM and 100 μM, respectively Fig. 2 A Schematic illustration of cyclic reaction of PTFCG@MH. B Glucose consumption kinetics catalyzed by different formulations and conditions. C The kinetics of pH change for PTFCG@MH and PTFCG in absence or presence of glucose. D The kinetics of oxygen generation for PTFCG@MH in absence or presence of H2O2 at different pH values. Cellular uptake We next tested the intracellular performance of the nano- system by using MDA-MB-231 cancer cells. To track the intracellular delivery, nanoparticles were labeled with a red fluorescent Nile red (NR), and the cell nuclei were stained blue by DAPI for localization (Fig. 3A). From confocal laser scanning microscopy (CLSM) images, a weak red signal was observed when the cells were treated with free NR, while the fluorescence was significantly intensified for nanoparticles. From the merged image, the With cyclic oxygen supply, we next explored the enhanced PDT effect by measuring the single oxy- gen (1O2) generation using singlet oxygen sensor green (SOSG) as a fluorescent indicator (Fig. 2F). In presence of H2O2, the production of 1O2 was obviously increased for PTFCG@MH, and the addition of glucose could also Liu et al. J Nanobiotechnol (2021) 19:149 Page 8 of 13 Fig. 3 A Cellular uptake of free NR and NR-labeled PTFCG@MH measured by CLSM. B Intracellular fluorescence quantification for different groups. C Flow cytometric analysis of nanoparticles uptake in MDA-MB-231 cells with pre-treatment of various inhibitors. D The quantification of uptake from flow cytometric analysis. P < 0.01, P < 0.001 Fig. 3 A Cellular uptake of free NR and NR-labeled PTFCG@MH measured by CLSM. B Intracellular fluorescence quantification for different groups. C Flow cytometric analysis of nanoparticles uptake in MDA-MB-231 cells with pre-treatment of various inhibitors. D The quantification of uptake from flow cytometric analysis. P < 0.01, P < 0.001 both PTFCG@MH and PTF@MH could scavenge the intracellular ROS to some extent as compared to the control, due to the catalase activity of MnO2. Upon irra- diation, each Ce6-based formulation showed enhanced green fluorescence inside cells based on both fluores- cent images (Fig. 4A) and quantified intensity (Fig. 4B). Notably, PTFCG@MH group emitted the strongest fluorescence, indicating the best PDT efficacy. This can partially be explained by the cyclic oxygenation of the nano-reactor for self-oxygen supply. To confirm this, we then measured the O2 balance by using a red fluorescent hypoxia detection kit. Both PTF@MH and PTFCG@ MH could relieve tumor hypoxia with red fluorescence decrease compared to non-treatment control (Fig. 4A, C), also attributable to self-oxygen supply of the MnO2. Upon addition of laser, the hypoxia was strongly exacer- bated for free Ce6 and PTFCG groups, ascribed to PDT- and GOx-based oxygen consumption. However, the PTFCG@MH group showed low hypoxia level even after laser irradiation. Cellular uptake Therefore, such nano-reactor is robust enough to maintain oxygen balance with enhanced PDT efficacy. fluorescence of nanoparticles mainly distributed in the cytoplasm, indicating endocytosis pathway for internali- zation. To demonstrate tumor targetability of such sur- face HA modified nanosystem, the cells were pretreated with free HA to saturate the CD44 binding, and in this case the intracellular nanoparticles signal decreased obvi- ously. We also quantified the contribution of HA-medi- ated internalization by measuring the intensity of each treatment, where the uptake was significantly decreased upon free HA pre-treatment (Fig. 3B). Using normal human umbilical vein endothelial cells (HUVEC) as con- trol, the results further demonstrated the targetability of the nanoparticles towards tumor cells with significantly higher fluorescence (Additional file 1: Figure S6). These results confirmed that our nanosystem was able to selec- tively recognize tumor cells via CD44 receptor for target- ing delivery. To further explore the delivery mechanism, various endocytosis inhibitors were employed, including chlorpromazine, nystatin and colchicine, which blocks clathrin-mediated endocytosis, caveolae-mediated endo- cytosis and micropinocytosis, respectively. Among then, chlorpromazine has the most significant impact on nano- particles uptake (Fig. 3C, D), indicating the main contri- bution of clathrin-mediated endocytosis. fi Besides cyclic oxygen supply, such nano-reactor was also designed to deplete both ATP and GSH. We next tested these properties by measuring the intracellular ATP and GSH levels using ATP assay kit and Ellman’s reagent, respectively. Because of catalytic glucose oxida- tion, free GOx could effectively decrease ATP level in a concentration dependent manner by blocking the energy supply (Fig. 4D). Likewise, the PTFCG@MH could also inhibit ATP generation, while the PTF@MH did not Enhanced anti‑tumor efficacy via hypoxia alleviation and ATP/GSH depletion After internalization, we next explored the intracellular functions of the nanosystem. To visualize the PDT effect, the ROS generation was probed by using 2′ 7’-dichloro- fluorescin diacetate (DCFDA) indicator. Interestingly, Liu et al. J Nanobiotechnol (2021) 19:149 Page 9 of 13 Fig. 4 A Fluorescence images of intracellular ROS generation and hypoxia level with different treatments. The quantitative analysis of B ROS generation and C hypoxia level. In each panel, G1: Control; G2: PTF@MH; G3: PTFCG@MH; G4: Ce6 + Laser; G5: PTFCG + Laser; G6: PTFCG@ MH + Laser. The intracellular D ATP and E GSH content of MDA-MB-231 cells incubated with various formulations. F Concentration dependent cytotoxicity of different formulations for MDA-MB-231 cells. G Fluorescence images of cells with different treatments after co-staining with calcein-AM and propidium iodide. P < 0.01, *P < 0.001 Fig. 4 A Fluorescence images of intracellular ROS generation and hypoxia level with different treatments. The quantitative analysis of B ROS generation and C hypoxia level. In each panel, G1: Control; G2: PTF@MH; G3: PTFCG@MH; G4: Ce6 + Laser; G5: PTFCG + Laser; G6: PTFCG@ MH + Laser. The intracellular D ATP and E GSH content of MDA-MB-231 cells incubated with various formulations. F Concentration dependent cytotoxicity of different formulations for MDA-MB-231 cells. G Fluorescence images of cells with different treatments after co-staining with l AM d d d d P *P Fig. 4 A Fluorescence images of intracellular ROS generation and hypoxia level with different treatments. The quantitative analysis of B ROS generation and C hypoxia level In each panel G1: Control; G2: PTF@MH; G3: PTFCG@MH; G4: Ce6+Laser; G5: PTFCG+Laser; G6: PTFCG@ Fig. 4 A Fluorescence images of intracellular ROS generation and hypoxia level with different treatments. The quantitative analysis of B ROS generation and C hypoxia level. In each panel, G1: Control; G2: PTF@MH; G3: PTFCG@MH; G4: Ce6 + Laser; G5: PTFCG + Laser; G6: PTFCG@ MH + Laser. The intracellular D ATP and E GSH content of MDA-MB-231 cells incubated with various formulations. F Concentration dependent cytotoxicity of different formulations for MDA-MB-231 cells. G Fluorescence images of cells with different treatments after co-staining with calcein-AM and propidium iodide. P < 0.01, *P < 0.001 Next, the in vitro cytostatic activity of nanosystem was evaluated by MTT assay. Without drugs loading, the PTF and PTF@MH showed satisfactory biocompatibil- ity even at high concentrations (Additional file 1: Figure S7). Enhanced anti‑tumor efficacy via hypoxia alleviation and ATP/GSH depletion The PTFCG, on the other hand, displayed a con- centration-dependent tumor ablation activity (Fig. 4F), ascribed to the starvation therapy for ATP depletion [40]. Upon laser irradiation, the antitumor effect was further enhanced. Notably, PTFCG@MH showed significantly better efficacy than PTFCG attributable to MnO2 doping for self-oxygen circulation, GSH depletion, as well as HA modification for targeting delivery. To confirm the tumor selectivity, MTT assay was also performed on HUVEC show any activity due to the absence of GOx loading. MnO2 is a well-known GSH depletor due to its capabil- ity to oxidize GSH into GSSG, accompanied by its reduc- tion into Mn2+ [28, 38]. As expected, the PTFCG@MH with MnO2 doping displayed high efficient GSH deple- tion, with over 80% GSH decrease at 5 μg/mL nanopar- ticles (Fig. 4E). Note that ATP is the basic energy source for tumor cells to acquire treatments resistance includ- ing PDT therapy [39], and GSH could directly scavenge 1O2 to alleviate the PDT efficacy. Therefore, with ATP and GSH dual-depletion activity, such nano-reactor was expected to enhance the PDT-based anti-tumor activity via distinct mechanisms. Liu et al. J Nanobiotechnol (2021) 19:149 Page 10 of 13 due to the fluorescence quenching of the nanosystem, further demonstrating its advantage for decreased pho- totoxicity. However, the signal at tumor tissue became intensified after 24 h circulation (Fig. 5A, black circle), indicating the EPR effect of the nanomedicine for pas- sive accumulation into tumor. We then quantified the results by collecting the main organs as well as tumor tis- sues for ex vivo fluorescence imaging (Fig. 5B). PTFCG@ MH exhibited ~ 3.7-fold higher intensity at tumor site than free Ce6, confirming the targetability of the nano- system towards tumor tissue. Meanwhile, we also noticed considerable accumulation of nanoparticles in liver and kidneys, the main organs to sequester and eliminate nanoparticles from body, and it was also observed in many other nanomedicines [41, 42]. Fortunately, tempo- ral controlled activation of PDT would enable selective damage toward tumor, thus minimizing the unwanted side-effects. cells, and as expected, higher cytotoxicity was observed for MDA-MB-231 cells (Additional file 1: Figure S8). We also explored the cell death pathway by co-staining the cells using calcein-AM (green fluorescence for live cells) and propidium iodide (red fluorescence for dead/late apoptotic cells). After different treatments, green fluores- cence was weakened while the red fluorescence became intensified inside cells (Fig. Enhanced anti‑tumor efficacy via hypoxia alleviation and ATP/GSH depletion 4G), indicating an apoptotic or necrotic cell death mechanism. In addition, the gen- eral anti-tumor activity for each treatment was highly consistent with the results from MTT assay. Abbreviations h d PDT: Photodynamic therapy; TME: Tumor microenvironment; GOx: Glucose oxidase; Ce6: Chlorin e6; PLGA: Poly (D, L-lactic-co-glycolic acid; MOF: Metal– organic framework; HA: Hyaluronic acid; GSH: Glutathione; PSs: Photosensitiz- ers; ROS: Reactive oxygen species; HIF-1α: Hypoxia inducible factor-1α; HSPs: Heat shock proteins; P-gp: P-glycoprotein; ABCG2: ATP-binding cassette super- family G member 2; TA: Tannic acid; NR: Nile red; PAH: Poly (allylamine hydro- chloride); SOSG: Singlet oxygen sensor green; MTT: 3-(4, 5-Dimethylthiazol- 2-yl)-2, 5-diphenyl tetrazolium bromide; DAPI: 4’, 6-Diamidino-2-phenylindole; NAC: N-acetylcysteine; DCFDA: 2′ 7’-Dichlorofluorescin diacetate; EDS: Energy dispersive X-Ray spectroscopy; CLSM: Confocal laser scanning microscopy. f Next, the tumor tissues were extracted for a series of characterizations. Based on the tumor weight evalua- tions (Fig. 5D), it was clearly seen that PTFCG@MH plus laser achieved the best efficacy, in consistent with the in vivo measurement. We also explored the patho- logical changes by H&E staining (Fig. 5E), in which the tumor with PTFCG@MH plus laser treatment displayed widened interstitial space, nuclear condensation, and large vacuoles, indicating the highest level of tumor apoptosis. We also evaluated the key pathological fea- ture of tumor hypoxia by immunofluorescence staining of HIF-1α. The solid tumor showed a relatively high-level HIF-1α expression due to its hypoxia microenvironment (Fig. 5F), and the immunofluorescence was even brighter after PTFCG treatment (with laser) because of the oxy- gen consumption by GOx catalysis and PDT. For com- parison, the tumor hypoxia was effectively relieved after PTFCG@MH (plus laser) treatment, due to oxygenation by MnO2. From quantified results, the HIF-1α level was decreased ~ 70% after PTFCG@MH treatment (Fig. 5G). Therefore, the PTFCG@MH could effectively modulate tumor hypoxia microenvironment and provide O2 sub- strate to improve PDT treatment outcome. Availability of data and materials All data generated or analysed during this study are included in this published article. Acknowledgements Not applicable. Acknowledgements Not applicable. Funding g This work was supported by Innovation-Driven Project of Central South Uni- versity (No. 20170030010004), National Natural Science Foundation of China (No. 21804144, U1903125, 82073799), and Hunan Provincial Clinical Medical Technology Innovation Guidance Project (2020SK50501). Finally, the biosafety of the nanosystem was examined. No obvious decrease of body weight was observed over the period of treatments (Additional file 1: Figure S9), and the H&E staining showed no pathological change of the major organs after treatments (Additional file 1: Fig- ure S10). These results indicate the biocompatibility of the nanosystem for in vivo applications. Authors’ contributions PL, JD, YL and WZ designed and conceptualized this study. PL, YZ, SH and XS performed the in vitro and in vivo experiments. PL, YY, YP QL and SH drafted the manuscript. All authors read and approved the final manuscript. In vivo performance of the nano‑reactor The laser was performed at 24 h after injec- tion. The therapeutic efficacy was monitored by measur- ing the tumor size every other day (Fig. 5C). Compared with PBS control, free Ce6 only showed marginal tumor growth inhibition, mainly due to its rapid clearance from mouse body with minimal accumulation into tumor tis- sue (Fig. 5A). While the nanoparticles could facilitate tumor targeting delivery of Ce6, the efficacy of PTFCG plus laser irradiation was still poor, due to the multiple resistance mechanisms of tumor against PDT therapy. For PTFCG@MH, by contrast, the growth of tumors was obviously suppressed with a strong growth-inhibitory activity (~ 76%). Since we only performed two dosages and the treatment was stopped at day 4, a slight tumor recurrence was observed at day 6. However, the overall tumor growth was significantly suppressed for PTFCG@ MH plus laser as compared with other treatment groups, demonstrating the superiority of the nanosystem for tumor therapy. Note that without laser irradiation, the therapeutic effect of PTFCG@MH was also rather lim- ited, verifying that the anti-tumor activity was mainly originated from the PDT effect. and day 4). The laser was performed at 24 h after injec- tion. The therapeutic efficacy was monitored by measur- ing the tumor size every other day (Fig. 5C). Compared with PBS control, free Ce6 only showed marginal tumor growth inhibition, mainly due to its rapid clearance from mouse body with minimal accumulation into tumor tis- sue (Fig. 5A). While the nanoparticles could facilitate tumor targeting delivery of Ce6, the efficacy of PTFCG plus laser irradiation was still poor, due to the multiple resistance mechanisms of tumor against PDT therapy. For PTFCG@MH, by contrast, the growth of tumors was obviously suppressed with a strong growth-inhibitory activity (~ 76%). Since we only performed two dosages and the treatment was stopped at day 4, a slight tumor recurrence was observed at day 6. However, the overall tumor growth was significantly suppressed for PTFCG@ MH plus laser as compared with other treatment groups, demonstrating the superiority of the nanosystem for tumor therapy. Note that without laser irradiation, the therapeutic effect of PTFCG@MH was also rather lim- ited, verifying that the anti-tumor activity was mainly originated from the PDT effect. Conclusions In conclusion, an intelligent cyclic nano-reactor, PTFCG@MH, was fabricated for enhanced PDT against solid tumor. The nanostructure was well-characterized, and the cyclic reactions were explicitly demonstrated in solution, including GOx-catalyzed glucose consump- tion, MnO2-mediated oxygenation and GSH depletion. The surface HA modification endowed the nano-reactor with improved colloidal stability and active targetability to facilitate its accumulation into tumor after intrave- nous injection. After being delivered into tumor cells, PTFCG@MH boosted the PDT efficacy via simultaneous ATP/GSH suppression and self-oxygen supply, result- ing in efficient tumor growth inhibition with no obvious side-effects. This cascade nano-reactor would promote the development of multifunctional nanoplatforms for improved cancer treatment by modulating unfavorable cancer microenvironment. In vivo performance of the nano‑reactor Finally, in vivo behavior of the nano-reactor was explored by using MDA-MB-231 tumor-bearing mice. By virtue of the intrinsic fluorescence of Ce6, the bio-distribution was visualized using a living imaging system. PTFCG@MH or free Ce6 was intravenously injected when the tumor volume reached ~ 100 mm3. At 1 h post-injection, Ce6 showed red fluorescence throughout the body (Fig. 5A), indicating non-specific distribution, while most of the fluorescence was cleared after 24 h, with major accumu- lation in liver. For comparison, the PTFCG@MH dis- played much weaker fluorescence at 1 h post-injection f To evaluate the in vivo tumor ablation activ- ity, the tumor bearing mice were randomly divided into five groups (n = 5), each intravenous injec- tion of PBS, Ce6 + Laser, PTFCG + Laser, PTFCG@ MH, and PTFCG@MH + Laser, respectively (100 μL, [Ce6] = 2.5 mg/kg), with totally two injections (day 0 Fig. 5 A In vivo (1 h vs 24 h post-injection) and ex vivo (24 h post-injection) fluorescent imaging of MDA-MB-231 tumor-bearing mice treated with free Ce6 and PTFCG@MH. B The quantified fluorescence intensity in major organs and tumor tissue at 24 h post injection. C Kinetics of tumor growth inhibition with various treatments. D Tumor mass and tumor images (inset) of the extracted tumor tissues after different treatments. E H&E staining and F HIF-1α immunofluorescence staining of the tumor tissues with different treatments. G The quantified HIF-1α level after different treatments. P < 0.01, *P < 0.001. In each panel, G1: PBS; G2: Ce6 + Laser; G3: PTFCG + Laser; G4: PTFCG@MH; G5: PTFCG@MH + Laser Fig. 5 A In vivo (1 h vs 24 h post-injection) and ex vivo (24 h post-injection) fluorescent imaging of MDA-MB-231 tumor-bearing mice treated with free Ce6 and PTFCG@MH. B The quantified fluorescence intensity in major organs and tumor tissue at 24 h post injection. C Kinetics of tumor growth inhibition with various treatments. D Tumor mass and tumor images (inset) of the extracted tumor tissues after different treatments. E H&E staining and F HIF-1α immunofluorescence staining of the tumor tissues with different treatments. G The quantified HIF-1α level after different treatments. P < 0.01, ***P < 0.001. In each panel, G1: PBS; G2: Ce6 + Laser; G3: PTFCG + Laser; G4: PTFCG@MH; G5: PTFCG@MH + Laser Liu et al. J Nanobiotechnol (2021) 19:149 Liu et al. J Nanobiotechnol (2021) 19:149 Page 11 of 13 and day 4). Ethics approval and consent to participate No applicable. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12951-021-00893-6. Additional file 1. Additional figures. Additional file 1. Additional figures. References Yu WQ, He XQ, Yang ZH, Yang XT, Xiao W, Liu R, Xie R, Qin L, Gao HL. Sequentially responsive biomimetic nanoparticles with optimal size in combination with checkpoint blockade for cascade synergetic treatment of breast cancer and lung metastasis. Biomaterials. 2019;217:119309. 6. Yu WQ, He XQ, Yang ZH, Yang XT, Xiao W, Liu R, Xie R, Qin L, Gao HL. Sequentially responsive biomimetic nanoparticles with optimal size in combination with checkpoint blockade for cascade synergetic treatment of breast cancer and lung metastasis. Biomaterials. 2019;217:119309. 28. Lin L-S, Song J, Song L, Ke K, Liu Y, Zhou Z, Shen Z, Li J, Yang Z, Tang W, Niu G, Yang H-H, Chen X. Simultaneous fenton-like ion delivery and glutathione depletion by MnO2-based nanoagent to enhance chemody- namic therapy. Angew Chem Int Edit. 2018;57:4902–6. 7. Akhavan O, Ghaderi E, Aghayee S, Fereydooni Y, Talebi A. The use of a glucose-reduced graphene oxide suspension for photothermal cancer therapy. J Mater Chem A. 2012;22:13773–81. 7. Akhavan O, Ghaderi E, Aghayee S, Fereydooni Y, Talebi A. The use of a glucose-reduced graphene oxide suspension for photothermal cancer therapy. J Mater Chem A. 2012;22:13773–81. 29. Dong Z, Feng L, Chao Y, Hao Y, Chen M, Gong F, Han X, Zhang R, Cheng L, Liu Z. Amplification of tumor oxidative stresses with liposomal fenton catalyst and glutathione inhibitor for enhanced cancer chemotherapy and radiotherapy. Nano Lett. 2019;19:805–15. 8. Mahmoudi K, Garvey KL, Bouras A, Cramer G, Stepp H, Raj JGJ, Bozec D, Busch TM, Hadjipanayis CG. 5-aminolevulinic acid photodynamic therapy for the treatment of high-grade gliomas. J Neuro-Oncol. 2019;141:595–607. 30. Chen Z, Shi T, Zhang L, Zhu P, Deng M, Huang C, Hu T, Jiang L, Li J. Mammalian drug efflux transporters of the ATP binding cassette (ABC) family in multidrug resistance: A review of the past decade. Cancer Lett. 2016;370:153–64. 9. Alsaab HO, Alghamdi MS, Alotaibi AS, Alzhrani R, Alwuthaynani F, Altho- baiti YS, Almalki AH, Sau S, Iyer AK. Progress in clinical trials of photody- namic therapy for solid tumors and the role of nanomedicine. Cancers. 2020;12:2793. 31. van Attikum H, Gasser SM. ATP-dependent chromatin remodeling and DNA double-strand break repair. Cell Cycle. 2005;4:1011–4. 10. Aniogo EC, George BPA, Abrahamse H. The role of photodynamic therapy on multidrug resistant breast cancer. Cancer Cell Int. 2019;19:91. 32. Liu T, Liu W, Zhang M, Yu W, Gao F, Li C, Wang S-B, Feng J, Zhang X-Z. References Ferrous-supply-regeneration nanoengineering for cancer-cell-specific ferroptosis in combination with imaging-guided photodynamic therapy. ACS Nano. 2018;12:12181–92. 11. Liang C, Zhang XL, Yang MS, Wang WJ, Chen P, Dong XC. Remod- eling tumor microenvironment by multifunctional nanoassem- blies for enhanced photodynamic cancer therapy. ACS Mater Lett. 2020;2:1268–86. 33. Liu P, Liu X, Cheng Y, Zhong S, Shi X, Wang S, Liu M, Ding J, Zhou W. Core- shell nanosystems for self-activated drug-gene combinations against triple-negative breast cancer. ACS Appl Mater Inter. 2020;12:53654–64. 12. Fan YT, Zhou TJ, Cui PF, He YJ, Chang X, Xing L, Jiang HL. Modulation of intracellular oxygen pressure by dual-drug nanoparticles to enhance photodynamic therapy. Adv Funct Mater. 2019;29:1806708. 34. Liu P, Shi X, Zhong S, Peng Y, Qi Y, Ding J, Zhou W. Metal-phenolic networks for cancer theranostics. Biomater Sci. 2021. https://doi.org/10. 1039/d1030bm02064h. 13. Dang J, He H, Chen D, Yin L. Manipulating tumor hypoxia toward enhanced photodynamic therapy (PDT). Biomater Sci. 2017;5:1500–11. 14. Cheng X, He L, Xu J, Fang Q, Yang L, Xue Y, Wang X, Tang R. Oxygen- producing catalase-based prodrug nanoparticles overcoming resistance in hypoxia-mediated chemo-photodynamic therapy. Acta Biomater. 2020;112:234–49. 35. Wu L, Zhang J, Watanabe W. Physical and chemical stability of drug nano- particles. Adv Drug Del Rev. 2011;63:456–69. 36. Hu C, Cun X, Ruan S, Liu R, Xiao W, Yang X, Yang Y, Yang C, Gao H. Enzyme- triggered size shrink and laser-enhanced NO release nanoparticles for deep tumor penetration and combination therapy. Biomaterials. 2018;168:64–75. 15. Liu P, Xie X, Shi X, Peng Y, Ding J, Zhou W. Oxygen-self-supplying and HIF- 1α inhibiting core-shell nano-system for hypoxia-resistant photodynamic therapy. ACS Appl Mater Inter. 2019;11:48261–70. 37. Stone JR, Yang S. Hydrogen peroxide: a signaling messenger. Antioxid Redox Sign. 2006;8:243–70. 16. Zeng W, Liu P, Pan W, Singh SR, Wei Y. Hypoxia and hypoxia inducible fac- tors in tumor metabolism. Cancer Lett. 2015;356:263–7. 38. Ding B, Shao S, Jiang F, Dang P, Sun C, Huang S, Ma Pa, Jin D, Al Kheraif AA, Lin J. MnO2-disguised upconversion hybrid nanocomposite: an ideal architecture for tumor microenvironment-triggered ucl/mr bioimaging and enhanced chemodynamic therapy. Chem Mater. 2019;31:2651–60. 38. Ding B, Shao S, Jiang F, Dang P, Sun C, Huang S, Ma Pa, Jin D, Al Kheraif AA, Lin J. MnO2-disguised upconversion hybrid nanocomposite: an ideal architecture for tumor microenvironment-triggered ucl/mr bioimaging and enhanced chemodynamic therapy. Chem Mater. 2019;31:2651–60. 17. Competing interest The authors declare no conflict of interest. The authors declare no conflict of interest. Consent for publication 18. Fuchs-Tarlovsky V. Role of antioxidants in cancer therapy. Nutrition. 2013;29:15–21. All authors agree to be published. 19. Chen G, Yang YY, Xu Q, Ling MJ, Lin HM, Ma W, Sun R, Xu YC, Liu XQ, Li N, Yu ZQ, Yu M. Self-amplification of tumor oxidative stress with degradable metallic complexes for synergistic cascade tumor therapy. Nano Lett. 2020;20:8141–50. Received: 10 February 2021 Accepted: 11 May 2021 Received: 10 February 2021 Accepted: 11 May 2021 22. Huo D, Jiang XQ, Hu Y. Recent advances in nanostrategies capable of overcoming biological barriers for tumor management. Adv Mater. 2020;32:1904337. 23. Han H, Hou Y, Chen X, Zhang P, Kang M, Jin Q, Ji J, Gao M. Metformin- induced stromal depletion to enhance the penetration of gemcitabine- loaded magnetic nanoparticles for pancreatic cancer targeted therapy. J Am Chem Soc. 2020;142:4944–54. References 1. Dolmans D, Fukumura D, Jain RK. Photodynamic therapy for cancer. Nat Rev Cancer. 2003;3:380–7. 1. Dolmans D, Fukumura D, Jain RK. Photodynamic therapy for cancer. Nat Rev Cancer. 2003;3:380–7. 24. Cheng YH, Cheng H, Jiang CX, Qiu XF, Wang KK, Huan W, Yuan A, Wu JH, Hu YQ. Perfluorocarbon nanoparticles enhance reactive oxygen levels and tumour growth inhibition in photodynamic therapy. Nat Commun. 2015;6:1–8. 2. Li X, Kwon N, Guo T, Liu Z, Yoon J. Innovative strategies for hypoxic-tumor photodynamic therapy. Angew Chem Int Edit. 2018;57:11522–31. 2. Li X, Kwon N, Guo T, Liu Z, Yoon J. Innovative strategies for hypoxic-tumor photodynamic therapy. Angew Chem Int Edit. 2018;57:11522–31. 3. Xu J, Yu S, Wang X, Qian Y, Wu W, Zhang S, Zheng B, Wei G, Gao S, Cao Z, Fu W, Xiao Z, Lu W. High affinity of chlorin e6 to immunoglobulin g for lntraoperative fluorescence image-guided cancer photodynamic and checkpoint blockade therapy. ACS Nano. 2019;13:10242–60. 3. Xu J, Yu S, Wang X, Qian Y, Wu W, Zhang S, Zheng B, Wei G, Gao S, Cao Z, Fu W, Xiao Z, Lu W. High affinity of chlorin e6 to immunoglobulin g for lntraoperative fluorescence image-guided cancer photodynamic and checkpoint blockade therapy. ACS Nano. 2019;13:10242–60. 25. Liu P, Xie X, Liu M, Hu S, Ding J, Zhou W. A smart MnO2-doped graphene oxide nanosheet for enhanced chemo-photodynamic combinatorial therapy via simultaneous oxygenation and glutathione depletion. Acta Pharm Sin B. 2021;11:823–34. y 4. Fan W, Huang P, Chen X. Overcoming the Achilles’ heel of photodynamic therapy. Nat Rev Cancer. 2016;45:6488–519. 26. Chen X, Liu Y, Wen Y, Yu Q, Liu J, Zhao Y, Liu J, Ye G. A photothermal- triggered nitric oxide nanogenerator combined with siRNA for precise therapy of osteoarthritis by suppressing macrophage inftammation. Nanoscale. 2019;11:6693–709. 5. Wang T, Wang D, Yu H, Wang M, Liu J, Feng B, Zhou F, Yin Q, Zhang Z, Huang Y, Li Y. Intracellularly acid-switchable multifunctional micelles for combinational photo/chemotherapy of the drug-resistant tumor. ACS Nano. 2016;10:3496–508. 5. Wang T, Wang D, Yu H, Wang M, Liu J, Feng B, Zhou F, Yin Q, Zhang Z, Huang Y, Li Y. Intracellularly acid-switchable multifunctional micelles for combinational photo/chemotherapy of the drug-resistant tumor. ACS Nano. 2016;10:3496–508. 27. Zhang Y-H, Qiu W-X, Zhang M, Zhang L, Zhang X-Z. MnO2 motor: a prospective cancer-starving therapy promoter. ACS Appl Mater Inter. 2018;10:15030–9. 6. Consent for publication All authors agree to be published. Consent for publication All authors agree to be published. Declarations Ethics approval and consent to participate No applicable. Liu et al. J Nanobiotechnol (2021) 19:149 Page 12 of 13 Page 12 of 13 Liu et al. J Nanobiotechnol (2021) 19:149 Liu et al. J Nanobiotechnol (2021) 19:149 Author details 1 20. Thomas AP, Lee AJ, Palanikumar L, Jana B, Kim K, Kim S, Ok H, Seol J, Kim D, Kang BH, Ryu JH. Mitochondrial heat shock protein-guided photody- namic therapy. Chem Commun. 2019;55:12631–4. 1 Xiangya School of Pharmaceutical Sciences, Central South University, Chang- sha 410013, Hunan, China. 2 Department of Pediatric Surgery, Hunan Children’s Hospital, Changsha 410004, Hunan, China. 3 Department of General Surgery, Peking Union Medical College Hospital, Beijing 100730, China. 21. Zhao L, Li J, Su Y, Yang L, Chen L, Qiang L, Wang Y, Xiang H, Tham HP, Peng J, Zhao Y. MTH1 inhibitor amplifies the lethality of reactive oxygen species to tumor in photodynamic therapy. Sci Adv. 2020. https://doi.org/10. 1126/sciadv.aaz0575. References Meng X, Deng J, Liu F, Guo T, Liu M, Dai P, Fan A, Wang Z, Zhao Y. Trig- gered all-active metal organic framework: ferroptosis machinery con- tributes to the apoptotic photodynamic antitumor therapy. Nano Lett. 2019;19:7866–76. 17. Meng X, Deng J, Liu F, Guo T, Liu M, Dai P, Fan A, Wang Z, Zhao Y. Trig- gered all-active metal organic framework: ferroptosis machinery con- tributes to the apoptotic photodynamic antitumor therapy. Nano Lett. 2019;19:7866–76. Liu et al. J Nanobiotechnol (2021) 19:149 Page 13 of 13 Page 13 of 13 42. Yang Z, Chen Q, Chen J, Dong Z, Zhang R, Liu J, Liu Z. Tumor-pH-respon- sive dissociable albumin-tamoxifen nanocomplexes enabling efficient tumor penetration and hypoxia relief for enhanced cancer photodynamic therapy. Small. 2018;14:1803262. 40. Ren J, Zhang L, Zhang J, Zhang W, Cao Y, Xu Z, Cui H, Kang Y, Xue P. Light- activated oxygen self-supplied starving therapy in near-infrared (NIR) window and adjuvant hyperthermia-induced tumor ablation with an augmented sensitivity. Biomaterials. 2020;234:119771. 39. Wan S-S, Liu M-D, Cheng Q, Cheng H, Zhang X-Z. A mitochondria-driven metabolic sensing nanosystem for oxygen availability and energy block- ade of cancer. Adv Ther. 2020;3:2000019. 41. Wang H, Chao Y, Liu J, Zhu W, Wang G, Xu L, Liu Z. Photosensitizer- crosslinked in-situ polymerization on catalase for tumor hypoxia modula- tion & enhanced photodynamic therapy. Biomaterials. 2018;181:310–7. 40. Ren J, Zhang L, Zhang J, Zhang W, Cao Y, Xu Z, Cui H, Kang Y, Xue P. Light- activated oxygen self-supplied starving therapy in near-infrared (NIR) window and adjuvant hyperthermia-induced tumor ablation with an augmented sensitivity. Biomaterials. 2020;234:119771. 42. Yang Z, Chen Q, Chen J, Dong Z, Zhang R, Liu J, Liu Z. Tumor-pH-respon- sive dissociable albumin-tamoxifen nanocomplexes enabling efficient tumor penetration and hypoxia relief for enhanced cancer photodynamic therapy. Small. 2018;14:1803262. 39. Wan S-S, Liu M-D, Cheng Q, Cheng H, Zhang X-Z. A mitochondria-driven metabolic sensing nanosystem for oxygen availability and energy block- ade of cancer. Adv Ther. 2020;3:2000019. 40. Ren J, Zhang L, Zhang J, Zhang W, Cao Y, Xu Z, Cui H, Kang Y, Xue P. Light- activated oxygen self-supplied starving therapy in near-infrared (NIR) window and adjuvant hyperthermia-induced tumor ablation with an augmented sensitivity. Biomaterials. 2020;234:119771. 41. Wang H, Chao Y, Liu J, Zhu W, Wang G, Xu L, Liu Z. Photosensitizer- crosslinked in-situ polymerization on catalase for tumor hypoxia modula- tion & enhanced photodynamic therapy. Biomaterials. 2018;181:310–7. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. 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https://openalex.org/W4389161575	http://revistascientificas.filo.uba.ar/index.php/matadero/article/download/13668/12099	es		La utopía encubierta: estrategias de localización de la aventura de ciencia ficción en El Eternauta (1957-1959) de Héctor G. Oesterheld y Francisco Solano López	El matadero	2,022	cc-by-sa	10,073	ARTÍCULOS ISSN 0329-9546 (impresa) / ISSN 2683-9687 (en línea) El Matadero 16 (2022): 39-55 http://doi.org/10.34096/em.n16.13668 La utopía encubierta: estrategias de localización de la aventura de ciencia ficción en El Eternauta (1957-1959) de Héctor G. Oesterheld y Francisco Solano López " Soledad Quereilhac Instituto de Historia Argentina y Americana “Dr. Emilio Ravignani”, Facultad de Filosofía y Letras, Universidad de Buenos Aires – Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina solquerei@gmail.com Fecha de recepción: 27/04/2022. Fecha de aceptación: 17/08/2022. Resumen El presente artículo indaga en las razones que llevaron a la historieta argentina El Eternauta, creada por Héctor German Oesterheld y Francisco Solano López, a convertirse en una obra de exitosa recepción durante su publicación original por entregas en Hora Cero Semanal, entre 1957 y 1959, y en las sucesivas reediciones posteriores, hasta convertirse hoy en un “clásico” de la ficción argentina. Se analiza el paso de Oesterheld por la revista Más Allá (1953-1957) y sus comienzos como cuentista, guionista de historietas y divulgador de temas científicos en sellos comerciales y, luego, en su propio sello, Frontera. El perfil profesional del autor fue la condición de emergencia de una forma particular de la historieta, donde se combinaron con eficacia convenciones de la industria cultural y un proyecto creativo individual. Se sostiene la hipótesis de que, en El Eternauta, tras la escena distópica, aflora como subtexto la utopía de una vida argentina con perspectivas de progreso, marcada por la sociabilidad masculina y la armonía de una familia tipo, el manejo de saberes técnico-científicos que recorren el amplio espectro de lo académico, lo fabril y el amateurismo, y los lazos de solidaridad y de filantropía que movilizan las acciones de la resistencia. La distopía permite hacer emerger, así, en posición de riesgo y de amenaza, esa construcción idealizada y afirmativa de la vida en común, de identificables rasgos nacionales. Palabras clave: Historieta argentina, imaginación técnica popular, utopía, ciencia ficción argentina, nacionalismo cultural. 39 ARTÍCULOS La utopía encubierta: estrategias de localización... Soledad Quereilhac The Hidden Utopia: Strategies for Nationalizing the Science Fiction Adventure in El Eternauta (1957-1959) by Héctor G. Oesterheld and Francisco Solano López Abstract This article explores the reasons that led the Argentine comic book El Eternauta, created by Héctor German Oesterheld and Francisco Solano López, to become a success during its original publication in Hora Cero Semanal (1957-1959), as well as in subsequent reprints, until becoming into a “classic” of Argentine fiction. The article analyzes Oesterheld’s work at the magazine Más Allá (1953-1957) and his beginnings as a short story writer, comic book scriptwriter, and disseminator of scientific knowledge in commercial publishing houses and, later, in his own company, Frontera. The author’s professional profile was the emergence condition of a particular form of Argentine comics, where cultural industry conventions and an individual creative project were effectively combined. This article sustains the hypothesis that, in El Eternauta, behind the dystopian scene, the utopia of an Argentinean life with prospects of progress emerges as subtext, defined by male sociability and the harmony of a nuclear family, the handling of technical-scientific knowledge that covers the broad spectrum of academia, industry, and amateurism, and the bonds of solidarity and philanthropy that mobilize the actions of the resistance. Thus, the dystopia allows that idealized and positive representation of the life in common to emerge in a position of risk and threat. Keywords: Argentine Comics, Science and Popular Imagination, Utopia, Argentine Science Fiction, Cultural Nationalism. ¿Por qué la historieta o el folletín ilustrado El Eternauta se ha convertido en un clásico? ¿Por qué caló tan hondo en los años de su publicación original (1957-1959) en la revista Hora Cero Semanal, de la editorial Frontera, creada y dirigida por el propio Héctor Germán Oesterheld? ¿Por qué cada una de sus reediciones siguió interpelando a tantos lectores del siglo XX y del XXI? ¿Qué hizo que Juan Salvo pasara a integrar el panteón de personajes de ficción que saltan al plano de lo real como figuras cuasi históricas, junto con Martín Fierro, Juan Moreira, Mafalda o La Maga? Muchos críticos y críticas han arriesgado valiosas respuestas sobre la perdurabilidad de El Eternauta y si bien todas aportan al diseño del complejo mosaico de causas, este nunca termina de completarse. Busco en este artículo aportar nuevas hipótesis sobre la particular interpelación de esta historieta a lo que parece ser una médula sensible de la cultura argentina, que trasciende varias generaciones en la medida en que se la entiende en términos idiosincráticos, identitarios o de nacionalismo cultural. En esa médula confluyen, por un lado, una imaginación técnica popular (Comastri 2014) de fuerte pregnancia y desarrollo en la década de cincuenta del siglo XX en Argentina, que configura también un tipo de consumo cultural; y por otro, una mirada sumamente positiva y celebratoria, no exenta de componentes utópicos, de un conjunto de prácticas, saberes y valores que integran ese universo llamado “costumbres argentinas”, y que toca particularmente a las clases medias y bajas trabajadoras. Haciendo uso de la distopía como recurso reconocible de la ciencia ficción, Oesterheld y Francisco Solano López logran diseñar a la vez, como subtexto, la utopía de una vida argentina en paz y tranquila con perspectivas de progreso, marcada por la sociabilidad masculina del truco y las trasnoches de buhardilla, la armonía de una familia “tipo” de esa ISSN 0329-9546 (impresa) / ISSN 2683-9687 (en línea) El Matadero 16 (2022): 39-55 http://doi.org/10.34096/em.n16.13668 40 ARTÍCULOS La utopía encubierta: estrategias de localización... Soledad Quereilhac época, el manejo de saberes técnico-científicos que recorren el amplio espectro de lo académico, lo fabril y el amateurismo, y los lazos de solidaridad y de filantropía que estructuran todas las heroicas acciones posteriores de la resistencia. En la superficie, El Eternauta narra una invasión alienígena que amenaza con una destrucción total; pero en un nivel más profundo del texto, respira una mirada utópica que recrea la vida cotidiana, los códigos comunes, los espacios reconocibles de la zona norte del conurbano bonaerense y de la capital federal, alentada por un imaginario técnico-científico optimista de impronta local, que contrasta notablemente con los temas negativos o catastrofistas que convoca la agenda internacional del armamentismo nuclear o la guerra biológica. En efecto, la distopía no surge por causa de elementos propios de la cultura argentina; no es nuestro mundo, tal como lo conocemos, el que ha engendrado el mal destructor. La invasión es completamente exterior, ajena, de parte de un (literal) “Ellos”. La escena distópica montada permite hacer emerger, entonces, en posición de riesgo y de amenaza, esa construcción idealizada y afirmativa de la vida en común, de identificables rasgos nacionales. Creo que esa operación tiene fuerte peso en la exitosa recepción, a lo largo de las décadas, de la historieta, así como en su decantación en un “clásico” de las ficciones argentinas. Antes que leer la destrucción alienígena de nuestro mundo, en El Eternauta leemos una versión celebratoria de nuestras costumbres y nuestra organización social, en un formato artístico –la historieta– que participa tanto de algunas convenciones del arte “culto” como del arte masivo y popular. Un mosaico de lecturas La mayoría de los estudios críticos sobre El Eternauta no deja de señalar la evidente presencia y uso de las convenciones de la ciencia ficción predominantemente anglosajona y popular de la edad de oro del género, vinculada a la aventura espacial, la predominancia de lo científico-técnico en las tramas y el privilegio de lo masculino en los personajes y en los conflictos. Laura Vázquez señala que “[s]u fórmula estuvo ligada al imaginario de divulgación científica presente en la literatura pulp norteamericana y en las tramas de las películas clase ‘b’ de la década del cincuenta” (2010, 134). Y agrega que “los temas tratados en la narrativa oesterheldiana delatan una ciencia ficción gestada no en la experiencia directa y la innovación tecnocientífica, sino en el consumo de noticias, filmes y narraciones extranjeros” (134). En efecto, si bien es cierto que la lluvia mortal, los gurbos, los Manos y los cascarudos remiten al (ahora) tradicional imaginario sci-fi, creo –como analizaré más adelante– que esa “experiencia directa” con lo tecnocientífico sí se traduce en otro elemento clave de la historieta: el universo de saberes y aun de valores de los personajes. En coincidencia con Vázquez, Martín Greco también pone en diálogo los elementos que van alimentando esta aventura por entregas con productos de consumo masivo, como las historietas, las revistas y el cine clase b de ciencia ficción, todos ellos de origen norteamericano. Y atribuye a la particular combinación saturada de estos tópicos la perdurabilidad de El Eternauta a lo largo de décadas: [P]ara hacer una buena historia alcanza con una sola situación arquetípica. El Eternauta, como Casablanca, desarrolla una trama que incluye colosales dosis de Arquetipos Eternos. Cuando todos los arquetipos irrumpen sin pudor alguno, dice Eco, ‘se alcanzan profundidades homéricas’. […] La perduración de El ISSN 0329-9546 (impresa) / ISSN 2683-9687 (en línea) El Matadero 16 (2022): 39-55 http://doi.org/10.34096/em.n16.13668 41 ARTÍCULOS La utopía encubierta: estrategias de localización... Soledad Quereilhac Eternauta tal vez obedezca a un fenómeno similar, a ese ‘recuento de clichés’, a su narratividad en estado salvaje, a su modo de cruzar múltiples obsesiones de la cultura de masas; no es una historieta, sino muchas; es una antología, un collage intertextual (2013, 13). Greco relativiza que los elementos locales de la historia hayan tenido importante influencia en su exitosa recepción, y elige leer en la saturación de todos los tópicos de la ciencia ficción de los años 50 –muy reconocibles, también, en los posters coloreados de las películas–, la clave del éxito. En dirección opuesta, Fernando García y Hernán Ostuni sostienen que el gran mérito de Oesterheld y Solano López es, justamente, haber trasladado a “nuestra geografía y nuestras costumbres el concepto de aventura anglosajona por excelencia” (2002, 127). Y agregan: “El Eternauta no es una historieta argentina sólo por estar realizada en el país. Hay un lenguaje, un tiempo narrativo, una concepción física e ideológica de nuestro rol en el mundo que la ubican definitivamente en estas tierras” (127). Este aspecto, sumado al pasaje de la supervivencia a la resistencia (al que los autores atribuyen virtudes proféticas sobre la militancia de los años setenta) y al planteo de “una alianza de clases para la solución de los problemas nacionales” (129), trazan las claves de su eficacia narrativa y su cimentación en un clásico. También Silvia Kurlat Ares elige la variable de la anticipación en su lectura (“el ‘nosotros’ que puebla las páginas del cómic preanuncia cómo y quiénes deberán hacerse cargo de la lucha por la liberación nacional”) y pondera los valores de la resistencia, la lealtad y la solidaridad como los que marcan el carácter de la aventura (2018, 74-75). Por lo general, quienes atribuyen virtudes anticipatorias a la historieta tienden a sobreimprimir momentos y ediciones diferentes: el viraje más claramente político y aun pedagógico de El Eternauta II, publicado en Skorpio a partir de diciembre de 1976, no está presente en El Eternauta de Hora Cero Semanal ni en su primera reedición en tres tomos de 1962 en la revista El Eternauta. También trazan ciertas homologaciones entre diferentes momentos de la trayectoria de Oesterheld y su vínculo con la política en general y la militancia en particular; es importante notar que en los años cincuenta, Oesterheld no adhería al peronismo, se reconocía liberal y su contacto con la militancia joven del peronismo se da mucho más tarde, fundamentalmente a través de la actividad de sus hijas en grupos católicos comprometidos (Nicolini y Beltrami 2016). Con frecuencia, salvo en el caso de Greco, las lecturas sobre la historieta sostienen que tanto la incorporación de los tópicos de ciencia ficción de la edad de oro como las estrategias de localización de la aventura constituyen –en su misma combinación– elementos estructurantes de su eficacia, pero en el análisis de este último punto tiende a primar más una concepción meramente espacial, cuando no topográfica y referencial, para dar cuenta del elemento argentino del texto: las imágenes de la cancha de River, de la plaza de los dos Congresos o las barrancas de Belgrano, los nombres visibles de las calles, las pintadas “Vote Frondizi”, el recuerdo de las protestas en Av. Callao por “la laica o la libre”, los carteles de YPF, entre otras referencias y marcas espaciales. Resultan más acertadas, por el contrario, observaciones como las de Laura Vázquez y Juan Sasturain que trascienden la mera noción de escenario, y enfatizan en el tipo de imaginario cultural y en cierta dimensión ideológica, históricamente fechada, que la historieta incorpora como parte del mundo de sus protagonistas y que, por cierto, estructura el tipo de aventura que ellos llevarán a cabo. La experiencia “pequeñoburguesa de un hombre común y padre de familia” se ensambla con materiales ideológicos como “el culto al progreso, la mística de la industrialización y el nacionalismo tecnológico”, cuyas contracaras, sobre todo a nivel internacional, eran “la protesta antinuclear, el pánico a la polución atómica y la conmoción por la escalada armamentista” (Vázquez 2010, 135). Esos materiales ISSN 0329-9546 (impresa) / ISSN 2683-9687 (en línea) El Matadero 16 (2022): 39-55 http://doi.org/10.34096/em.n16.13668 42 ARTÍCULOS La utopía encubierta: estrategias de localización... Soledad Quereilhac ideológicos se habían consolidado y expandido durante el gobierno peronista de 1946-1955, y continuaron vigentes durante el gobierno frondizista de 1958-1962. Para Sasturain, El Eternauta de 1957-59 es un texto que respira el clima ideológico y la propuesta social del desarrollismo que, con Frondizi, se propone como salida para los sectores medios y el país todo. La alianza de clases, el reconocimiento del papel fundamental del sector obrero, la presencia necesaria de los militares y la incorporación de los intelectuales son el marco en que el protagonista –¿qué otra cosa podía ser sino un pequeño industrial nacional? – sale de su casa a cumplir un deber histórico, un desafío (2010, 175). A estos componentes, Sasturain agrega también el elemento ficcional de la aventura, a la que se arrojan hombres y mujeres comunes que salen definitivamente transformados de esa experiencia. La experiencia de Más Allá El imaginario antes descripto sin dudas estructura el universo de valores y los perfiles de los personajes, pero también –y aquí quizás un elemento poco atendido– el de los lectores y las lectoras contemporáneas de la historieta, al tiempo que configura toda un área de la cultura argentina en la que confluyen el gusto por la divulgación científica y por las ficciones científicas o la ciencia ficción, la preferencia por las ciencias aplicadas, el amateurismo de taller casero y una concepción de “ciencia” como parte de la cultura, incluida la cultura popular. En este sentido, no es para nada menor, en primer lugar, la propia formación de Oesterheld como geólogo y su inicial (y corto) ejercicio de la profesión, primero como becario en YPF y luego en el laboratorio de minería del Banco de Crédito Industrial (Beltrami y Nicolini 2016, 21). En segundo lugar, su pasaje de estos trabajos hacia el oficio de escritor profesional en la industria cultural de las décadas de 1940 y 1950, cuando se incorpora a sellos como Códex y, luego, Abril, este último fundado por el emigrado italiano Cesare Civita. En esos ámbitos comenzó publicando textos de divulgación científica y de a poco también historietas, como la célebre Bull Rocket, en la revista Misterix. Pero lo que termina de sellar un contacto realmente cercano y fluido con un emergente grupo de lectores afines, tanto a lo técnico-científico como a la ficción y a la aventura, es su paso por la Más Allá, cuyos 48 números se publicaron entre junio de 1953 y junio de 1957, por editorial Abril. Su título completo rezaba Más Allá de la Ciencia y de la Fantasía. Revista mensual de aventuras apasionantes en el mundo de la magia científica y según algunos críticos, que retoman testimonios, Oesterheld pudo haberse desempeñado, durante un período al menos, como su director, además de colaborar con viñetas de divulgación científica y traducir algunos textos (Capanna, 2018; Grondona, 2018; Nicolini y Beltrami, 2016; Abraham, 2022).1 Además, Oesterheld publicó allí dos cuentos, “Inocente 1 En la versión de 2013 de su libro Las revistas argentinas de ciencia ficción, Abraham afirma que Oesterheld fue uno de los directores de la revista, si bien no cita su fuente de información; luego, en la reedición ampliada de su libro, de 2022, se retracta: dice haber sobreinterpretado declaraciones de Solano López en una entrevista de 2006. Allí, el dibujante había dicho que “Oesterheld era, además, medio guía y director, atrás, en bambalinas, de la revista Más Allá […]” (Saltal y Sabini 2006). También Capanna sostiene que Oesterheld fue director, pero lo hace como presunción: “A pesar de que la revista no daba a conocer la nómina de sus responsables presumimos que detrás de ella, por lo menos al comienzo, estuvo Héctor Germán Oesterheld.” (2018, 108). En el libro de Nicolini y Beltrami, los testimonios de Elsa Sánchez dan cuenta de la activa y variada labor de su marido en la revista, pero nunca dice que fuera su director. Lo cierto es que aún queda mucho por investigar en torno al equipo ISSN 0329-9546 (impresa) / ISSN 2683-9687 (en línea) El Matadero 16 (2022): 39-55 http://doi.org/10.34096/em.n16.13668 43 ARTÍCULOS La utopía encubierta: estrategias de localización... Soledad Quereilhac Maquiavelo Reforzado” (nº 29) y “Cuidado con el perro” (nº 3), este último con el pseudónimo de Héctor Sánchez Puyol.2 Más Allá surgió como una franquicia local de la norteamericana Galaxy Science Fiction, aunque sería erróneo reducirla a mero avatar de aquella. Más Allá representó la primera revista argentina dedicada a la ciencia ficción y a la divulgación científica que pudo sostenerse en el tiempo a lo largo de cuatro años sin interrupciones, y que gozó de una amplia comunidad de lectores y lectoras tanto en Argentina como en otros países de habla hispana. Por sus páginas circularon historias de autores centrales de la hard science fiction anglosajona, así como de algunas plumas que terminarían consolidándose como las más destacadas y creativas de un período posterior: el Bradbury de Crónicas marcianas, Theodore Sturgeon, Phillip K. Dick, Kurt Vonnegut, entre otros. Pero Más Allá fue también un ámbito donde algunos escritores argentinos pudieron dar a conocer sus relatos de ciencia ficción y donde también científicos e intelectuales del país encontraron espacio para sus publicaciones y para la labor de divulgación. El matemático Oscar Varsavsky colaboró con algunos cuentos (seudónimo “Abel Asquini”) y “según Boris Spivacow, […] íntimo amigo de Varsavsky, éste era el ‘alma’ del equipo, y respondía las cartas de lectores” (Grondona 2018). Pablo Capanna, pionero y lúcido estudioso de la ciencia ficción, fue otro colaborador junto con otras firmas locales como Juan Pedro Edmunds, Ignacio Covarrubias, Adolfo Pérez Zelaschi, Maximiliano Mariotti y Claudio Paz. Como dijimos, el contenido de la revista no se acotaba a la literatura; se dedicaba un importante espacio también a la divulgación científica de calidad. Las firmas de científicos extranjeros como Willy Ley, Kenneth Heuer y Wernher von Braun convivían con la del físico argentino José Westerkamp. Según Capanna, Westerkamp se encargaba también de diseñar el “Espaciotest” (cuestionario que evaluaba el conocimiento de los lectores sobre el espacio) y de responder las preguntas científicas del público en la sección fija para tal fin. También señala que un joven Mario Bunge se encargó durante un tiempo de redactar esas respuestas (Capanna 2007, 266; 2018, 110). A este lote de firmas se sumaban finalmente los ilustraores, tanto argentinos como extranjeros. El asiduo y variado intercambio que Más Allá propuso a sus lectores y lectoras fue ciertamente asombroso, y constituyó uno de los grandes logros de la publicación. La revista preguntaba a su público cuál había sido su narración favorita; armaba encuestas para conocer con precisión cómo se componía su público, en base a variables de género, edad, oficios, estudios; proponía un constante intercambio epistolar que a partir del número 17 se dividió en las seccione fijas: “Proyectiles dirigidos” y “Preguntas científicas”, en las que convivían dudas básicas con complejos planteos. Estos intercambios constituyeron un auténtico espacio de formación de un lectorado y una instancia de reflexión sobre el género de la fantasía científica. Es importante recordar que, en muchos editoriales, se ponderaba el valor de la literatura de anticipación con fundamento científico; y en las cartas de lectores se devolvía una mirada que adhería efectivamente a esa valoración.3 que intervino en la producción de Más Allá. 2 La revista presentó a Oesterheld, a propósito de su primera colaboración, como un escritor nacido gracias al incentivo de Más Allá. La sentencia no era del todo cierta, ya que Oesterheld escribía cuentos desde hace años (nº 3, agosto de 1953, 3). 3 “Señor director: El editorial de cada número sobrepasa en calidad y contenido a los anteriores. Es un magnífico compendio del principal fin de la F. C. [ficción científica]: hacernos capaces de manejar y dominar ideas y situaciones nuevas que a veces llegan a asustarnos, para tratarlas correctamente cuando nos enfrentamos con ellas. Omar González (La Plata)” (diciembre 1955, nº 31, 112). ISSN 0329-9546 (impresa) / ISSN 2683-9687 (en línea) El Matadero 16 (2022): 39-55 http://doi.org/10.34096/em.n16.13668 44 ARTÍCULOS La utopía encubierta: estrategias de localización... Soledad Quereilhac Más Allá integró un importante capítulo de esa historia de la imaginación científica y técnica que venía avanzando en la cultura argentina desde el último cuarto del siglo XIX. Fue la pariente futura de los personajes inventores de Roberto Arlt, de las fantasías científicas y las aventuras agrícola-tecnológicas de Horacio Quiroga, de las fusiones entre ocultismo y ciencia de Leopoldo Lugones (Quereilhac 2016), de las fantasías interplanetarias o evolucionistas de Eduardo L. Holmberg (Gasparini 2012), de los sujetos anónimos que multiplicaron las patentes de invención en las décadas de 1920 y 1930 (Sarlo 1992). Integró el espectro de la imaginación técnica popular de la era atómica, del Pulqui II y del affaire Ritcher (Comastri 2014, 2018), de las primeras revistas de ciencia ficción argentinas (Abraham 2013/2022), de proyectos de divulgación como Mundo Atómico (1950-1955). Fue la revista que escribió para todos aquellos lectores que se parecían mucho a los futuros protagonistas de El Eternauta. Me interesa, entonces, destacar el paso de Oesterheld por Más Allá, en años previos a su escritura de El Eternauta, pero contemporáneos a la de otras historietas, porque fue allí donde el autor entró en contacto con un público en base al cual luego modeló los personajes de su universo ficcional. El perfil y tipo de intereses que los lectores y, en menor medida, las lectoras de Más Allá ponían de manifiesto en sus cartas ingresa luego a El Eternauta como materia prima del mundo construido; son esos sujetos los que la historieta arroja a la aventura de la resistencia al invasor, en la cual se ponen en juego los saberes aprendidos en la divulgación científica de las revistas, en las historietas, en las narraciones de ciencia ficción, pero también en el taller, en la fábrica, en los cuarteles y en la universidad. En El Eternauta no todo se resuelve con valor y arrojo; antes bien, es el conocimiento técnico-científico el que permite la supervivencia, y el que provee los elementos para primero entender y luego accionar contra los diferentes ejércitos alienígenas. El narrador, Juan Salvo, insiste una y otra vez en que sin los conocimientos y la mentalidad práctica del profesor universitario de física Favalli, también aficionado a la electrónica, no hubieran podido sobrevivir siquiera a la primera noche: “Reconfortaba tener al lado a un hombre como Favalli, de cerebro práctico, entrenado para resolver problemas técnicos. A eso, para él, se reducía el problema de nuestra supervivencia, a un problema técnico” (Oesterheld y Solano López 1994, 14).4 De hecho, cuando debe decidirse quién saldrá primero al exterior, usando el traje aislante, Salvo insiste en ser él mismo, dado que se pregunta: “Si te pasa algo a ti, ¿cómo las arreglaré yo solo, con Lucas, para salvarlas [a Elena y Martita]? Tú eres el que sabe, Fava, el que puede resolver cualquier problema” (22). En efecto, a lo largo de historieta, Favalli es quien permanentemente razona y hace gala de un conocimiento general (que excede su especialización en física) muy cercano al espectro de tópicos que ocupaban revistas como Más Allá y, de manera algo diferente, en otra revista argentina de la época, Mundo Atómico. Por ejemplo, cuando Favalli, Juan Salvo y Alberto Franco están en la Avenida Callao y divisan uno de esos aparatos detectores de humanos, el primero observa: “Cuánto daría por desarmar 4 Mis citas a la historieta son de una edición de 1994 que, como muchas que incluyen la leyenda “versión original”, reproducen la versión que publicó en 1975 ediciones Record (el sello que también publicó, desde julio de 1974, la revista Skorpio). Esa edición tomó el texto original de las entregas de Hora Cero Semanal de 1957-1959, pero introdujo modificaciones de Oesterheld y de Solano López en la primera y, a veces, la última viñeta; esas eran las que, en las entregas de Hora Cero Semanal, llevaban el título de la historieta y un párrafo de “enganche” para el anuncio de “continuará”. Cabe recordar aquí que una primera reedición en volumen de la historieta completa fue la de Editorial Ramírez, que en 1962 adquiere todos los derechos de Frontera y publica en 3 tomos El Eternauta. A diferencia de ediciones Record, no contrata a Oesterheld y Solano para reemplazar las viñetas de comienzo y fin, y el resultado es, a todas luces, rudimentario. Agradezco a Martín Greco esta información. En otro orden, para una reconstrucción de la recepción de las diferentes versiones de la historieta, véase el trabajo de Sebastián Gago (2014). ISSN 0329-9546 (impresa) / ISSN 2683-9687 (en línea) El Matadero 16 (2022): 39-55 http://doi.org/10.34096/em.n16.13668 45 ARTÍCULOS La utopía encubierta: estrategias de localización... Soledad Quereilhac uno de esos aparatos… posiblemente registre ondas psicológicas. Como, sin darse cuenta, lo hacen los perros cuando descubren en el acto si uno es amigo o enemigo…” (252). Pericia técnica de taller propio (quiere desarmar él mismo el aparato), premisas pseudocientíficas (“ondas psicológicas”), equivalencias con otros fenómenos naturales propios de la divulgación (el comportamiento de los animales domésticos), todo confluye en los saberes del más instruido del equipo, y su máxima autoridad para interpretar y resolver todo lo que acontece. El repertorio de temas, las formas de razonar las relaciones entre temáticas y el alto grado de especulación encuentran correspondencias llamativas con el tipo de preguntas que los lectores y las lectoras de Mas Allá enviaban a la sección “Contestando a los lectores”, luego renombrada “Correspondencia. Respuestas de la sección científica”. Allí leemos: “¿Cómo se explica el gran poder destructor de la bomba atómica y de la bomba de hidrógeno? Joaquín García […] Evita” (abril de 1954, nº 11, 47), “¿Cómo debo hacer para construirme, hacerme construir o procurarme un telescopio de alcance apropiado para observaciones de aficionado? Carlos Turri […] Concepción de Uruguay, Entre Ríos” (abril de 1954, nº 11, 46), “Me gustaría ver descripta la locomoción y sobre todo la aeronáutica del futuro. Juan Cazzola […] Capital” (agosto de 1953, nº 3, 99), “¿Cómo se pueden cruzar los espacios interplanetarios? Néstor Nieva […] Rosario” (agosto de 1953, nº 3, 100). Este compendio que arma diálogo entre curiosidades y saberes expertos reaparece una y otra vez en El Eternauta. Cuando el grupo inicial se encuentra con los militares de Campo de Mayo, les preguntan cómo lograron sobrevivir a la nevada mortal y el cabo Amaya responde: “Uno de los soldados es estudiante de ingeniería. ¡Gracias a él estamos vivos! Y gracias también que en el depósito había de todo conseguimos comunicarnos con luces con las pocas unidades que se salvaron. Y aquí estamos” (72). La formación científico-técnica, otra vez universitaria como en el caso de Favalli, es una virtud altamente positiva en el mundo de Oesterheld y muestra sus resultados concretos ante la amenaza alienígena. Pero no son sólo académicos los instruidos. Cuando Salvo conoce a Alberto Franco, le pregunta cómo se llama y si es estudiante; ante lo cual Franco responde que es obrero fundidor. Luego Salvo agrega: –¿Cómo hiciste para salvarte? ¿Estabas con alguien que conocía de física? – No, estaba yo solo… y yo solo me hice el traje. Usted sabe, yo leo mucho… novelas, historietas del futuro, de ficción científica… –Te felicito. Te fabricaste el mejor traje aislante que he visto (75). Un lector de ciencia ficción, un lector de historietas, maneja un saber práctico aprendido en los libros de distribución masiva de la industria cultural. Como en otras puestas en abismo (la incorporación del guionista en el relato marco), la historieta incorpora a su propio lector y le asigna un desempeño heroico. Esa alta valoración del saber, de aplicación práctica, tiene un prestigio no mediado por las instituciones ni por los ámbitos de consagración. Ese prestigio sigue formas de valoración populares y de clase media que, antes que atender a especificidades, diplomas o grados, ve el conocimiento con perspectiva de progreso y de ascenso social. No es casual que cuando el grupo se encuentra con los obreros de una fábrica de productos químicos, sobrevivientes gracias a los trajes que diseñó el técnico a cargo, Medardo Sosa subraye que “el doctor […] era un hombre leído. Gracias a él estamos con vida” (91, subrayado mío). Esa expresión –“leído”– es similar a que utiliza Elsa Sánchez para referirse al propio Oesterheld: “Era un tipo cultísimo, leía varios idiomas, alemán, inglés, francés, era ISSN 0329-9546 (impresa) / ISSN 2683-9687 (en línea) El Matadero 16 (2022): 39-55 http://doi.org/10.34096/em.n16.13668 46 ARTÍCULOS La utopía encubierta: estrategias de localización... Soledad Quereilhac científico y todo eso le daba la posibilidad de escribir sobre cualquier tema” (Nicolini y Beltrami 2016, 16). Asimismo, cuando Elsa recuerda que Oesterheld no leía historietas antes de empezar a escribirlas, reconstruye el incentivo de Alberto Ongaro, quien ya trabajaba para Civita: “El italiano le dijo que se animara, que con su pluma y su cultura general podía escribir lo que se ocurriera” (24, subrayados míos). No es casual tampoco que originalmente, cuando Oesterheld concibió al personaje de Bull Rocket –quien protagonizaría su primer gran éxito– lo haya pensado como “un sabio, un técnico nuclear con conocimientos de todo tipo” (20). Civita demandó algunas correcciones, pero lo que importa en este dato de creación inicial es la permanente valoración del saber científico-técnico, su inclusión en la historieta como atributo de los personajes y la incorporación de elementos propios y/o altamente valorados de la comunidad de los lectores. Es ciertamente lúcida la interpretación que hace Hernán Comastri del vínculo de Oesterheld con lo que llama el “imaginario técnico popular” de los años cincuenta: […] resulta claro que la escritura de Oesterheld imprimió una perspectiva original en su obra mediante el recurso al saber y la divulgación científica, se incluyeran éstos de forma implícita o explícita. La imaginación técnica popular, por su parte, se hace presente en la obra de Oesterheld desde el momento mismo de la redacción, en tanto toda escritura supone, en su mismo acto, un receptor ideal, un otro con el que busca comunicarse. En este caso, es un público primordialmente juvenil, adulto antes que infantil, apasionado por la ciencia y la tecnología modernas, pero más por sus posibilidades materiales y lo espectacular de sus descubrimientos (lo que podríamos llamar: la aventura) que por lo que suponen como amenaza o por sus posibles consecuencias psicológicas y sociales. A modo de hipótesis, podría incluso plantearse una relación más directa de Oesterheld con la imaginación técnica popular, tal como la misma es expresada por las mismas clases populares, sin las intermediaciones del artista o el escritor (2014, 250). A esta aguda observación de Comastri puede sumarse también un uso de este imaginario técnico-popular como dador de verosimilitud, aun en el marco de una fantasía de invasión alienígena. Se reitera en muchos momentos que el laboratorio casero de Juan Salvo está provisto con un buen arsenal de herramientas, materiales y máquinas pequeñas que garantizan la supervivencia; los cortes de luz de años anteriores a la publicación de El Eternauta incentivaron la tenencia de “un equipo electrógeno que funciona con nafta” (29). La ferretería donde encuentran a Pablo, los cuarteles y las fábricas también proveen las armas, las herramientas y los materiales para el combate, al tiempo que tanto Favalli como Salvo, y en menor medida Franco, utilizan todo el tiempo sus conocimientos para interpretar los acontecimientos y pasar a una acción defensiva. También es verosímil el grado de escasez y cierta improvisación amateur en la forma de encarar la resistencia. Como apunta Laura Vázquez, citando al dibujante Carlos Meglia, si El Eternauta hubiera sido escrito en Estados Unidos seguramente Juan Salvo sería un tipo que trabajaba en la NASA y cuando va a salir por la nevada mortal, seguramente tiene un traje de la NASA […] Juan Salvo, no. Se lo hace con un overol, una máscara de pescar, se pone una capucha, unos guantes de albañil, se pone cinta para que no le entre y él sale a la calle. Y eso es lo que todos haríamos (2010, 139). El punto es importante, porque la historieta descuida otros aspectos de la verosimilitud, pero nunca el vinculado a las prácticas y saberes técnico-científicos. Descuida, por ISSN 0329-9546 (impresa) / ISSN 2683-9687 (en línea) El Matadero 16 (2022): 39-55 http://doi.org/10.34096/em.n16.13668 47 ARTÍCULOS La utopía encubierta: estrategias de localización... Soledad Quereilhac ejemplo, en su pasaje a libro, el año del marco de la enunciación: el Juan Salvo viajero en el tiempo se le presenta al guionista una noche de invierno de 1957, pero termina de contar su historia en 1959; publicada la historieta por entregas a lo largo de dos años en Hora Cero Semanal, se entiende la razón de hacer coincidir el tiempo de la trama con el tiempo de los lectores, para reforzar el efecto perturbador de una fantasía inserta en el mundo cotidiano. Pero en su pase a volumen esa estrategia no tiene razón de ser; es más, rompe la unidad de tiempo del relato. La trama transcurre a lo largo de cinco días corridos; si bien los personajes se alimentan en diferentes momentos, jamás duermen. Tampoco se resuelve dónde queda el Juan Salvo no-viajante, el que en 1957-1959 no sabe nada de la invasión aún, que se producirá recién en 1963. Si bien muchos detalles y omisiones son atribuibles a las condiciones de escritura –entregas semanales a lo largo de dos años, rediseño y alargue de la historia a medida que se verifica el éxito de recepción, entre otros–, también es cierto que lo que nunca falla es el tratamiento verosímil de los saberes y prácticas de los personajes, tan cercanos al universo de sus lectores. Un escritor profesional en busca de su arte Desde sus comienzos con el género de la historieta, Oesterheld demostró un interés particular por escribir sobre personajes argentinos, pero las condiciones de los sellos para los cuales trabajaba y el contexto de gran predominancia de las historietas extranjeras condicionaron –en parte– sus proyectos. A diferencia del escritor que crea en soledad, únicamente comprometido con su proyecto estético y sin ataduras previas a una demanda editorial o comercial (condicionado, en todo caso, por otros factores del campo literario propios de cada época), Oesterheld escribía en el marco de empresas comerciales que debían vender sus productos y llegar al mayor número de lectores. Fue al calor de esta producción en serie y a demanda que Oesterheld fue desarrollando un proyecto creativo propio, en diálogo permanente con la otra parte de la dupla creativa, los dibujantes: Hugo Pratt, Carlos Freixas, Francisco Solano López, Alberto Breccia, entre muchos otros. Un buen ejemplo es cuando, en el marco de su trabajo en Misterix, Oesterheld le propuso a Civita crear una historieta sobre un desertor del ejército argentino, un émulo de Martín Fierro. “Pero Civita, que intervenía en los inicios de cada tira, prefería un Western, aun cuando ese género ya había pasado de moda” (Nicolini y Beltrami 2016, 38). El resultado de esa negociación fue Sargento Kirk, un desertor del ejército norteamericano que actúa en el Lejano Oeste. Es recién cuando deja la editorial Abril y funda su propio sello, Frontera, junto con su hermano, Jorge, que Oesterheld puede dar mayor rienda a sus iniciativas creativas y concebir personajes argentinos. En el marco de un mercado de la historieta de amplísimo desarrollo y crecimiento desde los años cuarenta hasta comienzos de la década del sesenta,5 el guionista tiene claro un objetivo central: crear historietas de calidad. El primer número de Hora Cero Semanal, soporte de El Eternauta junto con otros títulos como Ernie Pike o Randall. The Killer, incluía en su contratapa una especie de manifiesto: “Defendamos la historieta”. Allí, se leía: La historieta es mala cuando se la hace mal. Negarla en conjunto, condenarla en globo, es tan irracional como negar el cine en conjunto porque hay películas malas. O condenar la literatura porque hay libros malos. […] 5 Para un detallado panorama del mercado de la historieta, véase Vázquez 2010, especialmente el capítulo 1, “Años dorados” (25-75). Para un panorama ampliado de la industria de revistas en los años cuarenta y cincuenta, véase la “Introducción” en Panella y Korn (2010, 9-14). ISSN 0329-9546 (impresa) / ISSN 2683-9687 (en línea) El Matadero 16 (2022): 39-55 http://doi.org/10.34096/em.n16.13668 48 ARTÍCULOS La utopía encubierta: estrategias de localización... Soledad Quereilhac Creemos estar en la línea de la historieta buena, entendiendo por buena la historieta fuerte, la historieta que sabe ser a la vez recia y alegre, violenta y humana […]. FRONTERA y HORA CERO son prueba de lo que decimos: los lectores saben ya qué distinto es el material que ofrecemos. Con HORA CERO SEMANAL entendemos habernos superado: estamos seguros de entregar un grupo de historietas de calidad tal como difícilmente se volverá a reunir. Las presentamos con legítimo orgullo de editores, sabiendo que con HORA CERO SEMANAL hacemos un nuevo aporte de valor al grupo de revistas que, dando la espalda al material importado, más barato, pero casi siempre inferior, prefieren abrir sus páginas al material argentino. Ese material que (alguna vez alguien tiene que decirlo) se ha conquistado, sin protecciones ni ayudas, un dignísimo lugar en la primera línea del mejor material que se produce en el mundo (año I, n° 1, 4 de septiembre de 1957, 16). Hay aquí dos puntos importantes: la reivindicación del género historieta como otra forma de arte –equiparable al cine o a la literatura– que no debe ser juzgada por sus peores exponentes ni rebajada a una forma de arte menor; y la valoración de la historieta escrita y dibujada en Argentina, cuya calidad demuestra ser equiparable o superior a los mejores exponentes extranjeros. Es a la luz de estas ideas que se gesta El Eternauta y es en esta historieta en donde Oesterheld logra hacer confluir sus intereses artísticos con las formas que demanda el mercado. Oesterheld siempre quiso ser escritor y sus primeros textos fueron cuentos; su vida laboral lo fue llevando hacia la historieta, pero, de alguna manera, el gran predominio del texto por sobre la imagen en El Eternauta, la perspectiva de una primera persona que relata oralmente los acontecimientos y que se da espacio para las reflexiones del yo (propias de la novela o el cuento), la estructura de relato enmarcado, la puesta en abismo del contexto de escritura (el guionista como personaje que escribirá lo que escuchó narrar a Salvo), la conjunción de temas fijos del género de ciencia ficción con un imaginario y una sensibilidad locales, entre otros rasgos, hablan del hallazgo de una forma particular y personal para la historieta, tan imbuida de elementos literarios como de convenciones gráficas y textuales de su género. El perfil profesional de Oesterheld, como sucedió antes con Horacio Quiroga, como sucedía de manera contemporánea con Rodolfo Walsh, fue el que condicionó, pero a la vez posibilitó, la emergencia de una obra de exitosa recepción entre lectores contemporáneos y posteriores. Es en esa carrera profesional que Oesterheld encontró su voz y su forma artística, que logró llevar la historieta a un registro de fuerte interpelación en su comunidad de lectores. En este sentido, es muy atinado el paralelismo que Sasturain detecta entre las trayectorias y las obras inaugurales de Oesterheld y de Rodolfo Walsh. Dice Sasturain: […] los memorables relatos que han escrito estos autores —desde el periodismo, desde el guion—, esas dos obras capitales y de las más originales de la narrativa argentina de la segunda mitad del siglo XX, El Eternauta y Operación Masacre — ambas de 1957–, se mantienen al margen del sistema literario de su tiempo, no son leídas/reconocidas entonces como literatura debido a varios factores. Uno es el medio soporte no habitual en el que se publican; otro, el circuito de consumo no calificado dentro del que son leídas; tercero, la originalidad (inclasificabilidad) de su forma, ya que no se parecen a nada conocido y no siguen ninguno de los modelos esperables —tradicionales o de vanguardia— en la narrativa de su tiempo en la Argentina y —final y crudamente, como deriva de los factores anteriores— la discutible condición de escritores, del guionista y el periodista que las firmaban. ISSN 0329-9546 (impresa) / ISSN 2683-9687 (en línea) El Matadero 16 (2022): 39-55 http://doi.org/10.34096/em.n16.13668 49 ARTÍCULOS La utopía encubierta: estrategias de localización... Soledad Quereilhac Pero tampoco es que los autores aspiraran, a priori, a eso. Trabajaron, en los dos casos, sobre la marcha, sin saber muy bien a dónde iban y en forma de entregas —una investigación en marcha, un folletín aventurero— porque no estaban ‘escribiendo un libro’ sino contando una historia que se les parecía, solo incitados por la necesidad personal (y funcional) de responder al desafío interior de aventurarse, y a las expectativas de un lector que los esperaba cada vez, cada semana, para saber cómo sigue lo que pasó/lo que pasará. (Sasturain 2010, 29). En esta línea, creo que otra de las claves de la perdurabilidad de El Eternauta es el hallazgo de una voz propia en el marco de un género lleno de condicionamientos. Y cuando hablo de voz propia no estoy pensando en términos de originalidad ni de arte vanguardista, rupturista o de innovación radical. Pienso en realidad en los términos en que Raymond Williams plantea el hallazgo de una forma de arte por parte del artista en tanto integrante de una comunidad y en tanto trasmisor de una experiencia colectiva a través de un lenguaje individual. El gran hallazgo de Oesterheld es esa forma de comunicar no sólo una aventura posible, sino una sensibilidad respecto de lo propio, una mirada sensible y recreativa del mundo conocido que aparece, así, redescubierto. En su libro La larga revolución, Williams pone el foco en la dimensión de la comunicación en el arte y su propuesta siempre me pareció una eficaz herramienta de lectura de la obra y la figura de Oesterheld. Lejos de proponer la existencia de un mensaje a decodificar, o de hablar en términos del esquema emisor-receptor, Williams elabora una concepción más estructural y profunda de la comunicación artística: cuando se logra transmitir un sentido aprehensible por la comunidad a la que pertenece el artista, ya se trate de un arte críptico o escurridizo a la comprensión, ya se trate de formas más convencionales y/o tradicionales. Williams jamás homologa el concepto de arte a sus formas más vanguardistas ni concibe al arte como portador de un “mensaje” cifrado u oculto, sólo revelable por el exégeta culto. Cuando recupera la dimensión comunicativa se refiere a que el artista puede comunicar su experiencia individual en una forma que resuena en lo colectivo. Sin esa comunicación operativa, de la cual participan tanto el artista como el espectador o lector, no puede haber arte. La idea de artista que trabaja Williams en este libro es la que lo presenta como la “voz de su comunidad”; lejos de una concepción individualista del artista y de la actividad creativa en general, Williams concibe al artista como aquel que halla la articulación de una forma y un contenido en los que se organiza una experiencia común, bajo una forma de arte personal, que no existía previamente así creada, pero que interpela un código reconocible. Agrega Williams: En muchas sociedades el arte tuvo la función de encarnar lo que podemos llamar significados comunes de la sociedad. El artista no describe nuevas experiencias, sino que encarna experiencias conocidas. Hay un gran peligro en el supuesto de que el arte solo actúa en las fronteras del conocimiento. Actúa en ellas, sobre todo en sociedades perturbadas y en rápido cambio. Pero también actúa en el centro mismo de las sociedades. A menudo, la sociedad expresa a través del arte su comprensión de ser justamente eso, una sociedad. El artista, en este caso, no es un explorador solitario sino la voz de su comunidad. Aun en nuestra compleja sociedad, algunos artistas parecen cercanos al centro de la experiencia común, mientras que otros están aparentemente en las fronteras, y sería un error suponer que esa diferencia es la existente entre “arte mediocre” y “gran arte” (Williams 2003, 43; subrayados míos). A lo largo de buena parte de su obra, la propuesta de Williams es entender el arte y la actividad cultural como prácticas concretas integradas al mundo social, y no como ISSN 0329-9546 (impresa) / ISSN 2683-9687 (en línea) El Matadero 16 (2022): 39-55 http://doi.org/10.34096/em.n16.13668 50 ARTÍCULOS La utopía encubierta: estrategias de localización... Soledad Quereilhac actividades meramente individuales, como de excepción o aureáticas, concebidas por fuera de toda producción material. En este libro puntual de 1965, piensa la “actividad creativa” como algo propio de lo humano, que a veces se canaliza en lo que llamamos arte. Esa especificidad impide decir que “el arte es un sustituto de otros tipos de comunicación, puesto que cuando es eficaz transmite notoriamente una experiencia que al parecer no es comunicable de otro modo” (46). De esta manera, no sólo lograr evadir el estudio del arte en función de una tajante división entre el gran arte y el arte popular, sino que puede poner el foco en los mecanismos creativos que una sociedad desarrolla en un período determinado. Creo, en función de esta propuesta, que Oesterheld ha logrado dar forma, en El Eternauta, a una experiencia social común en torno a las costumbres, los saberes y las prácticas técnico-científicas, las expectativas de progreso y las pulsiones utópicas nacionales que, a pesar de estar fechadas en los años cincuenta, han perdurado a lo largo de las décadas. Apelando a una invasión extraterrestre y nacionalizando el derecho a la aventura (hasta ese momento, patrimonio exclusivo de los norteamericanos, como dice Sasturain), Oesterheld logra hacer del género historieta la mejor forma de celebrar una Argentina posible: de solidaridad interclase, unida frente a un enemigo imperialista común, en la que académicos, profesionales, obreros industriales, dependientes de comercio y militares –todos hombres, por cierto–6 se defienden del invasor hasta el final, respetando una lógica de grupo y conjurando de cuajo la rivalidad de todos-contra-todos del comienzo de la nevada. “Hace apenas unas horas, los hombres nos cazábamos como fieras: nos asesinábamos apenas nos avistábamos. Cada sobreviviente era un enemigo. Ahora, cuando sabemos que los enemigos son seres extraños a la tierra, nos sentimos todos hermanos. Tenía que ocurrir semejante catástrofe para que los hombres aprendieran lo que no debían ignorar nunca” (91), dice Favalli, poniendo de manifiesto ese viraje hacia la solidaridad, la organización y la unión entre argentinos. También uno de los Manos, en el momento de su muerte, es el responsable de afianzar esa mirada utópica sobre el mundo de los humanos, que la invasión amenaza con exterminar; en la célebre escena de contemplación de una cafetera como obra de arte, el Mano exclama: “¿Se dan cuenta los hombres de todas las maravillas que los rodean? ¿Tienen idea de cuántos mundos habitados hay en el universo y de cuán pocos son los que han florecido en objetos como este? (160) Hacia el final, esa celebración de las “costumbres argentinas” se va proyectando cada vez más hacia la humanidad en general y es con un discurso de fuerte corte 6 El predominio de lo masculino en el mundo de Oesterheld es ciertamente asfixiante leído desde el siglo XXI. Al respecto, llama la atención una encuesta de la revista Más Allá. Ante la verificación de que sólo una de cada diez lectores era una mujer, la revista propuso a su público reflexionar sobre las posibles causas de esta disparidad. Todas las respuestas ofrecen un triste espectáculo de prejuicios machistas, como el siguiente: “Señor Director: A mi juicio, esta desproporción entre lectores y lectoras se debe a que, la elevada y ambiciosa fantasía, la sutil ironía, la formidable realidad y multitud de facetas de Más Allá no coincide con la fantasía, humor, intereses instructivos y rutina intelectual de la generalidad de nuestras mujeres; es un impacto emocional demasiado extraño a sus apetencias comunes, a su limitada realidad y a su modo quizá romántico de pensar, pero carente de esa rara mezcla de ilusión y espíritu científico para ver y juzgar nuestra civilización y sus posibilidades, que nos anima a los lectores de Más Allá. En otras palabras, Más Allá es algo demasiado bueno como para gustarle a las mujeres”. Hugo H. Chumbita, Sta. Rosa, La Pampa. (“Más allá y las mujeres”, febrero de 1957, nº 44, 117). En El Eternauta, el lugar de la esposa de Salvo, Elena, reproduce este desprecio machista de manera implícita: si bien es el objeto de amor absoluto de su esposo (junto con su hija, Martita), Elena es de una inutilidad pasmosa para la supervivencia, añora hacer compras por la Avenida Santa Fe en el medio de una nevada mortal y sus iniciativas se limitan a hacer una rica cena. Con todo, algo de esta fiesta de machismo busca ser enmendado en una historieta autoconclusiva publicada en Hora Cero Extra (nº 1, abril de 1958), en la que Oesterheld narra el tiempo en que Elena y su hija Martita pasan solas en la casa. Allí, dan asilo a un hombre que termina atacándolas, y Elena da muestra de sobrado coraje y entereza. ISSN 0329-9546 (impresa) / ISSN 2683-9687 (en línea) El Matadero 16 (2022): 39-55 http://doi.org/10.34096/em.n16.13668 51 ARTÍCULOS La utopía encubierta: estrategias de localización... Soledad Quereilhac filantrópico, espiritualista y “humanista” (a pesar de apelar a lo interplanetario y por ende a muchas especies) que la historieta cierra la primera etapa del viaje de Salvo. Aislados en la dimensión espaciotemporal “continum 4”, el Mano le dice al futuro Eternauta: Ya me entenderás Juan Salvo. Así como hay entre los hombres, por sobre los sentimientos de familia o de patria, un sentimiento de solidaridad hacia todos los seres humanos, descubrirás que también existe entre todos los seres inteligentes del universo, por más diferentes que sean, sentimientos de solidaridad, un apego a todo lo que sea espíritu, que une a los marcianos con los terrestres, a los ‘trípedos’ de Ruma del quinto planeta de Vega, con los ‘glóbulos’ de Laskaria, la patria de los ‘gurbos’… (347) El vínculo solidario y de igualdad interclase que cohesiona al grupo de sobrevivientes en la Tierra se proyecta, aquí, a una dimensión universal e interplanetaria: el apego al espíritu y el sentimiento de solidaridad es lo que une a todas aquellas formas de vida que se oponen a la destrucción imperialista de los “Ellos”. Hay aquí un claro “ideologema narrativo” (Jameson 1989): la proyección de un ideario y un imaginario utópico sobre los aspectos positivos de una vida cotidiana en común, sobre la serie de prácticas, conocimientos y vínculos que conforman el mundo tal como lo conocemos. Con la distopía invasora como escena de superficie, Oesterheld da cauce, en realidad, a una visión optimista del mundo de su presente, y con ello encauza ficcionalmente uno de los “impulsos utópicos” (Jameson 2005) que atravesó el imaginario de la Argentina industrialista y desarrollista de los años cincuenta. Cabe agregar que no es un dato menor aquí cuán excepcional era en la literatura argentina de hasta ese momento la figura de un héroe colectivo, compuesto por perfiles tan distintos en relación con su origen de clase.7 Sin dudas, esa transversalidad de clases también constituyó una clave de la exitosa recepción, en la medida en que también participa de una pulsión utópica.8 Final A lo largo de los dos años en los que Oesterheld fue publicando las entregas de El Eternauta en Hora Cero Semanal, los lectores y las lectoras accedieron a la gesta progresiva de un futuro clásico de la literatura argentina, si bien su estatuto “literario” 7 Greco señala en su artículo que es un rasgo característico de la ciencia ficción de la edad de oro norteamericana la emergencia de un héroe grupal en las tramas, esto es, “la reunión de individuos heterogéneos para constituir una suerte de personaje colectivo, en el que cada uno aporta según sus saberes y competencias” (2013, 11). Y cita a Sobchack en An introduction to film (1980), para agregar que lo que caracteriza a esta entidad colectiva “es ser abierta, democrática, cooperativa, en oposición a la histeria solitaria, que suele revelarse ineficaz, si no peligrosa, para el orden social” (2013, 11). Claramente El Eternauta sigue este patrón del género, pero resuelve el perfil de ese colectivo con características reconociblemente nacionales. 8 En esa misma década, Cortázar está escribiendo la serie de relatos fantásticos que tienen a la invasión como tópico y sensibilidad social predominante, pero con un sentido totalmente opuesto: “Casa tomada”, publicado originalmente en 1946 en la revista Los Anales de Buenos Aires, y posteriormente incluido en Bestiario (1951), junto con “Ómnibus”. También en “La banda” y “Las ménades”, incluidos en Final del juego (1956). Todos estos relatos despliegan escenas de invasión y/o de coacción de un otro social, de otro que de golpe y sin aviso irrumpe en ámbitos que anteriormente no le pertenecían, que les estaban vedados: una casona en Recoleta, un concierto, un cine de películas de autor, el transporte público. El ideologema de la invasión traduce aquí un malestar de clase evidente, un sentimiento de extrañamiento frente a los espacios conocidos y aun reconocidos como propios, que se ven transformados hasta la irrealidad por la presencia de otro social atemorizante o abyecto. ISSN 0329-9546 (impresa) / ISSN 2683-9687 (en línea) El Matadero 16 (2022): 39-55 http://doi.org/10.34096/em.n16.13668 52 ARTÍCULOS La utopía encubierta: estrategias de localización... Soledad Quereilhac no es el tradicional y aun muchos estudiosos de la historieta como género defenderán su legítimo derecho a ser considerada una forma de arte diferenciada de la literatura y las artes plásticas (García 2010). Para que El Eternauta fuera posible, Oesterheld tuvo que procurarse su propio ámbito de publicación, Frontera. A pesar de que, en el largo plazo, el proyecto, en tanto empresa comercial, no logró perdurar,9 fue el que posibilitó la emergencia de una forma de la historieta nacional que seguía un proyecto artístico de autor: personajes argentinos arrojados a la aventura en el marco de su ciudad y acorde a sus costumbres. Respetuoso de ciertas convenciones del mercado (por ejemplo, el uso del “tú y no del “vos”), Oesterheld pudo hacerles decir “al pelo”, “estoy groggy” o “suerte loca” a sus personajes. Pudo darse el gusto de narrar un largo combate en la cancha de River o en el subte de plaza Italia; o de concebir una invasión alienígena en otro país que no fuera, invariablemente, Estados Unidos. El encabezado de la historieta rezaba un título completo sumamente atractivo: Una cita con el futuro. El Eternauta. Memorias de un navegante del porvenir. Cita a cita, el público de Frontera participó de un ejercicio de imaginación utópica, camuflado tras el motivo distópico superficial de la invasión alienígena. La ideología progresista e industrialista, que llevaba implícitas nociones de soberanía económica, de alianza de clases y de desarrollo técnico-científico, respira por todos los poros de esta fantasía e insufla a toda la historia de una potencia cultural reconocible que trasciende su mero argumento. El Eternauta es ejemplo de cómo lo real histórico atraviesa la literatura y el arte en sus formas más constitutivas, en su dimensión simbólica profunda, independientemente de que esa realidad sea o no nombrada como referencia. Hasta la más fantástica historia sobre alienígenas lleva consigo las marcas discursivas –y por ende ideológicas– de su contemporaneidad. Acaso ese piso de “verdad histórica”, ese feliz hallazgo de una forma artística con una experiencia colectiva y compartida explique parte de la perdurabilidad de la obra. Como Juan Salvo, viajante en el tiempo y narrador, esta “voz de su comunidad” que encarnó Oesterheld sigue interpelando a los lectores de nuestro presente, los habitantes de su futuro. 9 Dice Solano López: “Hay que reconocer que Oesterheld tenía un talento incomparable para escribir guiones de historieta, pero dirigir una editorial es un oficio diferente. Su título universitario era de geólogo y se asoció con su hermano que era ingeniero agrónomo. Un geólogo y un ingeniero agrónomo dirigiendo una editorial, imaginate cómo les fue, les metieron el perro y se fundieron” (Saltal y Sabini 2006, 2). ISSN 0329-9546 (impresa) / ISSN 2683-9687 (en línea) El Matadero 16 (2022): 39-55 http://doi.org/10.34096/em.n16.13668 53 ARTÍCULOS La utopía encubierta: estrategias de localización... Soledad Quereilhac # Bibliografía Revistas » Hora Cero Semanal (1957-1959), disponible en https://ahira.com.ar/revistas/ hora-cero-suplemento-semanal/ » Hora Cero Extra, nº1, abril de 1958 » Más Allá (1953-1957), disponible en https://ahira.com.ar/revistas/mas-alla-dela-ciencia-y-de-la-fantasia/ » Mundo Atómico (1950-1955), disponible en https://ahira.com.ar/revistas/mundoatomico/ Bibliografía » Abraham, Carlos. 2013. “Más Allá (1953-1957)”. En Las revistas argentinas de ciencia ficción, 131-171. Temperley: Tren en movimiento, » Abraham, Carlos. 2022. “Más Allá (1953-1957)”. En Las revistas argentinas de ciencia ficción. Segunda edición, 141-226. Temperley: Tren en movimiento. » Capanna, Pablo. 2007. “La ciencia ficción argentina”. En Ciencia ficción. Utopía y mercado, 263-281. Buenos Aires: Cántaro. » Capanna, Pablo. 2018. “Argentina: la ciencia y la ficción”. El taco en la brea 7 (diciembre–mayo), 101–113. » Comastri, Hernán. 2014. “Bull Rockett, Héctor Germán Oesterheld y la imaginación técnica popular en la Argentina de mediados del siglo XX”. Anuario del Centro de Estudios Históricos “Prof. Carlos S. A. Segreti”, 14: 239–257. » Comastri, Hernán. 2018. “La apuesta por la energía atómica. Guerra Fría, políticas de Estado e imaginación técnica popular en el primer peronismo (19461955). En Saberes desbordados. Historias de diálogos entre conocimientos científicos y sentido común (Argentina, siglos XIX y XX), editado por J. Caravaca, C. Daniel y Mariano Ben Plotkin, 66-90. Buenos Aires: IDES. http://www.saberesdesbordados.com/ » Gago, Sebastián. 2014. “Fragmentos de las ‘memorias del porvenir’. Las lecturas de El Eternauta con el paso de los años”. En Siete intentos de escritura sobre Héctor Oesterheld. Géneros, intertextos y temas de la historieta argentina clásica, de Lucas Barone, 85-95. Córdoba: Universidad Nacional de Córdoba. » García, Santiago. 2010. La novela gráfica. Astiberri Ediciones: Bilbao. » Gasparini, Sandra. 2012. Espectros de la ciencia. Fantasías científicas de la. Argentina del siglo XIX. Buenos Aires: Santiago Arcos Editor. » Greco, Martín. 2013. “Cruces en la cultura de masas. El cine y la historieta de ciencia ficción en la década de 1950”. Actas del III Congreso Internacional “Artes en Cruce”, Departamento de Artes, Facultad de Filosofía y Letras, Universi- ISSN 0329-9546 (impresa) / ISSN 2683-9687 (en línea) El Matadero 16 (2022): 39-55 http://doi.org/10.34096/em.n16.13668 54 ARTÍCULOS La utopía encubierta: estrategias de localización... Soledad Quereilhac dad de Buenos Aires, agosto. » Grondona, Ana. 2018. “Más allá… del desarrollo. Ciencia, fantasía y proyectos nacionales en Oscar Varsavsky”. En Saberes desbordados, editado por J. Caravaca, C. Daniel y Mariano Ben Plotkin, 158-180. Buenos Aires: IDES. http://www. saberesdesbordados.com/ » Kurlat Ares, Silvia. 2018. “Entre El Eternauta y Ciudad: para un desideratum del imaginario de la cultura popular”. En La ilusión persistente. Diálogos entre la ciencia ficción y el campo cultural, 47-138. Pittsburg: Nuevo Siglo. » Nicolini, Fernanda y Beltrami, Alicia. 2016. Los Oesterheld. Buenos Aires: Sudamericana. » Oesterheld, Héctor G y Solano López, Francisco. 1994. El Eternauta. Buenos Aires: Record. » Panella, Claudio y Korn, Guillermo. 2010. Ideas y debates para la nueva Argentina: revistas culturales y políticas del peronismo: 1946-1955. La Plata: Universidad Nacional de La Plata. » Quereilhac, Soledad. 2016. Cuando la ciencia despertaba fantasías. Prensa, literatura y ocultismo en la Argentina de entresiglos. Buenos Aires: Siglo XXI. » Saltal, Marcelo y Sabini, Rafael. 2006. “El retorno de El Eternauta”. El Abasto 80, septiembre. » Sarlo, Beatriz. 1992. La imaginación técnica. Sueños modernos de la cultura argentina. Buenos Aires: Nueva Visión. » Sasturain, Juan. 2010. El Aventurador. Una lectura de Oesterheld. Buenos Aires: Aquilina. » Vazquez, Laura. 2010. El oficio de las viñetas. La industria de la historieta en la Argentina. Buenos Aires: Paidós. » Williams, Raymond (2003). La larga revolución. Buenos Aires: Nueva Visión. ISSN 0329-9546 (impresa) / ISSN 2683-9687 (en línea) El Matadero 16 (2022): 39-55 http://doi.org/10.34096/em.n16.13668 55
https://openalex.org/W2773295173	https://s-space.snu.ac.kr/bitstream/10371/138480/1/40902_2017_Article_138.pdf	English	null	Evaluation of bone healing using rhBMP-2 soaked hydroxyapatite in ridge augmentation: a prospective observational study	Maxillofacial Plastic and Reconstructive Surgery	2,017	cc-by	4,558	RESEARCH Open Access * Correspondence: 54211@snubh.org; kyk0505@snubh.org 1Department of Oral and Maxillofacial Surgery, Section of Dentistry, Seoul National University Bundang Hospital, 300 Gumi-dong, Bundang-gu, Seongnam, Gyunggi-do, South Korea Full list of author information is available at the end of the article © The Author(s). 2017 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. Evaluation of bone healing using rhBMP-2 soaked hydroxyapatite in ridge augmentation: a prospective observational study Hyun-Suk Kim1, Ju-Cheol Park2,3, Pil-Young Yun1 and Young-Kyun Kim1,3 Hyun-Suk Kim1, Ju-Cheol Park2,3, Pil-Young Yun1 and Young-Kyun Kim1,3 Maxillofacial Plastic and Reconstructive Surgery Maxillofacial Plastic and Reconstructive Surgery Maxillofacial Plastic and Reconstructive Surgery Kim et al. Maxillofacial Plastic and Reconstructive Surgery (2017) 39:40 DOI 10.1186/s40902-017-0138-9 * Correspondence: 54211@snubh.org; kyk0505@snubh.org 1Department of Oral and Maxillofacial Surgery, Section of Dentistry, Seoul National University Bundang Hospital, 300 Gumi-dong, Bundang-gu, Seongnam, Gyunggi-do, South Korea Full list of author information is available at the end of the article Abstract Background: The goal of this study is to evaluate complication and effectiveness of alveolar ridge augmentations using a hydroxyapatite-based alloplastic bony substitute with rhBMP-2. Methods: A total of 10 patients (4 males, 6 females; 58.5 ± 8.6 years) participated in this clinical research. Alveolar ridge augmentations were performed in edentulous (4 maxillary posterior, 5 mandibular posterior, and 1 mandibular anterior) regions. Anorganic bovine bone (ABB; Bio-Oss®, Geistlich Pharma AG, Wolhusen, Switzerland) was used as the bone graft material in the control group (n = 5)) while hydroxyapatite-based alloplastic bony substitute with rhBMP-2(HA+rhBMP-2; NOVOSIS®-Dent, CGBio Inc., Seongnam, Korea) was used in the experimental group (n = 5). In order to evaluate relative changes in bone volume and resorption rate of the bone graft material, CBCT radiographs were taken immediately and at 4 months after the bone graft in all subjects. Among the 10 patients, 8 received dental implants in Seoul National University Bundang Hospital, while the others received in local clinics. Bone specimens for further histomorphometric examinations were gained from these 8 patients using trephine burs during the implant placements. Clinical, radiographic, and histomorphometric evaluations were focused because of the small sample size. p p p examinations were gained from these 8 patients using trephine burs during the implant placements. Clinical, radiographic, and histomorphometric evaluations were focused because of the small sample size. Results: When CBCT radiographs were compared between immediately and at 4.07 ± 0.13 months after the bone graft, both alveolar bone widths (ABB 2.52 ± 0.18 mm, HA+rhBMP-2 1.75 ± 0.85 mm) and heights (ABB 1.68 ± 0.17 mm, HA+rhBMP-2 1.57 ± 0.28 mm) increased in the two groups. Resorption rates of transplanted bone graft material in the alveolar bone widths and heights were (ABB 29.7 ± 8.8%, HA+rhBMP-2 31.5 ± 7.4%) and (ABB 39.2 ± 21.8%, HA+rhBMP-2 52.6 ± 6.5%), respectively. Histomorphometrically, ABB group showed bone formation via osteoconduction and HA+rhBMP-2 group via osteoinduction. HA+rhBMP-2 group showed more bone formation around the bone graft materials than the ABB group. Postoperative complications were not found in all subjects. Conclusions: Our study had following conclusions: (1) Ridge augmentations using HA+rhBMP-2 could be clinically useful to supplement implant placements in edentulous regions. (2) Serious postoperative complications related to the graft material did not occur. Keywords: Alveolar ridge augmentation, Bone morphogenetic protein 2, Bone regeneration, Hydroxyapatite © The Author(s). © The Author(s). 2017 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. Abstract 2017 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. Background osteoinductive abilities important for osteogenesis [11]. Jung et al. showed that GBR with combination of the xenograft (Bio-Oss) with rhBMP-2 can enhance the maturation process of bone regeneration [12]. Adequate bone volume is one of the important factors to obtain osseointegration in dental implants. In 1986, Lekholm et al. reported that for the implant success, a minimum of 1 mm or more of the buccal and lingual bone was necessary surrounding the implant surface. Clinicians often encounter patients with deficient vertical or horizontal alveolar bones. Reasons may vary from trauma, periodontal disease, tooth extraction, and to tumor. Under such circumstances, the implant surface may not be entirely covered by the bone, and this could increase the risk of infection, gingival reces- sion, non-esthetic appearance, poor oral hygiene main- tenance, and peri-implantitis [1]. Similar to other growth factors, BMP-2 requires a carrier system that could provide optimal cellular and vascular growth, cellular attachment, and release kinetics [13, 14]. Highly soluble, BMP-2 requires robust scaffolds over long periods that act as drug carrier at the implant site to exert osteoinductive effects [15, 16]. Ideal scaf- folds should control-release growth factors and prevent degradations. Various materials have been proposed, and absorbable collagen sponge (ACS) has been the most documented carrier for rhBMP-2 because of its high binding and retention capacities for the rhBMP-2. According to a study by Hwang, the use of rhBMP-2 with ACS could result in accelerated bone formation compared to conventional bone grafting in postoperative bone defects [17]. However, collagen lacks osteoconduc- tivity as well as structural integrity in transplanted sites. Therefore, calcium phosphates, such as hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP), have been considered as suitable candidates for rhBMP-2 delivery system because of their space-providing properties [18]. The ideal bone graft material should have no immune response and include growth factors that facilitate rapid bone formation and re-vascularization. It should also be able to maintain space for new bone infiltration and readily available in clinics. Autografts are known to be the ideal material for the reconstruction of bone defects. Autografts have osteogen- esis, osteoconduction, and osteoinduction abilities that en- able rapid bone healing without inducting immune responses. They are, however, difficult to obtain in suffi- cient quantities without causing complications in the donor site and a large amount of the transplanted grafts often get absorbed. Background To overcome the problems, other bone substitutes such as allografts, xenografts, and alloplasts have been developed and used. However, allo- grafts and xenografts could be problematic due to the risk of infection and high price. The alloplasts are cheap and have no risk of infection, but they lack osteogenesis and osteoinduction abilities to form viable bone tissue [2]. The goal of this study is to evaluate effectiveness and complication of alveolar ridge augmentations using a hydroxyapatite-based alloplastic bony substitute with rhBMP-2. Methods Patients A total of 10 patients (4 males, 6 females; 58.5 ± 8.6 years) participated in this clinical research. Alveolar ridge augmentations were performed in edentulous (4 maxillary posterior, 5 mandibular posterior, and 1 mandibular anter- ior) regions. Bone morphogenetic protein (BMP), the leading osteoinductive growth factor, has been studied since 1995 and extensively in the 2000s. Animal studies focused on discovering roles of BMP in guided bone regeneration (GBR) when delivered with drug carriers [3, 4]. In general, osteoinductive capabilities should be given to osteoconductive bone graft materials for bone graft material development. Various proteins, such as BMP-2, 4, 7, and 14, have been reported to have osteoin- ductive abilities in animal experiments. Page 2 of 6 Page 2 of 6 Kim et al. Maxillofacial Plastic and Reconstructive Surgery (2017) 39:40 Page 2 of 6 Surgical procedure All patients received alveolar bone augmentation in the deficient ridge areas. Under local anesthesia using 1% lidocaine with 1:100,000 epinephrine (Huons, Hwasung, Korea), vertical and horizontal incisions were made in the mucoperiosteum of the labial or buccal sides of edentulous regions. A periosteal flap was elevated with a periosteal elevator, and a selection of bone graft mate- rials was placed underneath the highly cross-linked re- sorbable collagen membrane (Ossix Plus, Datum Dental Ltd., Telrad, Israel). Anorganic bovine bone (ABB; Bio-Oss®, Geistlich Pharma AG, Wolhusen, Switzerland) was used as the bone graft material in the control group (n = 5) while hydroxyapatite-based alloplastic bony sub- stitute with rhBMP-2(HA+rhBMP-2; NOVOSIS®-Dent, CGBio Inc., Seongnam, Korea) was used in the experi- mental group (n = 5). NOVOSIS®-DENT uses synthetic In particular, BMP-2 act as a growth and differenti- ation factor in the body and promotes the new bone for- mation by acting extensively at the entire stage of osteogenesis ranging from mesenchymal stem cells- osteoprogenitor-preosteoblast-osteoblast-osteocytic osteoblast-osteocyte [5]. BMP-2 also showed potential for bone regeneration through various studies including sinus augmentation [6, 7], alveolar bone preservation [8], bone augmenta- tion [9], and periodontal recovery [10]. Ike and Urist reported that BMP-2 contained in dentin exhibited Kim et al. Maxillofacial Plastic and Reconstructive Surgery (2017) 39:40 Page 3 of 6 Page 3 of 6 Page 3 of 6 Histomorphometric assessment Histomorphometric assessment Histomorphometric assessment grafting bone (hydroxyapatite) as multi-pore ceramic supporter to convey BMP-2 to the human body. Graft materials were prepared according to manufacturer’s in- structions. The mucoperiosteal flaps were then closed with 4–0 Vicryl (polyglactin; Ethicon Inc., Sommerville, NJ) using a simple interrupted suture technique. Bone specimens for further histomorphometric exami- nations were gained from the 8 patients who received implants in Seoul National University Bundang Hospital (control n = 4, experimental n = 4) using trephine burs during the implant placements. The acquired specimens were decalcified using 10% formic acid for 3 weeks, em- bedded in paraffin, and sagittal sections were obtained and then stained with hematoxylin and eosin (H&E) for histologic examinations. Because of a small sample size, histologic, radiographic, and clinical evaluations were focused. To determine the relative amount of bone for- mation, the new bone formation ratio was measured using the analySIS LS starter program. Case A 60-year-old female came to our department for restor- ation of left mandibular premolar and molar areas. On deficient alveolar ridge, ABB with resorbable collagen membrane was grafted. To radiographically evaluate the bone resorption and formation, as well as to provide support for graft materials, tenting screws were installed around the bone grafts (Fig. 1). This study was approved by the Institutional Review Board of Seoul National University Bundang Hospital (E-1501-282-001). Measurement of alveolar bone volume change To evaluate relative changes in bone volume and resorp- tion rate of the bone graft material, three dimensional measurements were obtained by cone-beam computed tomography (CBCT) immediately and at 4 months after the bone graft in all subjects (Fig. 2). The changes in bone width and height between the two groups were calculated, and Mann-Whitney U test (SPSS Inc., Chicago, IL, USA) was used to evaluate statistical significance. P values less than 0.05 were considered to be statistically significant. Postoperative complications were not found in all subjects. Postoperative complications were not found in all subjects. Surgical procedure The new bone formation was observed around the material in each of the 4 patient samples of each selected group, and the mean values of the areas were calculated. Statistical dif- ference between bone formation of the two groups were calculated using Mann-Whitney U test. Patients who underwent surgery took antibiotics (amoxi- cillin/clavulanate; Augmentin®, Ilsung Pharmaceuticals Co., Seoul, Korea) and a non-steroidal anti-inflammatory drug (talniflumate; Somalgen®, Kunwha Pharmaceutical Co., Seoul, Korea) for 5 days postoperatively. A 100 mL of 0.1% chlorhexidine mouth gargling (Hexamedine®, Bukwang Pharm, Ansan, Korea) was prescribed for oral hygiene maintenance. Sutures were stitched out between 1 and 2 weeks after the surgery. Histomorphometric findings of new bone formation Histomorphometric findings of new bone formation Bone formations were observed in both groups, but the appearance was different. Histomorphometrically, HA +rhBMP-2 group showed more bone formation around the bone graft materials than the ABB group (Fig. 3). Osteoconduction was observed around the material in the ABB group. In other words, ABB served as a bridge to bone formation as a scaffold. In the HA+rhBMP-2 group, osteoinduction occurred around the material and bone formation was observed. HA+rhBMP-2 group (35.2 ± 19.7%) showed more relative bone formation compared to the ABB group (28.9 ± 10.3%), but signifi- cant difference was not found (p = 0.886) (Fig. 4). A study by Burkus et al. showed that formation of anti-BMP-2 antibodies are low and transient in patients treated with rhBMP-2 [20]. Moreover, small formation of antibodies did not affect fusion success and had no visible affect clinical sequelae. Many studies support that BMP-2 is an effective osteoinducer, and there is no evidence that administra- tion of rhBMP-2 at the time of surgery links with an increased risk of cancer [21, 22]. However, rhBMP-2 could induce adverse clinical effects, including ectopic bone formation and tissue inflammation when used in high concentrations [23, 24]. Augmented bone volumes and resorption rates When CBCT radiographs were compared between imme- diately and at 4.07 ± 0.13 months after the bone graft, both alveolar bone widths (ABB 2.52 ± 0.18 mm, HA+rhBMP-2 1.75 ± 0.85 mm) and heights (ABB 1.68 ± 0.17 mm, HA +rhBMP-2 1.57 ± 0.28 mm) increased in the two test groups. Resorption rates of transplanted bone graft mater- ial in the alveolar bone widths and heights were (ABB 29.7 ± 8.8%, HA+rhBMP-2 31.5 ± 7.4%) and (ABB 39.2 ± 21.8%, HA+rhBMP-2 52.6 ± 6.5%), respectively (Table 1). Significant differences were not found in bone width and Among 10 patients, 8 received dental implants in Seoul National University Bundang Hospital, while the others received in local clinics. Fig. 1 Intraoral photographs of ridge augmentation using bone graft material and resorbable collagen membrane. a Deficient alveolar ridge. b Mucoperiosteal flap elevation. c Tenting screws installed around the bone grafts. d Bone graft placed. e Resorbable collagen membrane applied Fig. 1 Intraoral photographs of ridge augmentation using bone graft material and resorbable collagen membrane. a Deficient alveolar ridge. b Mucoperiosteal flap elevation. c Tenting screws installed around the bone grafts. d Bone graft placed. e Resorbable collagen membrane applied Page 4 of 6 Kim et al. Maxillofacial Plastic and Reconstructive Surgery (2017) 39:40 Fig. 2 CBCT, postoperative view. a Immediately after the bone graft. b Four months after the graft Fig. 2 CBCT, postoperative view. a Immediately after the bone graft. b Four months after the graft height resorptions between ABB and HA+rhBMP-2 groups (width p = 0.841, height p = 0.548). A study from Kim et al. showed that low-dose Escherichia coli–derived rhBMP-2 with HA is as effective as anorganic bovine bone xenografts in early stages for enhanced bone formation after maxillary sinus floor augmenta- tion without any major intraoperative or postoperative complications [7]. The soft tissue and residual graft areas showed no significant differences between the groups and rhBMP-2 antibody in the serum after BMP-2/H grafting did not increase significantly. Discussion Therefore, the result needs to be confirmed by the more number of the cases in the future study. the cases. Therefore, the result needs to be confirmed by the more number of the cases in the future study. (83% porosity, 300 μm pore size). NOVOSIS®-Dent pro- vides osteoconduction by using HA as a carrier and osteoinduction capability by utilizing BMP-2 to form a new bone at the bone defect site. Conclusions In this study, HA+rhBMP-2 showed relatively good bone formation compared with ABB. Although HA+rhBMP-2 had less volume of bone augmentation (widths ABB 2.52 ± 0.18 mm, HA+rhBMP-2 1.75 ± 0.85 mm; heights ABB 1.68 ± 0.17 mm, HA+rhBMP-2 1.57 ± 0.28 mm) and more resorptions over 4-month periods (widths ABB 29.7 ± 8.8%, HA+rhBMP-2 31.5 ± 7.4%, p = 0.841 and heights ABB 39.2 ± 21.8%, HA+rhBMP-2 52.6 ± 6.5%, p = 0.548), it showed relatively more bone formation in histomorphometric as- sessments (ABB 28.9 ± 10.3%, HA+rhBMP-2 35.2 ± 19.7%; p = 0.886). This suggests that HA+rhBMP-2 group may provide new bone formation through osteoinductive abil- ities provided by the osteogenic protein. This characteristic observation might be consistent with the in vivo and in vitro studies that the delivered rhBMP-2-activated dentin resorption that was associated with giant cells, ultimately promoting the bone formation and remodeling capacity in later stage [13, 25]. However, even though the average bone formation was higher in HA+rhBMP-2 group, the statistical difference was marginal because of the limited number of Bone graft material including rhBMP-2 showed good bone formation and remodeling capabilities. Within its limitation, this study suggested that ridge augmentations using rhBMP- 2 soaked HA could be clinically useful to supplement implant placements in edentulous regions. Additionally, ser- ious postoperative complications related to the graft material did not occur. Funding Funding There was no funding in support of this study. Availability of data and materials The dataset supporting the conclusions of this article is included within the article and Additional file 1. Authors’ contributions KHS participated in data collection and writing the manuscript. YPY participated in the study design and performed the statistical analysis. KYK participated in the study design and coordination and helped to draft the manuscript. PJC participated in histomophometric analysis of specimens. All authors read and approved the final manuscript. Fig. 4 Relative bone formation. HA+rhBMP-2 group showed more bone formation around the bone graft materials than the ABB group histologically, however, without statistical significance (p = 0.886) Consent for publication Fig. 4 Relative bone formation. HA+rhBMP-2 group showed more bone formation around the bone graft materials than the ABB group histologically, however, without statistical significance (p = 0.886) Fig. 4 Relative bone formation. HA+rhBMP-2 group showed more bone formation around the bone graft materials than the ABB group histologically, however, without statistical significance (p = 0.886) Competing interests The authors declare that they have no competing interests. Ethics approval and consent to participate Ethics approval and consent to participate This study was approved by the Institutional Review Board of Seoul National University Bundang Hospital (E-1501-282-001). pp p p This study was approved by the Institutional Review Board of Seoul National University Bundang Hospital (E-1501-282-001). Discussion Despite possessing good biocompatibility and osteocon- ductive potential, most of commercially available graft materials lack osteoinductive potential. Much research thus has been focused on graft materials mixed with ad- ditives that could promote osteogenic potentials such as bone morphogenetic protein (BMP). NOVOSIS®-Dent is a graft material used in combin- ation with rhBMP-2 and HA carrier in alveolar bone de- fect areas. In order for rhBMP-2 to exert its effects, it must act locally at the site where new bone formation is required, and for this reason, it is commonly used with carriers capable of releasing from local sites. The carrier of NOVOSIS®-Dent is HA, a material that occupies 65% of the bone and 98% of the dental enamel, and it pro- vides osteoconduction by providing a porous structure Kim et al. studies showed that demineralized dentin matrix (DDM) could act as an effective rhBMP-2 carrier [4, 13]. Kim also had reported that HA or DDM scaf- folds could be combined with rhBMP-2 and promote bone formation [19]. Table 1 Amount of bone augmentation Group Width Height T1 (mm) T4 (mm) Bone resorption (%) T1 (mm) T4 (mm) Bone resorption (%) ABB 3.7 ± 0.8 2.5 ± 0.2 29.7 ± 8.8 3.1 ± 1.4 1.7 ± 0.2 39.2 ± 21.8 HA+rhBMP-2 2.6 ± 1.2 1.8 ± 0.9 31.5 ± 7.4 3.3 ± 0.5 1.6 ± 0.3 52.6 ± 6.5 P value 0.841 0.548 Abbreviations: ABB anorganic bovine bone, HA hydroxyapatite, rhBMP-2 recombinant human bone morphogenetic protein-2, T1 immediately postoperative aug- mented bone, T4 augmented bone after 4 months after the graft Table 1 Amount of bone augmentation Abbreviations: ABB anorganic bovine bone, HA hydroxyapatite, rhBMP-2 recombinant human bone morphogenetic protein-2, T1 immediately postoperative aug- mented bone, T4 augmented bone after 4 months after the graft Kim et al. Maxillofacial Plastic and Reconstructive Surgery (2017) 39:40 Page 5 of 6 Fig. 3 Histologic findings after 4 months of bone healing. a ABB. b HA+rhBMP-2. New bone formations (arrows) are observed between the graft materials (stars) and adjacent bones after 4 months of bone healing. a ABB. b HA+rhBMP-2. New bone formations (arrows) are observed between the graft Fig. 3 Histologic findings after 4 months of bone healing. a ABB. b HA+rhBMP-2. New bone formations (arrows) are observed between the graft materials (stars) and adjacent bones the cases. Authors’ information All of the authors have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in this manuscript. This manuscript represents original works and is not being considered for publication elsewhere. Additional file Additional file 1: Case form and result of data. (XLSX 37 kb) Competing interests Th h d l h Page 6 of 6 Page 6 of 6 Kim et al. Maxillofacial Plastic and Reconstructive Surgery (2017) 39:40 Publisher’s Note 19. Kim YK (2014) Bone graft using two types of scaffolds and recombinant human bone morphogenetic protein-2: case series study. Oral Biology Research 38(2):127–134 Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 20. Burkus JK, Gornet MF, Glassman SD, Slosar PJ, Rosner MK, Deckey JE, Hatcher BM (2011) Blood serum antibody analysis and long-term follow-up of patients treated with recombinant human bone morphogenetic protein- 2 in the lumbar spine. Spine 36(25):2158–2167 Author details 1 1Department of Oral and Maxillofacial Surgery, Section of Dentistry, Seoul National University Bundang Hospital, 300 Gumi-dong, Bundang-gu, Seongnam, Gyunggi-do, South Korea. 2Department of Oral Histology, School of Dentistry, Seoul National University, Daehak-ro 101, Jongno-gu, Seoul 03080, South Korea. 3Department of Dentistry and Dental Research Institute, School of Dentistry, Seoul National University, Daehak-ro 101, Jongno-gu, Seoul 03080, South Korea. 21. Mines D, Gu Y, Kou TD, Cooper GS (2011) Recombinant human bone morphogenetic protein-2 and pancreatic cancer: a retrospective cohort study. Pharmacoepidemiol Drug Saf 20(2):111–118 22. Cooper GS, Kou TD (2013) Risk of cancer after lumbar fusion surgery with recombinant human bone morphogenic protein-2 (rh-BMP-2). Spine 38(21): 1862-1868 School of Dentistry, Seoul National University, Daehak-ro 101, Jongno-gu, Seoul 03080, South Korea. Received: 7 August 2017 Accepted: 20 November 2017 23. Zara JN, Siu RK, Zhang X, Shen J, Ngo R, Lee M, Wu BM (2011) High doses of bone morphogenetic protein 2 induce structurally abnormal bone and inflammation in vivo. Tissue Eng Part A 17(9–10):1389–1399 24. Wong DA, Kumar A, Jatana S, Ghiselli G, Wong K (2008) Neurologic impairment from ectopic bone in the lumbar canal: a potential complication of off-label PLIF/TLIF use of bone morphogenetic protein-2 (BMP-2). Spine J 8(6):1011–1018 References 1. Park SJ, Seon HG, Koh SW, Chee YD (2012) Retrospective clinical study on marginal bone loss of implants with guided bone regeneration. Maxillofac Plast Reconstr Surg 34(6):440–448 1. Park SJ, Seon HG, Koh SW, Chee YD (2012) Retrospective clinical study on marginal bone loss of implants with guided bone regeneration. Maxillofac Plast Reconstr Surg 34(6):440–448 25. Murata M, Um IW (2014) Advances in oral tissue engineering. Quintessence, Illinois 25. Murata M, Um IW (2014) Advances in oral tissue engineering. Quintessence, Illinois 2. Kim YK, Lee J, Um IW, Kim KW, Murata M, Akazawa T, Mitsugi M (2013) Tooth-derived bone graft. J Korean Assoc Oral Maxillofac Surg 39(3):103– 111. doi: 10.5125/jkaoms.2013.39.3.103 2. Kim YK, Lee J, Um IW, Kim KW, Murata M, Akazawa T, Mitsugi M (2013) Tooth-derived bone graft. J Korean Assoc Oral Maxillofac Surg 39(3):103– 111. doi: 10.5125/jkaoms.2013.39.3.103 3. Hwang ST, Han IH, Huh JB, Kang JK, Ryu JJ (2011) Review of the developmental trend of implant surface modification using organic biomaterials. J Adv Prosthodont 49(3):254–262 4. Um IW, Hwang SH, Kim YK, Kim MY, Jun SH, Ryu JJ, Jang HS (2016) Demineralized dentin matrix combined with recombinant human bone morphogenetic protein-2 in rabbit calvarial defects. J Korean Assoc Oral Maxillofac Surg 42(2):90–98 5. Cheng H, Jiang W, Phillips FM, Haydon RC, Peng Y, Zhou L, Szatkowski JP (2003) Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs). J Bone Joint Surg Am 85(8):1544–1552 6. Boyne PJ, Marx RE, Nevins M, Triplett G, Lazaro E, Lilly LC, Nummikoski P (1997) A feasibility study evaluating rhBMP-2/absorbable collagen sponge for maxillary sinus floor augmentation. Int J Periodontics Restorative Dent 17(1):11–25 7. Kim HJ, Chung JH, Shin SY, Shin SI, Kye SB, Kim NK, Kook MS (2015) Efficacy of rhBMP-2/hydroxyapatite on sinus floor augmentation: a multicenter, randomized controlled clinical trial. J Dent Res 94(suppl):158S–165S 7. Kim HJ, Chung JH, Shin SY, Shin SI, Kye SB, Kim NK, Kook MS (2015) Efficacy of rhBMP-2/hydroxyapatite on sinus floor augmentation: a multicenter, randomized controlled clinical trial. J Dent Res 94(suppl):158S–165S 8. Hanisch O, Tatakis DN, Boskovic MM, Rohrer MD, Wikesjö UM (1997) Bone formation and re-osseointegration in peri-implantitis defects following surgical implantation of rhBMP-2. Int J Oral Maxillofac Implants 12(5):604–610 8. Hanisch O, Tatakis DN, Boskovic MM, Rohrer MD, Wikesjö UM (1997) Bone formation and re-osseointegration in peri-implantitis defects following surgical implantation of rhBMP-2. Int J Oral Maxillofac Implants 12(5):604–610 9. References Howell TH, Fiorellini J, Jones A, Alder M, Nummikoski P, Lazaro M, Cochran D (1997) A feasibility study evaluating rhBMP-2/absorbable collagen sponge device for local alveolar ridge preservation or augmentation. Int J Periodontics Restorative Dent 17(2):125–139 10. Sigurdsson TJ, Nygaard L, Tatakis DN, Fu E, Turek TJ, Jin L, Wikesjö UM (1996) Periodontal repair in dogs: evaluation of rhBMP-2 carriers. Int J Periodontics Restorative Dent 16(6):525–537 11. Ike M, Urist MR (1998) Recycled dentin root matrix for a carrier of recombinant human bone morphogenetic protein. J Oral Implantol 24(3):124–132 11. Ike M, Urist MR (1998) Recycled dentin root matrix for a carrier of recombinant human bone morphogenetic protein. J Oral Implantol 24(3):124–132 12. Jung RE, Glauser R, Schärer P, Hämmerle CH, Sailer HF, Weber FE (2003) Effect of rhBMP-2 on guided bone regeneration in humans. Clin Oral Implants Res 14(5):556–568 12. Jung RE, Glauser R, Schärer P, Hämmerle CH, Sailer HF, Weber FE (2003) Effect of rhBMP-2 on guided bone regeneration in humans. Clin Oral Implants Res 14(5):556–568 13. Kim YK, Um IW, An HJ, Kim KW, Hong KS, Murata M (2014) Effects of demineralized dentin matrix used as an rhBMP-2 carrier for bone regeneration. J Hard Tissue Biol 23(4):415–422 13. Kim YK, Um IW, An HJ, Kim KW, Hong KS, Murata M (2014) Effects of demineralized dentin matrix used as an rhBMP-2 carrier for bone regeneration. J Hard Tissue Biol 23(4):415–422 14. Asahina I (2014) Bone morphogenetic proteins: their history and characteristics. J Hard Tissue Biol 23(3):283–286 14. Asahina I (2014) Bone morphogenetic proteins: their history and characteristics. J Hard Tissue Biol 23(3):283–286 15. Bessho K, Tagawa T, Murata M (1989) Purification of bone morphogenetic protein derived from bovine bone matrix. Biochem Biophys Res Commun 165(2):595–601 16. Sato K, Urist MR (1985) Induced regeneration of calvaria by bone morphogenetic protein (BMP) in dogs. Clin Orthop Relat Res 197:301–311 17. Hwang DY, On SW, Song SI (2016) Bone regenerative effect of recombinant human bone morphogenetic protein-2 after cyst enucleation. Maxillofac Plast Reconstr Surg 38(1):1–6 17. Hwang DY, On SW, Song SI (2016) Bone regenerative effect of recombinant human bone morphogenetic protein-2 after cyst enucleation. Maxillofac Plast Reconstr Surg 38(1):1–6 18. Geiger M, Li RH, Friess W (2003) Collagen sponges for bone regeneration with rhBMP-2. Adv Drug Deliv Rev 55(12):1613–1629
https://openalex.org/W2054289872	https://www.scielo.br/j/mioc/a/DVj49sphLkchJwj6GV6xM6p/?lang=en&format=pdf	English	null	Parvovirus B19 seroconversion in a cohort of human immunodeficiency virus-infected patients	Memórias do Instituto Oswaldo Cruz	2,012	cc-by	5,136	Kátia Martins Lopes de Azevedo1/+, Sérgio Setúbal1, Luiz Antonio Bastos Camacho2 Rita de Cássia Nasser Cubel Garcia3, Marilda Mendonça Siqueira4, Renata Freire Alves Pereira5, Solange Artimos de Oliveira1 1Disciplina de Doenças Infecciosas e Parasitárias, Hospital Universitário Antônio Pedro 3Departamento de Microbiologia e Parasitologia, Instituto Biomédico, Universidade Federal Fluminense, Niterói, RJ, Brasil 2Departamento de Epidemiologia, Escola Nacional de Saúde Pública 4Instituto Oswaldo Cruz-Fiocruz, Rio de Janeiro, RJ, Brasil 5Escola de Farmácia, Universidade do Grande Rio, Duque de Caxias, RJ, Brasil Erythrovirus B19 (B19V) infection may cause red cell aplasia in patients infected with human immunodeficiency virus (HIV). The introduction of highly active antiretroviral therapy (HAART) has improved the immune function of these patients by modifying the course of B19V infection. The purpose of this study was to estimate the frequency of B19 seroconversion in a cohort of HIV-infected patients and evaluate the occurrence of B19V-related anaemia during the seroconversion period. Adult HIV-infected patients were studied at a public hospital in Niterói, state of Rio de Janeiro, Brazil. IgG and IgM antibodies against B19V were detected by an enzyme-linked immunosorbent assay and B19 viraemia was assayed by polymerase chain reaction. Medical records were reviewed for any clinical evaluation of anaemia. Seroconversion was detected in 31.8% of the 88 individuals who began the study as anti- B19V IgG-negative. No clinical manifestations of B19V infection were detected during the period of seroconversion. Patients who seroconverted were 5.40 times more likely to have anaemia than those who did not [odds ratio 5.40 (95% confidence interval: 1.33-22.93)]. Anaemia was detected in eight patients. All patients recovered from anaemia by either beginning or continuing HAART, without requiring blood transfusions. In the HAART era, B19V infection may only be associated with a course of disease characterised by less severe chronic anaemia. This milder course of B19V-associated disease is likely due to the increased immune function of HAART-treated patients. Key words: human parvovirus B19 - seroconversion - HIV infection - anaemia Key words: human parvovirus B19 - seroconversion - HIV infection - anaemia Erythrovirus B19 (B19V) infection, classically linked to a common benign exanthematic disease of childhood, may cause red cell aplasia in immunocompromised pa tients, including those infected with human immunodefi ciency virus (HIV) (Young & Brown 2004). Anaemia is an independent predictive marker for clinical prognosis in HIV-infected patients (Mocroft et al. 1999, O’Brien et al. 2005). There are many causes of anaemia in HIV- infected patients, such as opportunistic infections and the use of antiretrovirals or antimicrobials (Abkowitz et al. 1997). 356 356 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 107(3): 356-361, May 2012 Parvovirus B19 seroconversion in a cohort of human immunodeficiency virus-infected patients Kátia Martins Lopes de Azevedo1/+, Sérgio Setúbal1, Luiz Antonio Bastos Camacho2, Rita de Cássia Nasser Cubel Garcia3, Marilda Mendonça Siqueira4, Renata Freire Alves Pereira5, Solange Artimos de Oliveira1 Kátia Martins Lopes de Azevedo1/+, Sérgio Setúbal1, Luiz Antonio Bastos Camach Rita de Cássia Nasser Cubel Garcia3, Marilda Mendonça Siqueira4, Renata Freire Alves Pereira5, Solange Artimos de Oliveira1 online \| memorias.ioc.fiocruz.br Financial support: CNPq (473432/2010-2, 471618/2008-0) + Corresponding author: kmlazevedo@id.uff.br Received 29 June 2011 Accepted 11 January 2012 SUBJECTS, MATERIALS AND METHODS Study population - Samples from a serum bank es tablished in 2001 were used in this study. These samples were from patients who received treatment for HIV in fection at the Infectious Diseases Department, Antônio Pedro University Hospital, Federal Fluminense Univer sity. This is a large tertiary public hospital in Niterói, state of Rio de Janeiro, Brazil. Niterói has approximately 441,078 inhabitants (IBGE 2010). The hospital also pro vides health care to other larger neighbouring munici palities and, to a lesser extent, to people from other parts of the state. A previous study had been conducted to es timate the B19V infection rate in the adult HIV-positive patients treated at the general medical outpatient care centre. The patients in that study received antiretroviral therapy and follow-up examinations to analyse CD4+ T cell counts and plasma HIV viral loads (Azevedo et al. 2009). Between November 2001-December 2003, single serum samples from 261 consecutive HIV-infected pa tients were tested using an enzyme immunoassay to detect anti-B19V IgG antibodies. The seroprevalence of B19V in this population was 62.8%. Patients who were originally anti-B19V IgG negative and then had anoth er serum sample collected between January 2004-July 2008 were included in the seroconversion study. The second serum sample was collected at least 12 months after the first serum sample. This study was planned to take advantage of the opportunity to detect B19V sero conversion during the B19V outbreak in Niterói that oc curred from 2004-2005 (Oliveira et al. 2005). PCR was conducted on the last serum sample that test ed negative for anti-B19V IgG antibodies and on the first serum sample that was anti-B19V IgG positive in all cases of seroconversion. Then, for the cases with no documented seroconversion, a PCR viraemia test was performed on the last serum sample. If the PCR assay was positive for B19V viral DNA, all subsequent samples in the serum bank were tested to determine the duration of B19V viraemia. Data analysis - Data were entered and analysed us ing the SPSS 17.0 software (Statistical Package for So cial Sciences, Inc, Chicago, IL, USA). Continuous vari ables were categorised. Differences in proportions were assessed using the chi-squared test. Odds ratios (OR) and their 95% confidence intervals (CI) were calculated to measure the association between anti-B19V IgG anti body conversion and risk factors, including demograph ic characteristics, CD4+ T cell counts, HIV viral load and haemoglobin level. Kátia Martins Lopes de Azevedo1/+, Sérgio Setúbal1, Luiz Antonio Bastos Camacho2 Rita de Cássia Nasser Cubel Garcia3, Marilda Mendonça Siqueira4, Renata Freire Alves Pereira5, Solange Artimos de Oliveira1 Appropriate treatment for anaemia depends on the identification of the cause, for example, anaemia caused by B19V infection is treatable with high intrave nous doses of human standard immunoglobulin (Frick hofen et al. 1990). risk for developing persistent anaemia if infected with B19V (Zuckerman et al. 1994). Epidemiological studies have shown that B19V infection occurs periodically in the form of outbreaks. These outbreaks represent an oc casion in which susceptible individuals are at a higher risk of contracting B19V infection (Oliveira et al. 1996). Prior to the highly active antiretroviral therapy (HAART) era, B19V infection in immunosuppressed HIV-infected patients manifested as persistent anaemia. The treatment for this anaemia was multiple blood trans fusions and high intravenous doses of human standard immunoglobulin. The introduction of HAART into clin ical practice has improved the immune function of HIV- infected individuals. This altered immune function has modified the course of B19V infection in these patients. Due to HAART, the course of B19V infection in HIV- infected patients is similar to that in immunocompetent individuals (Mylonakis et al. 1999, Arribas et al. 2000). Seroprevalence studies have shown that approxi mately 30% of adult HIV-infected patients have no anti- B19V IgG antibodies and are therefore susceptible to the infection (Gyllensten et al. 1994, van Elsacker-Neile et al. 1996, Azevedo et al. 2009). These patients remain at In the medical literature, there are only case reports about describing B19V seroconversion in HIV-positive patients and these have mostly occurred after the start ing of HAART treatments (Bremner & Cohen 1994, Ar ribas et al. 2000, Taguchi et al. 2001, Ware & Moore 2001, Clarke & Lee 2003, Watanabe et al. 2011). To better understand the importance of B19V infection in the HAART era, we conducted a study to estimate the frequency of B19V seroconversion in a cohort of HIV- infected patients. We also examined the occurrence of anaemia caused by B19V infection during the serocon online \| memorias.ioc.fiocruz.br Financial support: CNPq (473432/2010-2, 471618/2008-0) + Corresponding author: kmlazevedo@id.uff.br Received 29 June 2011 Accepted 11 January 2012 357 B19V seroconversion in HIV-infected patients • Kátia Martins Lopes de Azevedo et al. (PCR) using primers that amplify a 102-bp fragment of the NS1 gene. Briefly, viral DNA was extracted from serum samples using a QIAamp DNA Blood Mini Kit (QIAGEN, Brasil) according to the manufacturer’s in structions. SUBJECTS, MATERIALS AND METHODS Multivariate analysis (logistic regression) was used to adjust for the effect of covariates with substantial association in the univariate analysis. A 5% significance level was adopted for all results. The following definitions were used in this study: (i) the seroconversion period was defined as the time between the last negative anti-B19V IgG serum sample and the first positive anti-B19V IgG sample, (ii) anaemia was defined according to the World Health Organization criteria as a haemoglobin concentration below 13 g/dL in men and below 12 g/dL in women and (iii) severe anae mia was defined as a haemoglobin concentration below 7 g/dL (WHO 2001). Ethics - Written informed consent was obtained from all volunteers and the project was approved by the Hos pital Review Board (CMM/HUAP 134/05). Laboratory methods - Blood samples were collected and the serum samples were obtained by centrifugation of the clots. The serum samples were stored at -20ºC. Se rum samples were assigned a numerical code to conceal the identity of the study subjects. An enzyme-linked im munosorbent assay (Parvovirus B19 IgG and IgM Im munoassay; Biotrin International, Dublin, Ireland) was used to detect anti-B19V IgG and IgM antibodies in the serum samples according to the manufacturer’s instruc tions. First, the last available serum sample for each pa tient was tested for anti-B19V IgG antibodies. If sero conversion was detected, every serum sample for that patient was examined for anti-B19V IgG antibodies to determine the seroconversion period. In addition, every serum sample that tested positive for anti-B19V IgG an tibodies was also tested for anti-B19V IgM antibodies. Kátia Martins Lopes de Azevedo1/+, Sérgio Setúbal1, Luiz Antonio Bastos Camacho2 Rita de Cássia Nasser Cubel Garcia3, Marilda Mendonça Siqueira4, Renata Freire Alves Pereira5, Solange Artimos de Oliveira1 The PCR assay was performed using 12.5 pmol of the primers E1905F (nt 1905-1923) and E1987R (nt 2007-1987) as previously described by Nguyen et al. (2002) with some modifications. After an initial dena turation step at 94ºC for 10 min, amplification consisted of 35 cycles of incubations at 94ºC for 30 s, 55ºC for 30 s and 72ºC for 30 s, followed by a final extension of 7 min at 72ºC. The 102-bp PCR products were re solved on 1% agarose gel followed by ethidium bromide staining and the amplified DNA was visualised under ultraviolet light. Distilled milli-Q water was used as a negative control in all techniques and the recommended manipulations for the PCR procedures were performed as a precaution to avoid false-positive results. version period. In this generally benign viral infection, anaemia is a major clinical manifestation, particularly in immunocompromised subjects. RESULTS Chronic anaemia, which refers to anaemia that persists longer than five months, was observed in seven patients, but only one of them presented with severe anaemia (haemoglobin: 5.9 g/dL). Anaemia was also detected in two additional patients, but there were no sequential samples available to characterise its dura tion. Out of the 28 patients who seroconverted, 13 had no anaemia during the seroconversion period. In the remaining five cases, there were no data in the medi cal records regarding the occurrence of anaemia. All patients had recovered from anaemia by either begin ning or continuing HAART and none of them required immunoglobulins or blood transfusions. B19V infections are generally acute and self-limit ing. However, persistent infections have been demon strated in immunocompetent individuals by the detec tion of B19V DNA in blood or bone marrow samples that were collected years after infection. The mecha nisms of B19V persistence are not known (Cassinotti & Siegl 2000). Reactivation of persistent infection occurs in immunocompetent and, more often, in immunocom promised individuals (Cassinotti et al. 1997). Theoreti cally, it is possible that in HIV-infected individuals with severe immunodeficiency, the reactivation of persistent infection occurs more frequently. The main receptor for B19V is globoside P, which is present on erythroid cells. The viral replication in these cells causes destruction of the erythroid progenitors, resulting in a disruption of erythropoiesis and anaemia (Young & Brown 2004). Immunosuppressed patients are at risk of developing acute or chronic anaemia follow ing B19V infection due to a lack of protective antibod ies. Life-threatening anaemia can develop due to the in ability of these patients to mount an immune response to clear the viraemia. In HIV-infected patients, severe immunodeficiency may cause an impairment of the im mune response to B19V, leading to persistent viraemia and chronic anaemia (Abkowitz et al. 1997, Setúbal et al. 2003). The strong association of anaemia with B19V infection provides a relevant parameter for the clinical assessment of HIV-infected patients, particularly in pe riods of B19V outbreaks. The use of zidovudine was the main variable found to be correlated with anaemia. Zidovudine was used in 37 of the 88 cases in this study. However, although there was a strong association between the occurrence of anaemia and the use of this medication, it was not a statistically significant association (OR: 2.58; 95% CI: 0.67-9.96; p = 0.169) (data not shown). RESULTS Eighty-eight patients who tested negative for anti- B19V IgG antibodies were included in this study. There were 46 (52.3%) women (mean age: 38.6 years) and 42 (47.7%) men (mean age: 42.0 years). The interval be tween the last negative and the first positive serum sam ple (seroconversion period) varied from two-18 months (mean: 8.1 months; median: 7.0 months). Seroconversion to anti-B19V IgG antibodies was de tected in 28 (31.8%) of the patients, but anti-B19V IgM antibodies were present in only three of the patients who seroconverted. Seroconversion was more frequent in se rum samples that were collected in 2005 (9/28; 32.1%) and 2006 (6/28; 21.4%) than in those collected in 2007 and 2008. No clinical manifestations of B19V infection were detected in the medical records of these patients during the seroconversion period. The differences in B19V viraemia was evaluated by the detection of B19V-specific DNA by polymerase chain reaction Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 107(3), May 2012 358 B19V seroconversion across sex, age and educational categories were neither substantial nor statistically sig nificant. CD4+ T cell counts and HIV viral load did not appear to be associated with anti-B19V IgG seroconver sion. Patients who had seroconverted were found to be 5.40 (95% CI 1.33-22.93) times more likely to have anae mia in the last negative serum sample compared to those who did not seroconvert (Table); however, this increase in anaemia incidence was not found to be statistically significant in the first serum sample that tested positive for anti-B19V IgG antibodies. systematically gathering data on acute rash diseases over six years. During the study period, three distinct peaks of parvovirus infection were detected, suggesting that the disease appears to cycle in approximately four-five years. Another study identified an outbreak in Niterói, in 2004- 2005 (Oliveira et al. 2005). In the current study, B19V se roconversion was detected in nine patients in 2005 and in six patients in 2006 (32.1 and 21.4%, respectively), but it is possible that three of the cases in 2006 may have actually occurred in 2005. Due to the long intervals between the serum samples from these patients, the exact seroconver sion period could not be ascertained. During the period of seroconversion, transient anaemia was present in one patient and lasted for three months. RESULTS B19V viraemia, as measured by the detection of B19V DNA with PCR in the serum samples, was found in four of the 28 patients who had seroconverted. In two cases, viraemia was detected in only one serum sample, whereas in the remaining two cases, viraemia was de tected in a sequential serum sample that was collected three months later in one patient and nine months later in the other patient. Anaemia was identified in two of those four patients during the seroconversion period, but only one patient had severe chronic anaemia. The other two cases lacked any data regarding anaemia incidence in the medical records during that period. Viraemia was not detected in the last set of serum samples of the 60 patients who had not seroconverted. Anaemia is the most common haematologic abnor mality observed in HIV-infected patients (Koduri 2000). There are many causes of anaemia in these patients, in cluding the following: co-infection with mycobacteria, fungus, cytomegalovirus and B19V. Drugs, such as zi dovudine, trimethoprim sulfamethoxazole and antine oplastic drugs have also been shown to cause anaemia in HIV-infected patients. Finally, lymphoma and the direct effect of HIV on the function of accessory cells with in the bone marrow microenvironment can also cause anaemia (Abkowitz et al. 1997). DISCUSSION To our knowledge, this is the largest study to date that has examined B19V seroconversion in HIV-infected pa tients. In the medical literature, there are only reports of isolated cases, primarily after patients had begun HAART treatment (Bremner & Cohen 1994, Arribas et al. 2000, Taguchi et al. 2001, Ware & Moore 2001, Clarke & Lee 2003). The high rate of B19V seroconversion in the present study [28 (31.8%) out of 88 individuals] may be explained by the wide exposure of the population to this virus dur ing the epidemic. Epidemiological studies have shown that B19V infection increases periodically. Oliveira et al. (2003) identified a seasonal pattern of B19V infection by Patients who had seroconverted were 2.6 times more likely to develop anaemia (Table), which is similar to the value found by Calvet et al. (1999) in organ trans plant recipients. In the present study, the lack of a sig nificant association between anaemia and predisposing factors other than B19V seroconversion reinforces the importance of the role of B19V infection as a cause of anaemia in HIV-infected patients, particularly during periods of high viral activity. B19V seroconversion in HIV-infected patients • Kátia Martins Lopes de Azevedo et al. 359 B19V seroconversion in HIV-infected patients • Kátia Martins Lopes de Azevedo et al. DISCUSSION 359 Table Erythrovirus B19 (B19V)-IgG seroconversion among human immunodeficiency virus (HIV) infected-patients, according to demographic, clinical and laboratory parameters, Niterói, state of Rio de Janeiro, 2001-2008 Characteristic Seroconversion IgG B19V antibodies p OR (95% CI) Yes n (%) No n (%) Gender 0.950a Male 13 (31) 29 (69) - 0.93 (0.34-2.50) Female 15 (32.6) 31 (67.4) - 1.0 Age groups (years) 0.082a < 40 12 (29.3) 29 (70.7) - 0.80 (0.29-2.17) ≥ 40 16 (34) 31 (66) - 1.0 Educational level 0.911a Elementary 13 (34.2) 25 (65.8) - 0.91 (0.27-3.14) High school and college 8 (36.4) 14 (63.6) - 1.0 Unknown 7 (25) 21 (75) - - Clinical characteristics 0.522b Asymptomatic 4 (28.6) 10 (71.4) - 0.83 (0.20-3.32) Acquired immune deficiency syndrome 24 (32.4) 50 (67.6) - 1.0 CD4+ cell count/mm³ in the last serum sample B19V-IgG-negative 0.350a ≤ 200 8 (44.4) 10 (55.6) - 1.94 (0.58-6.43) > 200 19 (29.2) 46 (70.8) - 1.0 Unknown 1 (20) 4 (80) - - CD4+ cell count/mm³ in the first serum sample B19V-IgG-positive 0.694a ≤ 200 5 (41.7) 7 (58.3) - 1.58 (0.38-6.40) > 200 23 (31.1) 51 (68.9) - 1.0 Unknown 0 (0) 2 (100) - - Plasma HIV load (copies/mL) in the last serum sample B19V-IgG-negative 0.861a < 1.000 15 (31.2) 33 (68.8) - 0.96 (0.32-2.94) ≥ 1.000 9 (32.1) 19 (67.9) - 1.0 Unknown 4 (33.3) 8 (66.7) - - Plasma HIV load (copies/mL) in the first serum sample B19V-IgG-positive 0.903a < 1.000 15 (31.3) 33 (68.7) - 1.23 (0.38-4.07) ≥ 1.000 7 (26.9) 19 (73.1) - 1.0 Unknown 6 (42.9) 8 (57.1) - - Haemoglobin in the last serum sample B19V-IgG-negative 0.008a Anaemia 9 (60) 6 (40) - 5.40 (1.33-22.93) No anaemia 10 (21.7) 36 (78.3) - 1.0 Unknown 9 (33.3) 18 (66.7) - - Haemoglobin in the first serum sample B19V-IgG-positive 0.132b Anaemia 6 (50) 6 (50) - 2,57 (0,60-11,21) No anaemia 14 (28) 36 (72) - 1.0 Unknown 8 (30.8) 18 (69.2) - - a: Pearson chi-square test; b: Fisher exact test; CI: confidence interval; OR: odds ratio. Table Unknown a: Pearson chi-square test; b: Fisher exact test; CI: confidence interval; OR: odds ratio. 360 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 107(3), May 2012 B19V seroconversion was detected more frequently in patients whose CD4+ T cell counts were below 200 cells/ mm³, but this study lacked sufficient statistical power to make an unequivocal conclusion regarding this finding. It is possible that the use of HAART has contributed to a better immune response to B19V infection, even before a detectable increase in the number of CD4+ T cells in the blood is found. This is consistent with the known impact of restoring the immune response through the initiation of HAART, which could reduce or even eliminate the severe haematological changes caused by B19V infec tion (Arribas et al. 2000, Scapellato & Palumbo 2000, Ware & Moore 2001). These facts may explain the low frequency of severe anaemia observed in our study: only one out of seven patients had chronic anaemia during the seroconversion period. This study had limitations that are inherent a retro spective study that involves a review of medical record. One of the main limitations is the long intervals between blood collections. These long periods made it difficult to more precisely define the time of B19V seroconversion, especially for the evaluation of anaemia. However, these blood tests were performed because they were deemed necessary based on the physicians’ clinical assessments of these patients. Considering that only one case of se vere anaemia was observed during the seroconversion period, it is plausible to assume that even if anaemia had been identified through laboratory tests, it would most likely not have been of great magnitude. There were no reports in the medical records of complaints or changes found during the physical examinations that would be consistent with the presence of severe anaemia. p Raguin et al. (1997) determined the prevalence of B19V in 55 HIV-infected patients. Anti-B19V IgG anti bodies were detected in 53/55 (96%) of the serum samples and anti-B19V IgM antibodies were detected in only five (10%) patients who were also positive for anti-B19V IgG antibodies. Viral DNA was not detected in any of the 55 serum samples by PCR in that study. The prevalence of IgM-positive sera (10% - 5/53 patients) in that previous study was similar to that observed in our study (10.7% - 3/28 patients). However, Calvet et al. REFERENCES Abkowitz JL, Brown KE, Wood RW, Kovach NL, Green SW, Young NS 1997. Clinical relevance of parvovirus B19 as a cause of anae mia in patients with human immunodeficiency virus infection. J Infect Dis 176: 269-273. g p y The immune response may be altered by immuno suppression; consequently, genome detection has been largely applied in this clinical context (Kurtzman et al. 1989, Calvet et al. 1999). Calvet et al. (1999) found a fre quent dissociation between the molecular and serological markers of B19V infection in transplant patients; 60/109 PCR-positive specimens showed no evidence of recent infection and only two of the 24 patients with B19V infec tion had more than 40% of their samples test positive by either serology or PCR. In our study, viraemia was de tected in a single serum sample in two/four patients who seroconverted (1 patient seroconverted in 2005 and the other in 2006). In the other two patients, two sequential serum samples were positive for B19V DNA. Of these two patients, both had seroconverted in 2005. One patient had a PCR-positive sample three months after the previ ous PCR-positive sample and the other patient had a PCR- positive sample nine months later in 2006. Although some studies (Naides et al. 1993, Abkowitz et al. 1997) have sug gested the possibility of the persistence of B19V viraemia in HIV-infected patients, other authors demonstrated that this is unusual even among these immunocompromised individuals (Bremner & Cohen 1994, Raguin et al. 1997). In patients who are persistently positive for B19V DNA, acute or chronic clinical features may not be present when the levels of viraemia are low (Musiani et al. 1995b). In this study, the presence of B19V DNA was detected by PCR, which can detect low levels of viraemia for several months after the initial infection (Musiani et al. 1995a). Arribas JR, Peña JM, Echevarría JE 2000. Parvovirus B19-related anaemia in an HIV-infected patient: rapid control after produc tion of neutralizing antibodies during highly active antiretroviral therapy. Ann Intern Med 132: 1011. Azevedo KML, Setúbal S, Camacho LAB, Velarde LGC, Oliveira SA 2009. Seroepidemiological study of human parvovirus B19 among human immunodeficiency virus-infected patients in a medium-sized city in Rio de Janeiro, Brazil. Mem Inst Oswaldo Cruz 104: 901-904. Bremner JAG, Cohen BJ 1994. Parvovirus B19 as a cause of anaemia in human immunodeficiency virus-infected patients [letter]. J In fect Dis 169: 938-939. Table (1999) detected anti-B19V IgM antibodies in 12/24 (50%) patients who tested positive for B19V in a group of 62 transplanted patients. Compared with the results of the current study, the high proportion of IgM detected in that study may be associated with the shorter interval of collection and testing of serum samples by those authors. In conclusion, our findings show that in the HAART era, B19V infection may be restricted, subtle and only apparent in retrospective analyses. The presence of chronic anaemia, especially during B19V epidemics, should alert the physician to the possibility of B19V infection. Because this is a treatable cause of anaemia, identification of B19V infection could reduce the risk of multiple blood transfusions and other unnecessary therapeutic measures. Detection of DNA-B19V by PCR may indicate viraemia; however, our results suggest that viraemia is not necessarily associated with clinical man ifestations of B19V infection. REFERENCES Anaemia is an independent predictive marker for clinical prognosis in HIV-infected patients from across Europe. AIDS 13: 943-950. Scapellato PG, Palumbo AM 2000. Improvement of anaemia induced by parvovirus B19 in a patient with AIDS after combined antiret roviral therapy [letter]. Mayo Clin Proc 75: 215-217. Setúbal S, Jorge-Pereira MC, Sant’Anna ALM, Oliveira AS, Bazin AR, Nascimento JP 2003. Clinical presentation of parvovirus B19 infection in HIV-infected patients with and without AIDS. Rev Soc Bras Med Trop 36: 299-302. Musiani M, Zerbini M, Gentilomi G, Plazzi M, Gallinella G, Ven turoli S 1995a. Parvovirus B19 clearance from peripheral blood after acute infection. J Infect Dis 172: 1360-1363. Musiani M, Zerbini M, Gentilomi G, Rodrigo G, de Rosa V, Gibel lini D, Venturoli S, Gallinella G 1995b. Persistent B19 parvo virus infections in haemophilic HIV-1 infected patients. J Med Virol 46: 103-108. Taguchi H, Takahashi T, Goto M, Nakamura T, Iwamoto A 2001. Acute parvovirus B19 infection during anti-retroviral therapy. J Infect Chemother 7: 110-112. Mylonakis E, Dickinson BP, Mileno MD, Flanigan T, Schiffman FJ, Mega A 1999. Persistent parvovirus B19 related anaemia of seven years’ duration in an HIV-infected patient: complete remission associated with highly active antiretroviral therapy. Am J Hema tol 60: 164-166. van Elsacker-Neile AM, Kroon FP, van der Ende ME, Salimans MM, Spaan WJ, Kroes AC 1996. �� Prevalence of parvovirus B19 infec tion in patients infected with human immunodeficiency virus. Clin Infect Dis 23: 1255-1260. Ware AJ, Moore T 2001. Resolution of chronic parvovirus B19- induced anaemia by use of highly active antiretroviral therapy in a patient with acquired immunodeficiency syndrome. Clin Infect Dis 32: e122-123. Naides SJ, Howard EJ, Swack NS, True CA, Stapleton JT 1993. Par vovirus B19 infection in human immunodeficiency virus type 1-infected persons failing or intolerant to zidovudine therapy. J Infect Dis 168: 101-105. Watanabe D, Taniguchi T, Otani N, Tominari S, Nishida Y, Uehira T, Shirasaka T 2011. Immune reconstitution to parvovirus B19 and resolution of anaemia in a patient treated with highly active antiretroviral therapy. J Infect Chemother 17: 283-287. Nguyen QT, Wong S, Heegaard ED, Brown KE 2002. Identification and characterization of a second novel human erythrovirus vari ant A6. Virology 301: 374-380. O’Brien ME, Kupka R, Msamanga GI, Saathoff E, Huntern DJ, Fawzi WW 2005. Anaemia is an independent predictor of mortality and immunologic progression of disease among women with HIV in Tanzania. REFERENCES Calvet A, Pujol MO, Bertocchi M, Basgien O, Boissonnat P, Mornex JF 1999. Parvovirus B19 infection in thoracic organ transplant recipients. J Clin Virol 13: 37-42. Cassinotti P, Burtonboy G, Foppe M, Siegl G 1997. Evidence for per sistence of human parvovirus B19 DNA in bone marrow. J Med Virol 53: 229. Cassinotti P, Siegl E 2000. Quantitative evidence for persistence of human parvovirus B19 DNA in an immunocompetent individual. Eur J Clin Microbiol Infect Dis 19: 886-895. Clarke J, Lee JD 2003. Primary human parvovirus B19 infection in an HIV infected patient on highly active antiretroviral therapy. Sex Transm Infect 79: 336. Frickhofen N, Abkowitz JS, Safford M, Berry JM, Antunez-de-Mayolo J, Astrow A 1990. Persistent B19 parvovirus infection in patients infected with human immunodeficiency virus type 1 (HIV-1): a treatable cause of anaemia in AIDS. Ann Intern Med 113: 926-933. B19V seroconversion in HIV-infected patients • Kátia Martins Lopes de Azevedo et al. B19V seroconversion in HIV-infected patients • Kátia Martins Lopes de Azevedo et al. 361 Gyllensten K, Sönnerborg A, Jorup-Rönström C, Halvarsson M, Yun Z 1994. Parvovirus B19 infection in HIV-1 infected patients with anaemia. Infection 22: 356-358. Oliveira SA, Camacho LAB, Pereira ACM, Faillace TF, Setúbal S, do Nascimento JP 2003. Clinical and epidemiological aspects of human parvovirus B19 infection in an urban area in Brazil (Niterói city area, state of Rio de Janeiro, Brazil). Mem Inst Oswaldo Cruz 97: 965-970. IBGE - Instituto Brasileiro de Geografia e Estatística 2010. Census Data [Dados do Censo 2010]. Available from: censo2010.ibge. gov.br/dados_divulgados/index.php?uf=33. Oliveira SA, Marinho TAS, Silva ASV, Rodrigues NB, Faria RA, Se túbal S 2005. Estudo da frequência da infecção pelo parvovírus B19 em casos de doenças exantemáticas no período de 1994 a 2004. Braz J Infect Dis 9 (Suppl.): 73. Koduri 2000. Parvovirus B19-related anaemia in HIV-infected pa tients. AIDS Patient Care STDS 14: 7-11. Kurtzman GL, Cohen BJ, Field AM, Oseas R, Blaese RM, Young NS 1989. Immune response to B19 parvovirus and an antibody defect in persistent viral infection. J Clin Invest 84: 1114-1123. Raguin G, Leruez-Ville M, Gregoire V, Deplanche M, Leport C, Mori net F 1997. Low prevalence of active parvovirus B19 infection in HIV-infected patients. Eur J Clin Microbiol Infect Dis 16: 760-762. Mocroft A, Kirk O, Bartin SE, Dietrich M, Proença R, Colebun ders R 1999. REFERENCES J Acquir Immune Defic Syndr 40: 219-225. WHO - World Health Organization 2001. Iron deficiency anaemia. Assessment, prevention and control, WHO, Geneva, p. 33-46. Young NS, Brown KE 2004. Mechanisms of disease Parvovirus B19. N Engl J Med 350: 586-597. Oliveira SA, Brandão AB, Fernandes DG, Bettini LR, Carvalho AB, Pereira ACM 1996. �� Human parvovirus B19 infection: clini cal and epidemiological study of 24 cases. Rev Inst Med Trop Sao Paulo 38: 323-327. Zuckerman MA, Williams I, Bremmer J, Cohen B, Miller RF 1994. Persistent anaemia in HIV-infected individuals due to parvovirus B19 infection. AIDS 8: 1191-1192.
https://openalex.org/W2066328670	https://bmcbiotechnol.biomedcentral.com/counter/pdf/10.1186/1472-6750-7-88	English	null	Self-assembling Fmoc dipeptide hydrogel for in situ 3D cell culturing	BMC biotechnology	2,007	cc-by	6,700	BioMed Central BioMed Central Open Acce Research article Self-assembling Fmoc dipeptide hydrogel for in situ 3D cell culturing Thomas Liebmann, Susanna Rydholm, Victor Akpe and Hjalmar Brismar Address: Cell Physics, Department of Applied Physics, Royal Institute of Technology, S-106 91 Stockholm, Sweden Email: Thomas Liebmann - liebmann@kth.se; Susanna Rydholm - susannar@kth.se; Victor Akpe - victor@cellphysics.kth.se; Hjalmar Brismar - brismar@kth.se * Corresponding author Open Access Received: 31 August 2007 Accepted: 10 December 2007 Published: 10 December 2007 BMC Biotechnology 2007, 7:88 doi:10.1186/1472-6750-7-88 Published: 10 December 2007 BMC Biotechnology 2007, 7:88 doi:10.1186/1472-6750-7-88 This article is available from: http://www.biomedcentral.com/1472-6750/7/88 © 2007 Liebmann 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: Conventional cell culture studies have been performed on 2D surfaces, resulting in flat, extended cell growth. More relevant studies are desired to better mimic 3D in vivo tissue growth. Such realistic environments should be the aim of any cell growth study, requiring new methods for culturing cells in vitro. Cell biology is also tending toward miniaturization for increased efficiency and specificity. This paper discusses the application of a self-assembling peptide-derived hydrogel for use as a 3D cell culture scaffold at the microscale. Results: Phenylalanine derivative hydrogel formation was seen to occur in multiple dispersion media. Cells were immobilized in situ within microchambers designed for cell analysis. Use of the highly biocompatible hydrogel components and simplistic procedures significantly reduced the cytotoxic effects seen with alternate 3D culture materials and microstructure loading methods. Cells were easily immobilized, sustained and removed from microchambers. Differences in growth morphology were seen in the cultured cells, owing to the 3-dimentional character of the gel structure. Degradation improved the removal of hydrogel from the microstructures, permitting reuse of the analysis platforms. Conclusion: Self-assembling diphenylalanine derivative hydrogel provided a method to dramatically reduce the typical difficulties of microculture formation. Effective generation of patterned 3D cultures will lead to improved cell study results by better modeling in vivo growth environments and increasing efficiency and specificity of cell studies. Use of simplified growth scaffolds such as peptide-derived hydrogel should be seen as highly advantageous and will likely become more commonplace in cell culture methodology. 2D surface cultures to 3D suspension cultures [1-3]. This clear trend is a product of growth methodology improve- ments arising leading to more realistic and relevant anal- ysis results. In order to best mimic natural cell growth, in vitro cultures attempt to closely model the extracellular support structure naturally occurring in tissues. In vivo tis- sue growth involves cell production of extracellular matrix material, composed primarily of various proteins. Artifi- Background g Cell culture techniques are an integral part of cell analysis. Culture methods are often not a primary concern in growth studies, but rather a means to experimental results. As more understanding of its influences is revealed, the significance of cell growth methodology is receiving considerably more attention. For example, it is increasingly common to see a shift in growth studies from Page 1 of 11 (page number not for citation purposes) http://www.biomedcentral.com/1472-6750/7/88 BMC Biotechnology 2007, 7:88 nanotubes, forming a rigid scaffold within the aqueous dispersion medium. cially produced extracellular matrices commonly used consist of mixtures of proteins such as collagen, fibrin, elastin, fibronectins, and laminins. Such components are understood to play a role in cell anchoring and separa- tion, intercellular communication, and even nutrient feed and waste displacement. Recent studies reveal the possi- bility for more simplified extracellular matrix materials. Here we present a new approach and application of self- assembling hydrogels for 3D cell cultures in various struc- tures, including but not limited to microscale structures. The novelty is in the method of formation of the cell cul- ture, enabling simple and rapid self-assembly of a gel matrix in a wide range of cell analysis structures. Sponta- neous gel formation was used to immobilize liquid cell suspensions in situ by simply adding a concentrated Fmoc peptide solution to the cell dispersion. Rapid formation of the fibrous network led to hydrogel formation that could adapt to the size, shape and even complexity of the pat- terned container. This method eliminates the problematic loading of gels or highly viscous precursors into the designed container. With increasing need for specificity of culture patterns, whether large or microscale, a flexible method for cell culture formation will prove useful, with possibilities to adapt to a plethora of structural designs. Along with the trend of 3D growth, modern cell laborato- ries strive to improve culture analysis platforms through size reduction and design specificity. Miniaturization dra- matically increases the efficiency of studies by allowing higher throughput studies and reducing unnecessary and excessive material use. It also allows the experimental designer to develop patterns with significantly increased complexity, making concurrent parallel studies possible. This added complexity often requires formation of well defined culture volumes. Micropatterned cell analysis platforms enable the use of microfluidics for cell treat- ment and analysis, providing new methods for cell study with increased possibilities and improved productivity. Background However, this concept has proven rather problematic with previously used artificial extracellular matrices. The use of microstructures introduces significant barriers for creating stable cell suspensions confined within small compart- ments. Some studies have employed rapid gel formation of a continuous laminar flow of cells in solution through microchannels [4]. Others involve pumping of a temper- ature sensitive sol-gel, followed by induced gel formation via temperature change [1]. The many complexities asso- ciated with gelling laminar flows and precision volume pumping can be eliminated by incorporating dipeptide derivative hydrogels. The combined use of 3D growth and culture patterning incorporates the advantages of both methods for higher efficacy and increased productivity. An additional feature of the hydrogel makes it increas- ingly favorable for use within complex structures. Despite the stability of the assembled hydrogel samples, multiple stimuli were seen to initiate reversing of the sample and breakdown of the hydrogel structure. Previous studies with peptide nanotubes and peptide derivative hydrogels have shown response to various types of treatment, whether changing stability or degrading entirely [8-10]. The Fmoc diphenylalanine gel demonstrated sensitivity to mechanical forces, environmental changes, and enzyme digestion. Use of one or more of these treatments enabled degradation of the gel and facilitated removal from the culture system. This can be highly favorable for systems with confined or inaccessible culture compartments. Such is often the case in microscale gel cultures, where micro- fluidic treatment requires compartmentalization of the immobilized cells. Earlier studies have incorporated simplistic 9-fluorenyl- methyl carbamate (Fmoc) peptide hydrogel, first intro- duced by Xu et al. [5], as a growth substrate, adding ease of gel formation and experimental flexibility. The earliest growth study of Fmoc peptide hydrogel presented both surface (2D) and 3D growth [6], followed almost imme- diately by a second publication on a similar dipeptide- derived hydrogel used in 2D culturing [7]. The simplicity of the latter Fmoc hydrogel featured by Mahler et al. is a product of two dominant features. First, the hydrogel is a superabsorbent material, primarily consisting of water; stable gel formation was consistently seen with as low as 0.5 wt% peptide derivative. Additionally, the seemingly complex structure of this fibrous matrix is entirely self- assembling. Introduction of appropriate Fmoc peptide concentrations to a suitable environment induces rapid self-assembly of hollow nanotubes. This low concentra- tion Fmoc dipeptide solution is able to support a gel con- formation because of its stable network of interweaving Page 2 of 11 (page number not for citation purposes) Results and Discussion Hydrogel Formation y g Hydrogel samples were created from a stock gelling agent of either 25 or 100 mg/ml. Successful gelling was per- formed in purified MilliQ water with both stock solu- tions. Final Fmoc peptide concentrations ranged from 0.5 to 1.0 wt% with successful hydrogel results. Each sample within the range passed the inversion test; examples can be seen in Figure 1. The higher concentrations resulted in a gel with increased stability. Higher concentrations resulted in hydrogels that resisted deformation when mechanical force was applied. The lower concentrations were deformed with less applied stress (mixing with a pipet or vortex). Macroscale samples (1 ml) assembled stable hydrogels in less than 10 minutes, while less time was required for smaller sample volumes. Alternate dis- Page 2 of 11 (page number not for citation purposes) Page 2 of 11 (page number not for citation purposes) BMC Biotechnology 2007, 7:88 http://www.biomedcentral.com/1472-6750/7/88 Inversion test Figure 1 Inversion test. Inversion test for in situ assembled hydrogel samples in water. Samples contain 0.5 and 1.0 wt% peptide. The peptide source is Fmoc-Phe-Phe in DMSO (100 mg/ml). Inversion test Figure 1 Inversion test. Inversion test for in situ assembled hydrogel samples in water. Samples contain 0.5 and 1.0 wt% peptide. The peptide source is Fmoc-Phe-Phe in DMSO (100 mg/ml). Table 1: Hydrogel formation results Dispersion Medium Gelling Agent Concentration (mg/ml) Final Peptide Concentration (wt%) Hydrogel Assembly Result 1. PBS 10× 100 0.5 Unstable 2. PBS 1× 100 0.5 Unstable 3. PBS 1× 100 1.0 Partial 4. PBS 1× 25 0.5 Soft Gel 5. PBS 1× 25 1.0 Firm Gel 6. MEM 25 0.5 Unstable Result of in situ gel formation in multiple dispersion media and Fmoc dipeptide concentrations (macroscale). All gelling agent concentrations and final Fmoc dipeptide concentrations produced stable gels in de-ionized water. Result of in situ gel formation in multiple dispersion media and Fmoc dipeptide concentrations (macroscale). All gelling agent concentrations and final Fmoc dipeptide concentrations produced stable gels in de-ionized water. traditional gels used for cell immobilization are highly temperature sensitive and require thermal control to pre- vent early gel formation. The peptide-derived hydrogel presented here requires no temperature regulation and can be designed to form entirely within the structure designed for microscopy cell analysis (detail provided in methods section). persion media appropriate for cell studies were also tested for hydrogel assembly. Results and Discussion Hydrogel Formation For large scale samples (1–2 ml), varying media and Fmoc dipeptide concentrations were tested. The results are listed in Table 1. All of the samples prepared as culture scaffolds within microchip chambers were significantly smaller than the milliliter test volumes. Both gelling agent concentrations resulted in stable hydrogels in purified water. The smaller samples showed sufficient gelling results for dispersions 2 and 3 from Table 1. Final Fmoc peptide concentrations for in situ hydrogels may vary from the macroscale samples, as they are dependent on the extent of dispersion within the microchamber. All hydrogels were assembled at room temperature (~20°C) so temperature effects on hydrogel self-assembly was not addressed. In addition to simplified handling, in situ formation allows for more flexible experimental design. Since gel formation is initiated within the assay structure, the pre- cursor is an aqueous solution that can be dispensed into a wide variety of patterned platforms. In this way, the hydrogel can be confined within an assay chamber designed with high experimental specificity. This allows for more complex design possibilities without the concern of difficult gel loading. The useful feature of adapting to various confining structures was demonstrated by remov- ing a rigid gel sample formed within a conical mold. In the case of microscale gel formation, loading a micro- fluidic device with a highly viscous gel can be rather com- plex and difficult to consistently reproduce. Some Page 3 of 11 (page number not for citation purposes) Page 3 of 11 (page number not for citation purposes) BMC Biotechnology 2007, 7:88 http://www.biomedcentral.com/1472-6750/7/88 Hydrogel mold Figure 2 Hydrogel mold. Firm hydrogel formed in a conical mold. Green stain was applied to the bottom after complete gel formation, demonstrating diffusion into solid gel. Height of the sample is approximately 20 mm. To show the absorbing character of the hydrogel, a sample was stained after the stable gel was formed. Addition of a dye to the sample after removal from the mold revealed a slow diffusion into the gel. Diffusion rates were not deter- mined, but transport of the applied dye demonstrated the diffusion of dispersion medium. With a highly rigid struc- ture, the shape was maintained, determined entirely by the contours of the mold. A resulting stained gel is shown in Figure 2. Page 4 of 11 (page number not for citation purposes) Simulated Figure 4 g g Simulated loading of microchamber. a) entire view of microchip system. b) close-up of empty chamber. c) after injection of aqueous cell suspension. d) spontaneous self-assembly of nanotube gel matrix. e) immobilization of cells in 3D hydrogel. f) view of cells confined solely to assay chamber. environmental changes. By varying the cells suspended in each layer, the interaction between differing strains can also be observed. This opens new possibilities for 3D cell studies at a well defined interface. Immobilized cells Figure 5 Immobilized cells. COS-7 cells immobilized in a 3D hydro- gel within a microchamber. The scale bar represents 30 μm. Results and Discussion Hydrogel Formation Once the gel is created in a mold, additional layers can be formed by adding a new layer of dispersion medium and applying additional gelling agent. With formation of each layer, the assembling hydrogel binds to the previously formed layer, resulting with an adherent stack. With this method, patterned layers can be formed in a well defined volume and shape with the possibility to vary composi- tion between layers. A sample of gel layering is presented in Figure 3. In this figure, one layer was formed upon a previously set gel layer within a cylindrical mold. The lay- ers are distinguishable by the varied dispersion media colors, with some diffusion seen across the layer bound- ary. The possibility to generate layered patterns of hydro- gel samples could also prove advantageous for experimental studies. The boundary condition at the layer interface can be utilized to examine cell response to Hydrogel mold Figure 2 Hydrogel mold. Firm hydrogel formed in a conical mold. Green stain was applied to the bottom after complete gel formation, demonstrating diffusion into solid gel. Height of the sample is approximately 20 mm. Hydrogel layering Figure 3 Hydrogel layering. Sample of hydrogel layering. Layers are distinguished by colored dye in dispersion media. Each layer inter- face is indicated by a dashed line. Hydrogel layering Figure 3 Hydrogel layering. Sample of hydrogel layering. Layers are distinguished by colored dye in dispersion media. Each layer inter- face is indicated by a dashed line. Page 4 of 11 (page number not for citation purposes) Page 4 of 11 (page number not for citation purposes) BMC Biotechnology 2007, 7:88 http://www.biomedcentral.com/1472-6750/7/88 Simulated loading of microchamber Figure 4 Simulated loading of microchamber. a) entire view of microchip system. b) close-up of empty chamber. c) after injection of aqueous cell suspension. d) spontaneous self-assembly of nanotube gel matrix. e) immobilization of cells in 3D hydrogel. f) view of cells confined solely to assay chamber. Cell Immobilization Hydrogel samples were used to immobilize cells in cul- ture plates as well as in various microstructures. We dem- onstrated the ease of generating well defined 3D cell suspensions within patterned silicon microstructures. Hydrogel was formed from aqueous cell suspensions within chambers designed for microfluidic treatment and confocal analysis. The three different cell types used for suspension studies were selected because they are typi- cally used in mammalian studies as human models; Astro- cyte (two strains), MDCK and COS 7 cells were used. Figure 4 presents a simulation of the microchip loading process. A sample of cell immobilizing hydrogel formed within a microstructure is seen in Figure 5. The fluorescing cells are seen suspended within the chamber designed with 5 μm vertical pillars (seen as dark circles in the figure). Immobilized cells Figure 5 Immobilized cells. COS-7 cells immobilized in a 3D hydro- gel within a microchamber. The scale bar represents 30 μm. Immobilized cells Figure 5 Immobilized cells. COS-7 cells immobilized in a 3D hydro- gel within a microchamber. The scale bar represents 30 μm. g Immobilized cells. COS-7 cells immobilized in a 3D hydro- gel within a microchamber. The scale bar represents 30 μm. Figure 6 shows a comparison of cells grown on a glass sur- face with a projection of cells suspended in 3D within the in situ assembled hydrogel. The 3D image was generated Page 5 of 11 (page number not for citation purposes) Page 5 of 11 (page number not for citation purposes) BMC Biotechnology 2007, 7:88 http://www.biomedcentral.com/1472-6750/7/88 Morphology comparison Figure 6 Morphology comparison. 2D surface growth on glass (left) and 3D growth in hydrogel (right). Scale bar in 2D image repre- sents 50 μm. Images are of MDCK cell cultures. Morphology comparison Figure 6 Morphology comparison. 2D surface growth on glass (left) and 3D growth in hydrogel (right). Scale bar in 2D image repre- sents 50 μm. Images are of MDCK cell cultures. Morphology comparison Figure 6 Morphology comparison. 2D surface growth on glass (left) and 3D growth in hydrogel (right). Scale bar in 2D image repre- sents 50 μm. Images are of MDCK cell cultures. assembly) Fmoc diphenylalanine hydrogels reported that limited or no net proliferation was seen after the first three days of cultivation, while measurable growth was evident after 7 days [6]. We performed additional growth studies with all of our cell strains. Cell Immobilization Over the first few days of cul- turing, relatively little growth was detected (none in most test samples) while cell viability remained. Viability was verified by administering calcein-AM to the hydrogel cul- ture. Viable cells were observable after calcein-AM uptake and internal modification to fluorescent calcein. Figure 7 presents an example of cell proliferation and/or migration in an in situ assembled hydrogel plug. This comparison of identical planes within the microchamber is between 4 and 5 days of incubation, for left and right images respec- tively. A majority of the cells remain in a fixed position, while some migration is seen with cells shifting in and out of the focal plane. There is a positive net change in cell number density shown between the images, resulting from a net flow into the focal plane and/or growth of new cells. Additional testing should be performed to differen- tiate the cell density change owing to migration from that of proliferation. from a stack of laser confocal microscopy scans of 10 μm increments over total thickness of 160 μm. The length and width of the image scale to 230 μm each. Cells grown on a 2D substrate tend to elongate on the surface and grow significantly larger than the spherical cells suspended in hydrogel, with typical lengths of 50 μm and 10–20 μm for surface and bulk cultures, respectively. This demonstrates the rather significant morphology difference between the dimensionally different methods of cell culture. As seen in naturally occurring human tissues, cells often grow in a 3D network. Given this spatial orientation, cells interact with both their surroundings and each other in a way fitting to their environment. Cellular function is likely to be significantly different when provided different stresses and orientation. The morphology difference dem- onstrated here is a closer to realistic tissue conformations. More realistic cell function, as affected by realistic cell morphology, should give rise to more relevant experimen- tal results. For better understanding of the cell immobili- zation and morphology changes, the cell-hydrogel interaction should be further examined. As cell attach- ment is considered significant to metabolic activity and general cell function, examination of cell-hydrogel inter- action would improve understanding of the cell's level of tolerance for the hydrogel environment. Page 6 of 11 (page number not for citation purposes) Gel Reversing Hydrogel samples were additionally tested for reversibil- ity. Introduction of mechanical stress was shown to dis- rupt the gel network and reduce the gel stability. Sheer forces were applied by pipette or vortex mixing to degrade the gel. Once the initial gel was disrupted, a fully stable, An initial 3D growth study demonstrating cultivation of Chondrocytes in preassembled (as opposed to in situ Page 6 of 11 (page number not for citation purposes) Page 6 of 11 (page number not for citation purposes) BMC Biotechnology 2007, 7:88 http://www.biomedcentral.com/1472-6750/7/88 Cell proliferation/motility Figure 7 Cell proliferation/motility. Some migration is noticeable as COS 7 cells shift in or out of the focal plane. Most of the origi- nal cells are seen to maintain their initial position and new cells are present (indicated by arrows). Scale bar represents 25 μm. The left image is after 4 days of incubation, and the right image is after 5 days of incubation. Cell proliferation/motility Figure 7 Cell proliferation/motility. Some migration is noticeable as COS 7 cells shift in or out of the focal plane. Most of the origi- nal cells are seen to maintain their initial position and new cells are present (indicated by arrows). Scale bar represents 25 μm. The left image is after 4 days of incubation, and the right image is after 5 days of incubation. cohesive gel was unattainable. Hydrogel formation was seen to be pH sensitive. It follows that the stability of the gel is also a condition of the environment pH. A stable sample modified from neutral pH to above pH 8.0 would no longer pass the inversion test. Further elevation to pH 9.0 was utilized to ensure extensive removal from struc- tures (described in next paragraph). More detail of the sta- bility was revealed with microscopic examination of the samples. Suspended microspheres (15 μm diameter) were monitored over a 10 minute period after an elevation of the pH. Hydrogel reversing was also examined via enzy- matic digestion. A gel sample (5 mg/ml Fmoc dipeptide) was treated with the digestion solution (final proteinase K concentration of 10 μg/ml) and compared to a control treated with an equivalent volume of water. Some macro- scale reversing effects were noted after incubation at 37°C for one hour. Inversion testing showed partial degrada- tion of macroscale samples. Reversing was also performed in the presence of microspheres. Gel Reversing Figure 8 shows the acquired microscopic results for both pH and enzymatic reversing. Both pH and enzyme treatment increased mobility of suspended particles. removal from a microchip. Nearly all traces of the gel have vanished after a 16.5 minute perfusion of the reversing solution. Extensive removal from micropatterned culture chambers was less attainable with preassembled hydro- gels as they were more difficult to confine exclusively to the microchamber. Any overfilling limited the ability to remove stable gel from the microstructures. Complete removal of other 3D matrix gel was extremely difficult and reuse of the microstructures was not feasible. Conclusion d i Introduction of Fmoc peptide hydrogels to cell culture methodology has shown potential to both simplify exper- imental procedures and increase protocol flexibility. In situ hydrogel formation eliminates the complexities asso- ciated with gel handling. With gelling, the samples become more difficult to handle and apply to the actual observation/analysis platform. The peptide-derivative hydrogel simplified both handling and loading of the gel to the microstructures. With easier gel formation, it is plausible to design and construct more complex culture systems. This opens the door to new possibilities and new approaches to cell biology. Further gel reversing was performed within microstruc- tures via continuous pumping (specific protocol in Meth- ods section). Active flow of the reversing solution increased breakdown of the hydrogel by combining flow- generated sheer stress, pH modification (pH 9.0 and above), and enzyme digestion. This method was sufficient for significant removal of hydrogel plugs within micro- structures. Figure 9 shows confocal images of hydrogel One intriguing possibility that this hydrogel enables is the study of cells at an interface. With the ability to easily con- trol the cell type, cell density and composition of each given hydrogel layer, interactions can be studied at a well defined interface. This culture method allows one to study cellular interaction in a controlled manner that can be adapted to a variety of scales and designs. Page 7 of 11 (page number not for citation purposes) Page 7 of 11 (page number not for citation purposes) BMC Biotechnology 2007, 7:88 http://www.biomedcentral.com/1472-6750/7/88 Hydrogel Reversibility Figure 8 Hydrogel Reversibility. Time resolved confocal scan of 15 μm spheres suspended in situ with pH and enzyme triggered reversing. Color indicates time; red is the start time, green is at 5 minutes, blue is at 10 minutes. Lack of color differentiation indicates limited mobility. Hydrogel Reversibility Figure 8 Hydrogel Reversibility. Time resolved confocal scan of 15 μm spheres suspended in situ with pH and enzyme triggered reversing. Color indicates time; red is the start time, green is at 5 minutes, blue is at 10 minutes. Lack of color differentiation indicates limited mobility. Another significant benefit of the hydrogel as a culture method is the improved relevance. The goal of mimicking in vivo cell growth required a more realistic spatial growth arrangement than conventional 2D surface growth. Immobilization of suspended cells allows for a 3D disper- sion of cells throughout the gel. Page 8 of 11 (page number not for citation purposes) Conclusion d i This arrangement more closely models the in vivo growth of cells seen in tissues. Microcopy also revealed that cells suspended in a hydro- gel sample tend to adopt a 3D structure, rather than the elongated conformation seen in surface cultures. The sup- port provided by a 3D gel results in more spherical cell growth. This may have significant implications in cell studies as more relevant and realistic results are desired. Hydrogel Removal Figure 9 Hydrogel Removal. Removal of a stable hydrogel plug from a microchip assay chamber with perfusion of enzymatic removal solution. Time interval between frames is 90 sec- onds. Each frame is labeled chronologically, with 'a' as the ini- tial frame. The assay chamber is 500 μm wide. With a trend toward miniaturization of cell studies, microstructures become more common culture platforms. As micropatterning can carry a significant expense, the ability to reuse the microstructures can be economically preferable. Traditional gels used in 3D cell culture can be extremely difficult to remove from microstructures due to the increased surface interactions. Hydrogel reversibility by means of applied force, environmental change, or enzymatic digestion facilitates breakdown of the rigid gel structure. Removal of the hydrogel culture can then allow for repeated experiments within a single device. Hydrogel reversing was observed, but could be improved with fur- ther studies. Increased specificity of enzyme activity could dramatically increase the rate and extent of Fmoc peptide digestion. With improved reversal, total removal could be achieved with non-intrusive methods, possibly facilitating downstream recovery of cells for additional testing. Hydrogel Removal Figure 9 Hydrogel Removal. Removal of a stable hydrogel plug from a microchip assay chamber with perfusion of enzymatic removal solution. Time interval between frames is 90 sec- onds. Each frame is labeled chronologically, with 'a' as the ini- tial frame. The assay chamber is 500 μm wide. Use of the hydrogel as a cell culture medium showed many additional benefits. Living cells suspended within the dispersion medium were easily immobilized with addition of the gelling agent. Immobilization of the cells allows for easy monitoring over extended period of time. Microscopic studies demonstrated that the immobilized cells were not restricted from migration or possible prolif- eration, while maintaining high viability. Page 8 of 11 (page number not for citation purposes) BMC Biotechnology 2007, 7:88 http://www.biomedcentral.com/1472-6750/7/88 Microchip platform and Perfusion System Figure 10 Microchip platform and Perfusion System. Conclusion d i Left image is the microchip platform designed for a confocal microscope stage. Right image is the tubing configuration for continuous perfusion that was performed via a syringe pump and the tubing configuration presented here. Holes in cover-plate (right) coincide with inlet and outlet holes in the microchannels. Microchip platform and Perfusion System Figure 10 Microchip platform and Perfusion System. Left image is the microchip platform designed for a confocal microscope stage. Right image is the tubing configuration for continuous perfusion that was performed via a syringe pump and the tubing configuration presented here. Holes in cover-plate (right) coincide with inlet and outlet holes in the microchannels. Methods Gel Formation only examined the use of 25 mg/ml gelling agents for a final hydrogel concentration of 10 mg/ml. Diffusion of dispersion medium in layers was observed by adding a colored dye in one layer of hydrogel. For general diffusion observation, a drop of dye was applied to a stable get and allowed to diffuse into the sample. The self-assembling hydrogel samples were made by first creating a gelling agent. Lyophilized Fmoc-Phe-Phe dipeptide (Bachem) was weighed and dissolved in dime- thyl sulfoxide (DMSO). Deviating from the preliminary methods provided by Mahler et al., DMSO was selected to replace 1,1,1,3,3,3-hexafluoro-2-propanol because it is less detrimental to cell viability. The gelling agent was used in concentrations of 25 and 100 mg/ml. A small stock solution was created to run experiments for each day so as to avoid any pre-aggregation of dipeptide deriva- tives. The gelling agent was applied to the desired disper- sion medium by pipette addition to a final concentration of either 5 or 10 mg/ml; the higher concentration corre- sponded to increased gel stability. The gel was created in situ within the desired analysis structure. All structures were fabricated via silicon etching/masking procedures by the Microsystems Technology Group in the School of Electrical Engineering, Royal Institute of Technology, Swe- den. Transparent glass cover slides were bonded to the etched surface to create channels and chambers for micro- scopic observation. Gel Reversing Mechanical reversing was performed by applying sheer forces to a stable gel. Large samples were destabilized by vortex or pipette mixing. Environmental pH changes were also used to reverse assembled gel stability. NaOH (0.5M) was added to elevate the matrix solution above pH 9. Enzyme reversing was performed with proteinase K (Sigma-Aldrich). Reversing in microstructures was per- formed by actively applying a flow of reversing solution: 0.05 mg/ml proteinase K, 1.0 wt% SDS, pH between 9 and 10, and temperature control at 37°C. Microchambers were all flanked by parallel channels (Figure 4) where the enzyme reversing solution was perfused. Precision pumps were fitted to drive the solution from a syringe, through flexible tubing, into the inlets of the parallel microchan- nels, and out through the outlets. The microchip stage and perfusion tubing setup is shown in Figure 10. Layer formation was performed by repetition of the gel forming procedure. The initial layer was first generated in a generic mold and allowed to stabilize. Upon stabile hydrogel formation, the additional dispersion media was added to the mold, forming a fluid layer above the stable hydrogel. An appropriate amount of gelling agent was directly added to the fluid layer. Layers were only formed in purified water, but it can be assumed that all successful dispersion media can also be used in layer formation. We Page 9 of 11 (page number not for citation purposes) Detecting Gel Transitions To confirm gel formation, two protocols were employed. The first was a macroscopic observation of the gel stabil- ity. In the presence of a rigid conformation, the hydrogel should be sufficiently stable to undergo and inversion test without conformational changes. Inversion testing was performed on 2 ml samples in a 15 ml Falcon Tube. After appropriate stabilization time, the sample was inverted Page 9 of 11 (page number not for citation purposes) Page 9 of 11 (page number not for citation purposes) http://www.biomedcentral.com/1472-6750/7/88 BMC Biotechnology 2007, 7:88 phosphate buffered saline solution (1× PBS). The final cell pellet was then immobilized in the peptide derivative extracellular matrix by first suspending in PBS and adding the gelling agent to initiate hydrogel self-assembly. A typ- ical cell number density used was between 5 and 12 mil- lion cells/ml of final gel volume. Nutrients were supplied to the immobilized cell suspensions by perfusion as soon as the hydrogel was stabilized. For long-term culturing, the microstructures were removed from the microchip platform and submerged in culture media under 37°C. Cells were viewed by staining with 1.0 μM calcein-AM (Invitrogen). If the cells were to be stained before in situ culture formation, calcein-AM was added to the disper- sion medium. For in situ staining within the microcham- ber, calcein-AM was added to a perfusion solution of growth medium and perfused through the microchannels. Cells were maintained in stain solution for 30 minutes to 1 hour at 37°C before confocal observation. and monitored for deformation. A sample undergoing incomplete gel transition shows deformation in the form of flow toward the lid (lowest point of the inverted tube). A complete and stable hydrogel transition was character- ized by an inversion test resulting in zero visible deforma- tion. A successful inversion test confirmed stable macroscopic hydrogel formation. The second protocol utilized to verify gel stability required observation at the microscopic level. Optically distin- guishable foreign material was added to the gel and mon- itored by confocal microscopy. The 'impurity' behavior was then used to infer hydrogel characteristics. Fluores- cent microspheres (15 μm Focal Check microspheres, Molecular Probes) were suspended in the dispersion medium of choice just prior to addition of the gelling agent. The microspheres were then induced with 488 nm laser excitation on a Zeiss LSM 5 Pascal confocal micro- scope over a 10 minute detection time interval. Detecting Gel Transitions Confocal tracking of the microspheres presented the level of stabil- ity of the hydrogel matrix in the form of sphere immobil- ity. Immobilization of the suspended microspheres over an extended time period was a sufficient indication of a stable hydrogel matrix. Acknowledgements We would like thank Thomas Frisk (Microsystems Technology Group in the School of Electrical Engineering, Royal Institute of Technology, Sweden) for providing microstructures. Additional thanks to Gunaratna Rajarao (Applied Environmental Microbiology, Royal Institute of Technology, Swe- den) for her assistance and support. Funding for this project was provided by the Swedish Research Council and the European Community-funded CellPROM project under the 6th Framework Program, contract no. NMP4- CT-2004-500039. Authors' contributions TL was the primary researcher for this project. He also designed the project and wrote the manuscript. In addi- tion to assisting with cell handling, SR provided instruc- tion and guidance for inclusion of the microsystems and microfluidics. VA provided general assistance and guid- ance in chemical synthesis. HB was the guiding supervisor for both the experimental work and the manuscript writ- ing. All authors have read and approved the manuscript. Stable hydrogel samples were monitored for sensitivity to multiple reversing methods. The above gel transition methods were utilized to demonstrate reverse transitions from stable a stable hydrogel matrix to a less stable liquid solution. If a stable hydrogel sample can be modified such that it no longer satisfies the discussed requirements for a stable gel, the sample has reverted from a gel to a liquid phase solution. More explicitly, a sample that fails the inversion test or shows a dramatic increase in microscopic movement upon modification is no longer considered a stable hydrogel. References The cell lines examined in hydrogel cultures include COS- 7, MDCK, and two strains of Astrocytes (CTX TNA2 and DI TNC1), all obtained from ECACC. Each cell line was cultured according to the ECACC recommendations. Astrocytes were grown to confluency in a liquid culture of MEM (Gibco) and the remaining cell lines were grown in DMEM (Sigma). Cells were grown with an addition of 10% fetal bovine serum (Gibco), 1.0% penicillin/strepto- mycin (Sigma) and 1.0% L-glutamine (Sigma). MDCK cells were grown with an additional treatment of 1.0% non-essential amino acids (Sigma). Incubation was per- formed at 37°C with 5% CO2 and 100% humidity. The cells were harvested from the culturing petri dishes by trypsinization. The harvested cells were spun down to a pellet by centrifugation for 5 minutes at 1700 rpm. Cells were then washed twice before a final spin down. Staining treatments and washing were performed with a standard References 1. Frisk T, Rydholm S, Andersson H, Stemme G, Brismar H: A Concept For Miniaturized 3-D Cell Culture Using an Extracellular Matrix Gel. Electrophoresis 2005, 26:4751-4758. 2. Paguirigan A, Beebe DJ: Gelatin Based Microfluidic Devices for Cell Culture. Lab on a Chip 2006, 6:407-413. 3. Albrecht DR, Underhill DH, Wassermann TB, Sah RL, Bhatia SN: Probing the Role of Multicellular Organization in Three- Dimensional Microenvironments. Nature Methods 2006, 5:369-375. 4. Minseok KS, Yeon JH, Park JK: A Microfluidic Platform for 3- Dimensional Cell Culture and Cell-Based Assays. Biomedical Microdevices 2007, 9:25-34. 5. Zhang Y, Gu H, Yang Z, Xu : Supramolecular Hydrogels Respond to Ligand-Receptor Interaction. JACS 2003, 125:13680-13681. 6. Jayawarna V, Ali M, Jowitt TA, Miller AF, Saiani A, Gough JE, Ulijn RV: Nanostructured Hydrogels for Three-Dimensional Cell Cul- ture Through Self-Assembly of Fluorenylmethoxycarbonyl- Dipeptides. Advanced Materials 2006, 18:611-614. 7. Mahler A, Reches M, Rechter M, Cohen S, Gazit E: Rigid, Self- Assembled Hydrogel Composed of a Modified Aromatic Dipeptide. Advanced Materials 2006, 18:1365-1370. 1. Frisk T, Rydholm S, Andersson H, Stemme G, Brismar H: A Concept For Miniaturized 3-D Cell Culture Using an Extracellular Matrix Gel. Electrophoresis 2005, 26:4751-4758. 1. Frisk T, Rydholm S, Andersson H, Stemme G, Brismar H: A Concept For Miniaturized 3-D Cell Culture Using an Extracellular Matrix Gel. Electrophoresis 2005, 26:4751-4758. p 2. Paguirigan A, Beebe DJ: Gelatin Based Microfluidic Devices for Cell Culture. Lab on a Chip 2006, 6:407-413. p 2. Paguirigan A, Beebe DJ: Gelatin Based Microfluidic Devices for Cell Culture. J , 10. Reches M, Gazit E: Casting Metal Nanowires with Discrete Self-Assembled Peptide Nanotubes. Science 2003, 300:625-627. BMC Biotechnology 2007, 7:88 y g y 9. Toledano S, Williams RJ, Jayawarna V, Ulijn R: Enzyme-Triggered Self-Assembly of Peptide Hydrogels via Reversed Hydrolysis. JACS 2006, 128:1070-1071. 8. Qiu Y, Park K: Environment-Sensitive Hydrogels for Drug Delivery. Advanced Drug Delivery Reviews 2001, 53:31-339. 9. Toledano S, Williams RJ, Jayawarna V, Ulijn R: Enzyme-Triggered Self-Assembly of Peptide Hydrogels via Reversed Hydrolysis. JACS 2006, 128:1070-1071. 10. Reches M, Gazit E: Casting Metal Nanowires with Discrete Self-Assembled Peptide Nanotubes. Science 2003, 300:625-627. 8. Qiu Y, Park K: Environment-Sensitive Hydrogels for Drug Delivery. Advanced Drug Delivery Reviews 2001, 53:31-339. References Lab on a Chip 2006, 6:407-413. p 3. Albrecht DR, Underhill DH, Wassermann TB, Sah RL, Bhatia SN: Probing the Role of Multicellular Organization in Three- Dimensional Microenvironments. Nature Methods 2006, 5:369-375. 4. Minseok KS, Yeon JH, Park JK: A Microfluidic Platform for 3- Dimensional Cell Culture and Cell-Based Assays. Biomedical Microdevices 2007, 9:25-34. 5. Zhang Y, Gu H, Yang Z, Xu : Supramolecular Hydrogels Respond to Ligand-Receptor Interaction. JACS 2003, 125:13680-13681. 6. Jayawarna V, Ali M, Jowitt TA, Miller AF, Saiani A, Gough JE, Ulijn RV: Nanostructured Hydrogels for Three-Dimensional Cell Cul- ture Through Self-Assembly of Fluorenylmethoxycarbonyl- Dipeptides. Advanced Materials 2006, 18:611-614. p p , 7. Mahler A, Reches M, Rechter M, Cohen S, Gazit E: Rigid, Self- Assembled Hydrogel Composed of a Modified Aromatic Dipeptide. Advanced Materials 2006, 18:1365-1370. p p 7. Mahler A, Reches M, Rechter M, Cohen S, Gazit E: Rigid, Self- Assembled Hydrogel Composed of a Modified Aromatic Dipeptide. Advanced Materials 2006, 18:1365-1370. Page 10 of 11 (page number not for citation purposes) Page 10 of 11 (page number not for citation purposes) BMC Biotechnology 2007, 7:88 http://www.biomedcentral.com/1472-6750/7/88 8. Qiu Y, Park K: Environment-Sensitive Hydrogels for Drug Delivery. Advanced Drug Delivery Reviews 2001, 53:31-339. 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https://openalex.org/W3003284395	http://old.scielo.br/pdf/rbent/v64n1/1806-9665-rbent-64-1-e2019100.pdf	English	null	Identification key for drosophilid species (Diptera, Drosophilidae) exotic to the Neotropical Region and occurring in Brazil	Revista Brasileira de entomologia/Revista brasileira de entomologia	2,020	cc-by	6,795	* Corresponding author Email: rotidon@unb.br (R.Tidon) Identification key for drosophilid species (Diptera, Drosophilidae) exotic to the Neotropical Region and occurring in Brazil Keven Yuzuki1, Rosana Tidon1*  1Universidade de Brasília, Instituto de Ciências Biológicas, Brasília, DF, Brasil. A R T I C L E I N F O Thirteen species of drosophilid exotic to the Neotropical Region are recorded in Brazil, and some of them are highly invasive and threaten significantly fruit cultures. We provide an illustrated key for identifying these species, and briefly discuss their taxonomic status, distribution, and occurrence in the Neotropics. The key should not only support newcomers to the study of Drosophila but also facilitate their identification by those interested in insects associated with cultivated areas. Article history: Received 20 October 2019 Accepted 07 December 2019 Available online 31 January 2020 Associate Editor: Claudio Carvalho Article history: Received 20 October 2019 Accepted 07 December 2019 Available online 31 January 2020 Associate Editor: Claudio Carvalho Keywords: biological invasions, Drosophila, invasive species, Scaptodrosophila, Zaprionus. Keywords: biological invasions, Drosophila, invasive species, Scaptodrosophila, Zaprionus. Keywords: biological invasions, Drosophila, invasive species, Scaptodrosophila, Zaprionus. https://doi.org/10.1590/1806-9665-RBENT-2019-100 © 2020 Sociedade Brasileira de Entomologia Published by SciELO - Scientific Electronic Library Online.. This is an open-access arti Commons Attribution License (type CC-BY), which permits unrestricted use, distribution and reproduction in any medium, provided the origina © 2020 Sociedade Brasileira de Entomologia Published by SciELO - Scientific Electronic Library Online.. This is an open-access article distributed under the terms of the Creative Commons Attribution License (type CC-BY), which permits unrestricted use, distribution and reproduction in any medium, provided the original article is properly cited. Revista Brasileira de Entomologia 64(1):e2019100, 2020 Revista Brasileira de Entomologia 64(1):e2019100, 2020 de Entomologia Published by SciELO - Scientific Electronic Library Online.. This is an open-access article distributed under the terms of the Creat ), which permits unrestricted use, distribution and reproduction in any medium, provided the original article is properly cited. p // g/ / © 2020 Sociedade Brasileira de Entomologia Published by SciELO - Scientific Electronic Library O Commons Attribution License (type CC-BY), which permits unrestricted use, distribution and reproduction in a https://doi.org/10.1590/1806-9665-RBENT-2019-100 © 2020 Sociedade Brasileira de Entomologia Published by SciELO - Scientific Electronic Library Online.. This is an open-access article distributed under the terms of the Creative Commons Attribution License (type CC-BY), which permits unrestricted use, distribution and reproduction in any medium, provided the original article is properly cited. Introduction 1F)..……………......……….… Drosophila busckii 3. Dark brown fly. Carina longitudinally grooved (Fig. 1G). Basal scutellar setae divergent (Fig. 1H). Wing crossveins slightly shaded (Fig. 1I). Costal index about 3.0. Abdominal tergites 2-6 completely dark .............................................……Drosophila virilis - Basal scutellar setae convergent (Fig. 1J) …..................……........4 4 Presence of cuneiform setae on the inner side of profemur (Fig. 2A) .…......…....................................................................................5 - Absence of cuneiform setae on the inner side of profemur ….. .............................................................................................................…....6 5 Face region with a silvery-shining pigmentation (Fig. 2B), this area is more easily visualized in dry preserved males. Pleura with a wide, slightly brownish stripe (Fig 2C). Costal index about 3.1 .................................................................Drosophila nasuta - Male protarsomeres 1 and 2 with a dense brush of long hairy setae (Fig. 2D). Wings with crossveins and tips of longitudinal veins slightly shaded (Fig. 2E), costal index about 4.4. Abdominal bands medially interrupted with triangular marginal bands (Fig. 2F) ............................................................Drosophila immigrans 6 Presence of prescutellar setae on mesonotum (Fig. 2G). Light hyaline wings. Sex combs absent (Scaptodrosophila genus)…..…....…...................................................................………….7 - Absence of prescutellar setae on mesonotum. Males with sex combs on protarsus (melanogaster species group) ……….………………8 7 Light yellow fly. Costal index about 1.5. Abdomen deep dark (Fig. 2H) ….........…….............…....…...Scaptodrosophila latifasciaeformis - Dark brown fly. Costal index about 2.1 Large brown abdominal bands ………..……………….…Scaptodrosophila lebanonensis 8 Male protarsus with two small rows of 3-4 peg-like setae forming a sex comb (Fig. 2I). Male wings with a large subdistal black spot (Fig. 3A), costal index about 3.5. Females with a large serrated oviscapt (Fig 3B) ................................Drosophila suzukii - Male protarsus with a single row of peg-like setae forming a sex comb (Fig. 3C). Costal index 2.4. Male epandrial posterior lobe small and nearly triangular (Fig. 3D). Female abdomen with about 2.6. Subapical setae on fourth and fifth abdominal tergites arising from blackish spots……...........…… Zaprionus indianus identification resources that can be easily used by non-specialists. The primary dichotomous key for drosophilids in Brazil (Freire-Maia and Pavan, 1949) attended several generations of researchers, and certainly contributed for spreading the study of these flies in the country. However, it does not include the recently introduced species and needs to be taxonomically upgraded (e.g. Drosophila mirim was synonymized as Scaptodrosophila latifasciaeformis). Here, we provide an illustrated identification key for 13 exotic drosophilid species recorded in Brazil and briefly discuss their taxonomic status, distribution, and occurrence in the Neotropics. Introduction The Taxonomic Catalog of the Brazilian Fauna (TCBF) records 305 drosophilid species in this country (Tidon et al., 2019). Most are neotropical and ecologically restricted to a particular type of vegetation. Thirteen of these species, however, are exotic to the Neotropical Region and widely distributed in the world. Some (e.g., Drosophila melanogaster Meigen and D. simulans Sturtevant) possibly arrived in Brazil in the 16th century, transported by slave ships from Africa. Others reached the country later, throughout trade ships and airplanes. From the late 20th century, four new arrivals in the Neotropics were accurately recorded in the earlier stages of invasion: D. malerkotliana Parshad and Paika (Val and Sene, 1980), Zaprionus indianus Gupta (Vilela, 1999), D. nasuta Lamb (Vilela and Goñi, 2015) and D. suzukii Matsumura (Deprá et al., 2014). consequences for biodiversity (Millennium Ecosystem Assessment, 2005). Moreover, invasive species can also cause economic troubles. In Brazil, annual losses to major crops caused by alien species are estimated to about US$1.6 billion (ca. 16% caused by dipterans), and for USA and India this value is even higher (Oliveira et al., 2013). Therefore, it is crucial to understand the biology and distribution of exotic species (Sakai et al., 2001). In Brazil, drosophilid communities have been studied by several independent research groups, covering the Amazonian and Atlantic forests (Medeiros and Klaczko, 2004; Penariol and Madi-Ravazzi, 2013; Coutinho-Silva et al., 2017; Santa-Brígida et al., 2017), the Cerrado biome (Tidon, 2006; Blauth and Gottschalk, 2007), Caatinga (Rohde et al., 2010; Garcia et al., 2014; Oliveira et al., 2016), araucarias (Cavasini et al., 2014), pampas (Poppe et al., 2016) and urban sites (Ferreira and Tidon, 2005; Gottschalk et al., 2007). Exotic species are recorded in all drosophilid assemblages, and their relative abundance depends on many factors, as vegetation type, the season of the year, and disturbance (Mata et al., 2015). The impacts of biological invasions have been widely recognized since the seminal book The ecology of invasions by animals and plants (Elton, 1958). Ecological interactions between invasive and native species, like predation and competition, often affect the population dynamics (births, deaths, migration) of native species and bring severe negative Taxonomic identification based on morphological characters is an effective way of determining many drosophilid species, especially the non-natives in a particular region. However, there is a lack of K. Yuzuki, R. Tidon / Revista Brasileira de Entomologia 64(1):e2019100, 2020 2-9 about 2.6. Introduction Subapical setae on fourth and fifth abdominal tergites arising from blackish spots……...........…… Zaprionus indianus - Yellowish fly with three dark-brown stripes across the mesonotum, median one forked in posterior half (Fig. 1D). Pleura with two horizontal stripes (Fig. 1E). Costal index about 3.1. Abdomen with dark bands interrupted in the middle and narrowed at the sides (Fig. 1F)..……………......……….… Drosophila busckii 3. Dark brown fly. Carina longitudinally grooved (Fig. 1G). Basal scutellar setae divergent (Fig. 1H). Wing crossveins slightly shaded (Fig. 1I). Costal index about 3.0. Abdominal tergites 2-6 completely dark .............................................……Drosophila virilis - Basal scutellar setae convergent (Fig. 1J) …..................……........4 4 Presence of cuneiform setae on the inner side of profemur (Fig. 2A) .…......…....................................................................................5 - Absence of cuneiform setae on the inner side of profemur ….. .............................................................................................................…....6 5 Face region with a silvery-shining pigmentation (Fig. 2B), this area is more easily visualized in dry preserved males. Pleura with a wide, slightly brownish stripe (Fig 2C). Costal index about 3.1 .................................................................Drosophila nasuta - Male protarsomeres 1 and 2 with a dense brush of long hairy setae (Fig. 2D). Wings with crossveins and tips of longitudinal veins slightly shaded (Fig. 2E), costal index about 4.4. Abdominal bands medially interrupted with triangular marginal bands (Fig. 2F) ............................................................Drosophila immigrans 6 Presence of prescutellar setae on mesonotum (Fig. 2G). Light hyaline wings. Sex combs absent (Scaptodrosophila genus)…..…....…...................................................................………….7 - Absence of prescutellar setae on mesonotum. Males with sex combs on protarsus (melanogaster species group) ……….………………8 7 Light yellow fly. Costal index about 1.5. Abdomen deep dark (Fig. 2H) ….........…….............…....…...Scaptodrosophila latifasciaeformis - Dark brown fly. Costal index about 2.1 Large brown abdominal bands ………..……………….…Scaptodrosophila lebanonensis 8 Male protarsus with two small rows of 3-4 peg-like setae forming a sex comb (Fig. 2I). Male wings with a large subdistal black spot (Fig. 3A), costal index about 3.5. Females with a large serrated oviscapt (Fig 3B) ................................Drosophila suzukii - Male protarsus with a single row of peg-like setae forming a sex comb (Fig. 3C). Costal index 2.4. Male epandrial posterior lobe small and nearly triangular (Fig. 3D). Female abdomen with about 2.6. Subapical setae on fourth and fifth abdominal tergites arising from blackish spots……...........…… Zaprionus indianus - Yellowish fly with three dark-brown stripes across the mesonotum, median one forked in posterior half (Fig. 1D). Pleura with two horizontal stripes (Fig. 1E). Costal index about 3.1. Abdomen with dark bands interrupted in the middle and narrowed at the sides (Fig. Material and Methods We analyzed specimens of 11 among the 13 non-neotropical drosophilids occurring in Brazil and collected data of Drosophila virilis and Scaptodrosophila lebanonensis from the literature (Table 1). The illustrations were hand-made utilizing a camera lucida coupled to a Leica MZ75 stereoscope and followed traditional China ink scientific illustration methods reassembling those from Bächli et al. (2004). The morphological terms used in the dichotomous key are also based mainly on Bächli et al. (op cit.). The dichotomous key does not consider the degree of phylogenetic correlation, but rather the difficulty of identifying each specimen. Thus, the species with the most striking morphological characteristics (Z. indianus with their bright stripes and D. busckii with their mesonotum patterns) were placed at the first steps of the key. Those specimens that required the rigorous analysis of bristles or sex combs (Scaptodrosophila genus and melanogaster species group) were left for the end section of the key. The characters addressed here to identify females of D. melanogaster and D. simulans are useful for lab routine or identifying flies in the field, but insufficient for a taxonomic study because the black pigmentation on each tergite is highly variable according to genotype and grow temperature (Moreteau et al., 1995). For more accurate techniques such as egg-shape inspection or wing and thorax size ratio see Moreteau et al. (op cit.). Identification key for non-neotropical drosophilid species occurring in Brazil 1. Presence of stripes on mesonotum…………….…...…………….2 - Absence of stripes on mesonotum…………...…………………...3 2. Yellowish fly, with two silvery-white stripes bordered by black across the head (Fig. 1A). Four stripes across the mesonotum and two in the scutellum (Fig. 1B). Two reduced prescutellar setae. Profemur with a row of strong setae, each one arising from a small tubercle with another setula (Fig. 1C). Costal index Table 1 Non-neotropical species of Drosophilidae recorded in Brazil. Genus Subgenus Species Source Drosophila Dorsilopha D. busckii Coquillett collection Drosophila D. immigrans Sturtevant collection D. nasuta Lamb lab strain D. virilis Sturtevant literature* Sophophora D. ananassae Doleschall lab strain D. kikkawai Burla collection D. malerkotliana Parshad and Paika lab strain D. melanogaster Meigen lab strain D. simulans Sturtevant lab strain D. suzukii Matsumura collection Scaptodrosophila S. lebanonensis Wheeler literature** S. latifasciaeformis Duda collection Zaprionus Z. indianus Gupta collection * description and Miller et al. (2017) ** description and Bächli et al. (2005). Table 1 K. Yuzuki, R. Tidon / Revista Brasileira de Entomologia 64(1):e2019100, 2020 its large dark bands on the sixth tergite running to its ventral margin (Fig. 3E).………......................…Drosophila melanogaster - Male protarsus with a single row of peg-like setae, forming a sex comb (Fig. 3C). Costal index 2.3. Male epandrial posterior lobe very large and roundish with an amber like color (Fig. 3F). Female abdomen pigmentation border line making an angle with the sixth tergite margin (Fig. 3G) …..................................... .................................................................…......Drosophila simulans - Male protarsus with several transverse rows of short setae the ventral surface, forming an indistinct sex comb (Fig. 3 Costal index 1.5 .............................................Drosophila ananass - Male protarsus with three small rows of 2-3 peg-like set forming a sex comb (Fig. 3I). Costal index 1.7............................ ..................................................................... Drosophila malerkotlia - Male protarsus with two rows of peg-like setae, forming a s comb (Fig. 3J). Costal index 1.9................... Drosophila kikkaw K. Yuzuki, R. Tidon / Revista Brasileira de Entomologia 64(1):e2019100, 2020 3-9 its large dark bands on the sixth tergite running to its ventral margin (Fig. 3E).………......................…Drosophila melanogaster - Male protarsus with a single row of peg-like setae, forming a sex comb (Fig. 3C). Costal index 2.3. Male epandrial posterior lobe very large and roundish with an amber like color (Fig. 3F). Female abdomen pigmentation border line making an angle with the sixth tergite margin (Fig. 3G) …..................................... .................................................................…......Drosophila simulans ................................................................…......Drosophila simulans comb (Fig. 3J). Costal index 1.9................... Drosophila kikkawai Zaprionus indianus, Drosophila busckii and D. virilis. A. Head of Z. indianus in frontal view, showing the two silvery stripes bordered by black. B. Thorax of Z. indianus in w, showing the four silvery stripes bordered by black. C. Profemur of Z. indianus, showing the row of characteristic strong setae. D. Thorax of D. busckii in dorsal view ts characteristic dark stripes, the middle one bifurcating at the base. E. Thorax of D. busckii in lateral view with its characteristic dark stripes. F. Abdomen of D. busckii he bands interrupted in the middle. G. Head of D. virilis in frontal view, showing its carina with a longitudinal groove at midline. H. Divergent basal scutellar setae wing, showing the shaded crossveins. J. Convergent basal scutellar setae setae. Bar = 1mm. Illustrations of D. virilis were based on published images without scale al., 2017). Figure 1. Zaprionus indianus, Drosophila busckii and D. virilis. A. Head of Z. Table 1 indianus in frontal view, showing the two silvery stripes bordered by black. B. Thorax of Z. indianus in dorsal view, showing the four silvery stripes bordered by black. C. Profemur of Z. indianus, showing the row of characteristic strong setae. D. Thorax of D. busckii in dorsal view, showing its characteristic dark stripes, the middle one bifurcating at the base. E. Thorax of D. busckii in lateral view with its characteristic dark stripes. F. Abdomen of D. busckii, showing the bands interrupted in the middle. G. Head of D. virilis in frontal view, showing its carina with a longitudinal groove at midline. H. Divergent basal scutellar setae. I. D. virilis wing, showing the shaded crossveins. J. Convergent basal scutellar setae setae. Bar = 1mm. Illustrations of D. virilis were based on published images without scale (Miller et al., 2017). K. Yuzuki, R. Tidon / Revista Brasileira de Entomologia 64(1):e2019100, 2020 4-9 ure 2. Drosophila nasuta, D immigrans, Scaptodrosophila latifasciaeformis, and D. suzukii. A. Profemur with a row of cuneiform setae. B. Head of D. nasuta in frontal view, showing characteristic silvery pigmentation in the face region. C. Thorax of D. nasuta in lateral view, showing the large brownish stripe on half dorsal area of pleura. D. D. immigrans otarsomeres, showing the brush like thin setae. E. D. immigrans wing, showing the shaded cross veins and ends of longitudinal veins. F. D. immigrans abdomen, showing the nds interrupted in the middle. G. Prescutellar setae. H. S. latifasciaeformis dark abdomen. I. D. suzukii protarsus with sex combs. Bar = 1mm. Figure 2. Drosophila nasuta, D immigrans, Scaptodrosophila latifasciaeformis, and D. suzukii. A. Profemur with a row of cuneiform setae. B. Head of D. nasuta in frontal view, show the characteristic silvery pigmentation in the face region. C. Thorax of D. nasuta in lateral view, showing the large brownish stripe on half dorsal area of pleura. D. D. immig protarsomeres, showing the brush like thin setae. E. D. immigrans wing, showing the shaded cross veins and ends of longitudinal veins. F. D. immigrans abdomen, showing bands interrupted in the middle G Prescutellar setae H S latifasciaeformis dark abdomen I D su ukii protarsus ith se combs Bar 1mm Figure 2. Drosophila nasuta, D immigrans, Scaptodrosophila latifasciaeformis, and D. suzukii. A. Profemur with a row of cuneiform setae. B. Head of D. Table 1 nasuta in frontal view, showing the characteristic silvery pigmentation in the face region. C. Thorax of D. nasuta in lateral view, showing the large brownish stripe on half dorsal area of pleura. D. D. immigrans protarsomeres, showing the brush like thin setae. E. D. immigrans wing, showing the shaded cross veins and ends of longitudinal veins. F. D. immigrans abdomen, showing the bands interrupted in the middle. G. Prescutellar setae. H. S. latifasciaeformis dark abdomen. I. D. suzukii protarsus with sex combs. Bar = 1mm. Figure 2. Drosophila nasuta, D immigrans, Scaptodrosophila latifasciaeformis, and D. suzukii. A. Profemur with a row of cuneiform setae. B. Head of D. nasuta in frontal view, showing the characteristic silvery pigmentation in the face region. C. Thorax of D. nasuta in lateral view, showing the large brownish stripe on half dorsal area of pleura. D. D. immigrans protarsomeres, showing the brush like thin setae. E. D. immigrans wing, showing the shaded cross veins and ends of longitudinal veins. F. D. immigrans abdomen, showing the bands interrupted in the middle. G. Prescutellar setae. H. S. latifasciaeformis dark abdomen. I. D. suzukii protarsus with sex combs. Bar = 1mm. K. Yuzuki, R. Tidon / Revista Brasileira de Entomologia 64(1):e2019100, 2020 5-9 K. Yuzuki, R. Tidon / Revista Brasileira de Entomologia 64(1):e2019100, 2020 5-9 Figure 3. Drosophila suzukii, D. melanogaster, D. simulans, D. ananassae, D. malerkotliana and D. kikkawai. A. D. suzukii male spotted wing. B. D. suzukii female abdomen, showing the serrated oviscapt. C. D. melanogaster and D. simulans protarsus sex comb. D. D. melanogaster male epandrial posterior lobe. E. D. melanogaster female abdomen in lateral view, showing the large dark bands on the sixth tergite running to its ventral margin. F. D. simulans male epandrial posterior lobe. G. D. simulans female abdomen in lateral view, showing the pigmentation border line making an angle with the sixth tergite margin. H. D. ananassae protarsus sex combs. I. D. malerkotliana protarsus sex combs. J. D. kikkawai protarsus sex combs. Bar = 1mm. Figure 3. Drosophila suzukii, D. melanogaster, D. simulans, D. ananassae, D. malerkotliana and D. kikkawai. A. D. suzukii male spotted wing. B. D. suzukii female abdomen, showing the serrated oviscapt. C. D. melanogaster and D. simulans protarsus sex comb. D. D. melanogaster male epandrial posterior lobe. E. D. Subgenus Drosophila Fállen This is the largest subgenus of Drosophila, supporting about 80% of its ca. 2000 species. Therefore, this subgenus is organized in 47 species groups, which are an informal taxonomic rank (Sturtevant, 1942) that organize the diversity within large clades of drosophilids. Finally, a species that is currently meriting the status of cosmopolitan is D. suzukii, known as the spotted-wing Drosophila due to the dark spot on the male wings. Probably native of East Asia, this species has spread to western regions: it was found in Hawaii in the early 1980s and more recently in North America and Europe, where it has reached invasive pest status (Asplen et al., 2015). The female has a long and narrow ovipositor used to infest soft-skinned fruit crops and causing enormous economic damage on commercial plantations; hence, the spotted-wing Drosophila has become an agricultural pest in several countries worldwide (Walsh et al., 2011). In Brazil, this cold-adapted species was first detected in the southern region in February 2013 (Deprá et al., 2014). As conjectured by Vilela and Mori (2014), who recorded this species in Southeast Brazil, D. suzukii is probably expanding its territory to other South American areas through the trade of cultivated soft skin fruits and the use of small wild fruits as breeding sites. Although Southern Brazil is the most climatically favorable area for D. suzukii development (Benito et al., 2016), it has already been found in natural vegetations of the tropical Brazilian Savanna, at low abundances (Leão et al., 2017). The immigrans species group, probably of oriental origin, currently contains about 100 nominal species and two of them are recorded in Brazil. The cosmopolitan Drosophila immigrans is abundant in the Palearctic and Nearctic Regions (David and Tsacas, 1981) and has been collected in Brazilian natural and modified environments since the pioneer studies of Pavan (1959). In this country, the abundance of D. immigrans seems to be low at warmer areas (Sene et al., 1980) but increases in the colder southern regions (Hochmüller et al., 2010). Drosophila nasuta, on the other hand, is widespread and abundant in the tropical parts of Africa (David and Tsacas, op cit.) and has been recently recorded in the Neotropics (Vilela and Goñi, 2015). In forest patches located in the Brazilian savanna, where D. immigrans remains rare (data not shown), D. nasuta can reach 20% relative abundance of drosophilids during Summer months (Leão et al., 2017). Subgenus Dorsilopha Sturtevant This subgenus of oriental origin (Toda, 1986) consists of only four species, including the cosmopolitan Drosophila busckii. While many common drosophilids prefer decaying fruit, D. busckii larvae seem to have a broader niche (Atkinson and Shorrocks, 1977) and were recorded feeding on many different vegetables (Valadão et al., 2019), animal excrement and meat (Sturtevant, 1921). This species is very abundant in temperate places, where its competitive ability is apparently higher (David and Tsacas, 1981). In Brazil, D. busckii is rare in natural habitats but can be found in disturbed areas (Sene et al., 1980). The oriental species D. kikkawai supports a cosmopolitan status (Pinheiro and Valente, 1993), but it is not clear when it was introduced in South America and Africa and nor if this process was mediated by humans (Ashburner, 1989). In Brazil, this species is found in both natural (Sene et al., 1980) and urban environments (Costa et al., 2003), usually in low abundances (but see Cavasini et al., 2014). Drosophila malerkotliana, also native of the Orient, was described from the Malerkotla region in India by Parshad and Paika, in 1964. Besides being widespread in the Orient, it also occurs in Africa and Americas, where it was introduced in recent decades (Val and Sene, 1980; Sene et al. 1980; David and Tsacas 1981; Castrezana et al., 2010). In Brazil, it occurs in both natural environments (Medeiros and Klaczko, 2004; Torres and Madi-Ravazzi, 2006; Tidon, 2006; Schmitz et al., 2010), and urbanized sites (Gottschalk et al., 2007). Discussion This subgenus currently contains 344 species organized in nine species groups. The Drosophila melanogaster species group, the largest of this subgenus, contains almost 200 species widely distributed in the Oriental Region and adjacent areas, but some lineages reached the African continent and radiated. Six species of this group are recorded in Brazil. Potentially colonizer drosophilids had attracted the attention of researchers for over five decades (Dobzhansky, 1965; Lewontin, 1965; David and Tsacas, 1981; Parsons, 1983), and it is well established that these flies can be used as models in studies of biological invasions (Gibert et al., 2016). The species registered here are widely distributed not only in South America but also in other regions of the world (Bächli, 2019). The section below provides information about the origin (David and Tsacas, 1981), distribution (Brake and Bächli, 2008; Bächli, 2019), and general characteristics of each species. The sibling African species Drosophila melanogaster and D. simulans probably are the most studied examples of widespread drosophilids (Capy et al., 2004). D. melanogaster first colonized Eurasia (10,000‑ 15,000 years ago) and later spread to America and Australia. It is generally admitted that this human commensal species travel easily with fruit shipments, mostly as larvae or pupae on rotting material. D. simulans expanded worldwide more recently, probably a few centuries ago, establishing in natural and disturbed environments throughout the invaded areas. In Brazil, both species arrived probably as a result of the slave trade. Currently, D. simulans is a widespread species in all sorts of environments, whereas D. melanogaster is rare in natural habitats (Sene et al., 1980). Subgenus Drosophila Fállen These findings support the hypothesis raised by David and Tsacas (op cit.) that D. immigrans is cold-adapted, whereas D. nasuta is a tropical species. The virilis species group consists of about thirteen described species that are typically found in a boreal distribution (Markow and O’Grady, 2006). The group appears to have originated in Asia as a species of the temperate deciduous forest associated predominantly with riparian communities (Throckmorton, 1975). Only Drosophila virilis Sturtevant has been recorded worldwide in wine production areas as well as in breweries (Bächli et al., 2004). Genus Drosophila Fállen The genus Drosophila includes approximately half of the 4,000 species in the family Drosophilidae. Its members have adapted to and radiated in a variety of niches (Markow and O’Grady, 2008), and currently divided into nine subgenera (O’Grady and DeSalle, 2018). Drosophila ananassae, probably native of the Orient, is a peridomestic species occurring in tropical and subtropical areas of the world. In Brazil, it is rare in natural environments but can be found in open types of vegetation (Sene et al., 1980). We have been collecting this species in urban orchards, near Brasília. Table 1 melanogaster female abdomen in lateral view, showing the large dark bands on the sixth tergite running to its ventral margin. F. D. simulans male epandrial posterior lobe. G. D. simulans female abdomen in lateral view, showing the pigmentation border line making an angle with the sixth tergite margin. H. D. ananassae protarsus sex combs. I. D. malerkotliana protarsus sex combs. J. D. kikkawai protarsus sex combs. Bar = 1mm. K. Yuzuki, R. Tidon / Revista Brasileira de Entomologia 64(1):e2019100, 2020 6-9 doi.org/10.1007/BF00345697. Bächli, G., 2019. TaxoDros: The Database on Taxonomy of Drosophilidae. Available in: https://www.taxodros.uzh.ch/search/dist_reg.php (accessed 8 February 2019). Bächli, G., Haring, E., Vilela, C. R., 2005. On the phylogenetic relationships of Scaptodrosophila rufifrons and S. lebanonensis (Diptera, Drosophilidae). Bull. la Société Entomol. Suisse. 78, 349-364. Bächli, G., Vilela, C., Escher, S. A., Saura, A., 2004. The Drosophilidae (Diptera) of Fennoscandia and Denmark Fauna Entomologica Scandinavica, vol. 39. Brill: Nederland, pp. 1-362. Genus Zaprionus Benito, N. P., Lopes-da-Silva, M., Santos, R. S. S., 2016. Potential spread and economic impact of invasive Drosophila suzukii in Brazil. Pesqui. Agropecu. Bras. 51, 571-578. https://doi.org/10.1590/S0100- 204X2016000500018. Zaprionus is an Afrotropical genus that extended its distribution to the Australian, Oriental and Palearctic regions. Zaprionus indianus constitutes one of the most successful colonizing species of this genus (Chassagnard and Tsacas, 1993), probably because of its broad niche‑width characteristics: it utilizes diverse food resources and displays adaptation to variable climatic conditions (Parkash and Yadav, 1993). The first record of Z. indianus in Brazil was in São Paulo State (Vilela, 1999) and, after that, the species was found in many Brazilian regions (Galego and Carareto, 2010). In natural areas where environmental conditions are similar to those observed in its original area in Africa, Z. indianus dominates drosophilid assemblages during the wet season (Tidon et al., 2003). It also predominates in fig plantations: among 125,00 drosophilids captured in São Paulo State, 83,339 were identified as Z. indianus (Roque et al., 2017). This species is highly adaptable (Mata et al., 2010) and deserves to be monitored. Blauth, M. L., Gottschalk, M. S., 2007. A novel record of Drosophilidae species in the Cerrado biome of the State of Mato Grosso, west- central Brazil. Drosoph. Inf. Serv. 90, 90-96. Brake, I., Bächli, G., 2008. Drosophilidae (Diptera). World Catalog of Insects, v. 9. Apollo Books, Stenstrup. Capy, P., Gibert, P., Boussy, I., 2004. Drosophila melanogaster, Drosophila simulans: So Similar, So Different. Springer, Netherlands. Castrezana, S., Faircloth, B. C., Gowaty, P. A., 2010. Drosophila collection in Los Angeles, California. Drosoph. Inf. Serv. 93, 91. Cavasini, R., Buschini, M., Machado, L., Mateus, R., 2014. Comparison of Drosophilidae (Diptera) assemblages from two highland Araucaria forest fragments, with and without environmental conservation policies. Braz. J. Biol. 74, 761-768. https://doi.org/10.1590/1519- 6984.00113. Final Remarks Chassagnard, M.-T., Tsacas, L., 1993. Le Sous-genre Zaprionus S. Str.: définition de groupes d’espèces et révision du sous-groupe vittiger (Diptera: Drosophilidae). Ann. Soc. Entomol. Fr. 29, 173-194. Most drosophilids approached in this study are widely distributed thought natural populations in many Brazilian biomes, and two species in particular, Drosophila suzukii and Zaprionus indianus, can cause great impact on cultivated areas. The identification key provided here should not only support newcomers to the study of Drosophila but also the professional that will be dealing with the elimination of the pests affecting plantation, who generally will not be Drosophila experts. Costa, B., Rohde, C., Valente, V., 2003. Temperature, urbanization and body color polymorphism in South Brazilian populations of Drosophila kikkawai (Diptera, Drosophilidae). Iheringia Ser. Zool. 93, 381-393. https://doi.org/10.1590/S0073-47212003000400005. Coutinho-Silva, R. D., Montes, M. A., Oliveira, G. F., Carvalho-Neto, F. G., Rohde, C., Garcia, A. C. L., 2017. Effects of seasonality on drosophilids (Insecta, Diptera) in the northern part of the Atlantic Forest, Brazil. Bull. Entomol. Res. 107, 634-644. https://doi.org/10.1017/ S0007485317000190. Acknowledgments We are grateful to B.F.D. Leão and F. Roque for collecting the specimens and taxonomic advices, to C.R. Vilela and C.H. Lue for suggestions on earlier versions of this manuscript, and to two anonymous referees for their valuable recommendations. This paper was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, grant number 88887.136269/2017-00) and by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant numbers 309973/2017-1 and 441581/2016-1). David, J. R., Tsacas, L., 1981. Cosmopolitan, subcosmopolitan and widespread species: different strategies within the Drosophilidae family (Diptera). C. R. Séances Soc. Biogeogr. 57, 11-26. Deprá, M., Poppe, J. L., Schmitz, H. J., De Toni, D. C., Valente, V. L. S., 2014. The first records of the invasive pest Drosophila suzukii in the South American continent. J. Pest Sci. 87, 379-383. https://doi. org/10.1007/s10340-014-0591-5. Dobzhansky, T., 1965. “Wild” and “Domestic” species of Drosophila. In: Baker, H.G., Stebbins, G.L. (Eds.), The Genetics of Colonizing Species. Academic Press, London, pp. 533-551. Genus Scaptodrosophila Duda The genus Scaptodrosophila, initially described as a subgenus of Drosophila, was formally elevated to generic rank by Grimaldi (1990). Probably originated in tropical Asia, Scaptodrosophila currently includes K. Yuzuki, R. Tidon / Revista Brasileira de Entomologia 64(1):e2019100, 2020 7-9 (Drosophila suzukii): a global perspective and future priorities. J. Pest Sci. 88, 469-494. https://doi.org/10.1007/s10340-015-0681-z. almost 300 species distributed in Asia, New Guinea, Australia, and Africa, with few species in the Americas and Europe. Two species of this genus have reached the Neotropical Region. Scaptodrosophila latifasciaeformis is a widespread species in tropical and subtropical areas of the world. In Brazil, it was described by Dobzhansky and Pavan (1943) as Drosophila mirim and has been collected in all types of vegetations (Sene et al., 1980), including mangroves in the southern region of the country (Schmitz et al., 2010). S. lebanonensis, later described as Drosophila galloi by Lourenço and Mourão (1992) and synonymized by Bächli et al. (2005), was recorded in southern Neotropical region in 1960 and we have no notice of other records in the Neotropical region after then. Atkinson, W., Shorrocks, B., 1977. Breeding site specificity in the domestic species of Drosophila. Oecologia 29, 223-232. https:// References Dobzhansky, T., Pavan, C., 1943. Studies on Brazilian species of Drosophila. Bol. Fac. Filos. Ciências e Let. da Univ. São Paulo. Biol. Geral 36, 7-72. Ashburner, M., 1989. Drosophila: A Laboratory Handbook, Cold Spring Harbor, New York. Elton, C. S., 1958. The Ecology of Invasions by Plants and Animals. University of Chicago Press, Chicago. Asplen, M. K., Anfora, G., Biondi, A., Choi, D. S., Chu, D., Daane, K. M., Gibert, P., Gutierrez, A. P., Hoelmer, K. A., Hutchison, W. D., Isaacs, R., Jiang, Z. L., Kárpáti, Z., Kimura, M. T., Pascual, M., Philips, C. R., Plantamp, C., Ponti, L., Vétek, G., Vogt, H., Walton, V. M., Yu, Y., Zappalà, L., Desneux, N., 2015. Invasion biology of spotted wing Drosophila Ferreira, L. B., Tidon, R., 2005. Colonizing potential of Drosophilidae (Insecta, Diptera) in environments with different grades of urbanization. Biodivers. Conserv. 14, 1809-1821. https://doi. org/10.1007/s10531-004-0701-4. K. Yuzuki, R. Tidon / Revista Brasileira de Entomologia 64(1):e2019100, 2020 8-9 Drosophila melanogaster and D. simulans (Diptera Drosophilidae). Ann. Soc. Entomol. Fr. 31, 249-257. Freire-Maia, N., Pavan, C., 1949. Introdução ao Estudo da Drosophila. Cultus 1, 3-61. Drosophila melanogaster and D. simulans (Diptera Drosophilidae). Ann. Soc. Entomol. Fr. 31, 249-257. O’Grady, P. M., DeSalle, R., 2018. Phylogeny of the genus Drosophila. Genetics 209, 1-25. https://doi.org/10.1534/genetics.117.300583. Galego, L. G. C., Carareto, C. M. A., 2010. Scenario of the spread of the invasive species Zaprionus indianus Gupta, 1970 (Diptera, Drosophilidae) in Brazil. Genet. Mol. Biol. 33, 767-773. https://doi. Oliveira, C. M., Auad, A. M., Mendes, S. M., Frizzas, M. R., 2013. Economic impact of exotic insect pests in Brazilian agriculture. J. Appl. Entomol. 137, 1-15. https://doi.org/10.1111/jen.12018. org/10.1590/S1415-47572010005000080. Garcia, A. C. L., Oliveira Silva, D. M. I., Monteiro, A. G. F., Oliveira, G. F., Montes, M. A., Rohde, C., 2014. Abundance and richness of cryptic species of the willistoni group of Drosophila (Diptera: Drosophilidae) in the biomes Caatinga and Atlantic Forest, Northeastern Brazil. Ann. Entomol. Soc. Am. 107, 975-982. https://doi.org/10.1603/AN14052. Oliveira, G. F., Garcia, A. C. L., Montes, M. A., Jucá, J. C. L. A., Valente, V. L. S., Rohde, C., 2016. Are conservation units in the Caatinga biome, Brazil, efficient in the protection of biodiversity? An analysis based on the drosophilid fauna. J. Nat. Conserv. 34, 145-150. https://doi. org/10.1016/j.jnc.2016.10.006. Gibert, P., Hill, M., Pascual, M., Plantamp, C., Terblanche, J. S., Yassin, A., Sgrò, C. M., 2016. Drosophila as models to understand the adaptive process during invasion. Biol. References Invasions. 18, 1089-1103. https://doi. org/10.1007/s10530-016-1087-4. Parkash, R., Yadav, J. P., 1993. Geographical clinal variation at seven esterase-coding loci in Indian populations of Zaprionus indianus. Hereditas 119, 161-170. https://doi.org/10.1111/j.1601-5223.1993.00161.x. Parsons, P. A., 1983. Ecobehavioral genetics: habitats and colonists. Annu. Rev. Ecol. Syst. 14, 35-55. Gottschalk, M. S., De Toni, D. C., Valente, V. L. S., Hofmann, P. R. P., 2007. Changes in Brazilian Drosophilidae (Diptera) assemblages across an urbanisation gradient. Neotrop. Entomol. 36, 848-862. https:// doi.org/10.1590/S1519-566X2007000600005. Pavan, C., 1959. Relações entre populações naturais de Drosophila e o meio ambiente. Bol. Fac. Filos. Ciências e Let. da Univ. São Paulo. 221, 1-81. Grimaldi, D. A., 1990. A Phylogenetic, revised classification of genera in the Drosophilidae (Diptera). Bull. Am. Mus. Nat. Hist. 197, 1-139. Penariol, L. V., Madi-Ravazzi, L., 2013. Edge-interior differences in the species richness and abundance of drosophilids in a semideciduous forest fragment. Springerplus 2, 1-7. https://doi.org/10.1186/2193- 1801-2-114. Hochmüller, C. J. C., Lopes-da-Silva, M., Valente, V. L. S., Schmitz, H. J., 2010. The drosophilid fauna (Diptera, Drosophilidae) of the transition between the Pampa and Atlantic Forest biomes in the State of Rio Grande do Sul, Southern Brazil: First Records. Pap. Avulsos Zool. 50, 286-295. Pinheiro, B. E., Valente, V. L. S., 1993. Drosophila kikkawai, a subcosmopolitan or a cosmopolitan species? Drosoph. Inf. Serv. 72, 146-150. Poppe, J. L., Schmitz, H. J., Valente, V. L. S., 2016. Changes in the structure of Drosophilidae (Diptera) assemblages associated with contrasting environments in the Pampas biome across temporal and spatial scales. Ann. Entomol. Soc. Am. 109, 567-573. https:// doi.org/10.1093/aesa/saw033. Leão, B. F. D., Roque, F., Deus, P., Tidon, R., 2017. What happens when exotic species arrive in a new area? The case of drosophilids (Diptera) in the Brazilian Savanna. Drosoph. Inf. Serv. 100, 65-69. Lewontin, R. C., 1965. Selection for colonizing ability. In: Baker, H.G., Stebbins, G.L. (Eds.), The Genetics of Colonizing Species. Academic Press, London, pp. 77-91. Rohde, C., Silva, D. M. I. O., Jucá, J. C. L. A., Montes, M. A., Garcia, A. C. L., 2010. Espécies invasoras da família Drosophilidae (Diptera, Insecta) em ambientes da Caatinga de Pernambuco. An. Acad. Pernambucana. Cienc. Agron. 7, 227-240. Lourenço, M. F., Mourão, C. A., 1992. Drosophila galloi sp. n. the first occurrence of the victoria group (Subgenus Scaptodrosophila) in the Neotropical Region (Diptera Drosophilidae). Rev. Bras. Biol. 52, 575-578. Roque, F., Matavelli, C., Lopes, P. H. S., Machida, W. S., Von Zuben, C. References J., Tidon, R., 2017. Brazilian fig plantations are dominated by widely distributed drosophilid species (Diptera: drosophilidae). Ann. Entomol. Soc. Am. 110, 521-527. https://doi.org/10.1093/aesa/sax044. Markow, T. A., O’Grady, P., 2006. Drosophila: A Guide to Species Identification and Use. Elsevier, San Diego. Sakai, A. K., Allendorf, F. W., Holt, J. S., Lodge, D. M., Molofsky, J., With, K. A., Baughman, S., Cabin, R. J., Cohen, J. E., Ellstrand, N. C., McCauley, D. E., 2001. The population biology of invasive species. Annu. Rev. Ecol. Syst. 32, 305-332. https://doi.org/10.1146/annurev. ecolsys.32.081501.114037. Markow, T. A., O’Grady, P., 2008. Reproductive ecology of Drosophila. Funct. Ecol. 22, 747-759. https://doi.org/10.1111/j.1365-2435.2008.01457.x. Mata, R. A., Roque, F., Tidon, R., 2015. Measuring the variability of the drosophilid assemblages associated with forests of the Brazilian Savanna across temporal and spatial scales. Nat. Conserv. 13, 166- 170. https://doi.org/10.1016/j.ncon.2015.11.005. Santa-Brígida, R., Schmitz, H. J., Martins, M. B., 2017. Drosophilidae (Insecta, Diptera) in the State of Pará (Brazil). Biota Neotrop. 17, 1-9. https://doi.org/10.1590/1676-0611-bn-2016-0179. Mata, R. A., Tidon, R., Côrtes, L. G., Marco, P., Diniz-Filho, J. A. F., 2010. Invasive and flexible: niche shift in the drosophilid Zaprionus indianus (Insecta, Diptera). Biol. Invasions 5, 1231-1241. https:// doi.org/10.1007/s10530-009-9542-0. Schmitz, H. J., Hofmann, P. R. P., Valente, V. L. S., 2010. Assemblages of drosophilids (Diptera, Drosophilidae) in mangrove forests: community ecology and species diversity. Iheringia Ser. Zool. 100, 133-140. Sene, F. M., Val, F. C., Vilela, C. R., Pereira, M. A. Q. R., 1980. Preliminary data on the geographical distribution of Drosophila species within morphoclimatic domains of Brazil. Pap. Avulsos Zool. 33, 315-326. Medeiros, H. F., Klaczko, L. B., 2004. How many species of Drosophila (Diptera, Drosophilidae) remain to be described in the forests of São Paulo, Brazil?: Species lists of three forest remnants. Biota Neotrop. 4, 1-12. https://doi.org/10.1590/S1676-06032004000100005. Sturtevant, A. H., 1921. The North American Species of Drosophila. Carnegie Inst., Washington. Millenium Ecosystem Assessment, 2005. Ecosystems and Human Well-Being: Biodiversity Synthesis. World Resources Institute, Washington, DC. Sturtevant, A. H., 1942. The Classification of the genus Drosophila, with descriptions of nine species. Univ. Tex. Publ. 4213, 5-51. Throckmorton, L. H., 1975. The Phylogeny, Ecology and Geography of Drosophila. In: King, R.C. (Ed.), Handbook of Genetics. v. 3. Plenum Publishing Corporation, New York, pp. 421-469. Miller, M. E., Marshall, S. A., Grimaldi, D. A., 2017. A review of the species of Drosophila (Diptera: Drosophilidae) and genera of Drosophilidae of Northeastern North America. Can. J. Arthropod Identif. 31, 1-208. https://doi.org/10.5063/F11J97Q0. References Tidon, R., 2006. Relationships between drosophilids (Diptera, Drosophilidae) and the environment in two contrasting tropical vegetations. Biol. J. Linn. Soc. Lond. 87, 233-247. https://doi.org/10.1111/j.1095- 8312.2006.00570.x. Moreteau, B., Pétavy, G., Gibert, P., Morin, J. P., Munoz, A., David, J. R., 1995. New discriminating traits between females of two sibling species: K. Yuzuki, R. Tidon / Revista Brasileira de Entomologia 64(1):e2019100, 2020 9-9 playing the field and specialising in generalism? Ecol. Entomol. 44 (6), 721-737. https://doi.org/10.1111/een.12769. Tidon, R., Gottschalk, M. S., Schmitz, H. J., Martins, M. B., 2019. Drosophilidae, Catálogo Taxonômico da Fauna do Brasil. Available in: http://fauna. jbrj.gov.br/fauna/faunadobrasil/2 (accessed 4 February 2019). Vilela, C. R., 1999. Is Zaprionus indianus Gupta, 1970 (Diptera, Drosophilidae) currently colonizing the Neotropical Region? Drosoph. Inf. Serv. 82, 37-39. Tidon, R., Leite, D. F., Leão, B. F. D., 2003. Impact of the colonisation of Zaprionus (Diptera, Drosophilidae) in different ecosystems of the Neotropical Region: 2 years after the invasion. Biol. Conserv. 112, 299-305. https://doi.org/10.1016/S0006-3207(02)00322-1. Vilela, C. R., Goñi, B., 2015. Is Drosophila nasuta Lamb (Diptera, Drosophilidae) currently reaching the status of a cosmopolitan species? Rev. Bras. Entomol. 59, 346-350. https://doi.org/10.1016/j. Toda, M. J., 1986. Drosophilidae (Diptera) in Burma. I: the subgenus Dorsilopha Sturtevant of the genus Drosophila, with descriptions of two new species. Kontyu 54, 282-290. Vilela, C. R., Mori, L., 2014. The invasive spotted-wing Drosophila (Diptera, Drosophilidae) has been found in the city of São Paulo (Brazil). Rev. Bras. Entomol. 58, 371-375. https://doi.org/10.1590/ S0085-56262014000400004. Torres, F. R., Madi-Ravazzi, L., 2006. Seasonal variation in natural populations of Drosophila spp. (Diptera) in two woodlands in the State of São Paulo, Brazil. Iheringia Ser. Zool. 96, 437-444. https:// doi.org/10.1590/S0073-47212006000400008. Walsh, D. B., Bolda, M. P., Goodhue, R. E., Dreves, A. J., Lee, J., Bruck, D. J., Walton, V. M., O’Neal, S. D., Zalom, F. G., 2011. Drosophila suzukii (Diptera: Drosophilidae): invasive pest of ripening soft fruit expanding its geographic range and damage potential. J. Integr. Pest Manag. 40, 55-64. https://doi.org/10.1603/IPM10010. Val, F. C., Sene, F. M., 1980. A newly introduced Drosophila in Brazil (Diptera, Drosophilidae). Pap. Avulsos Zool. 33, 293-298. Valadão, H., Proença, C. E. B., Kuhlmann, M. P., Harris, S. A., Tidon, R., 2019. Fruit-breeding Drosophilids (Diptera) in the Neotropics:
https://openalex.org/W2290985175	http://shura.shu.ac.uk/11641/3/Porritt%20Protocol%20feasibility%20study%20self%20help%20CBT%20dental%20anxiety.pdf	English	null	Protocol for a feasibility study of a self-help cognitive behavioural therapy resource for the reduction of dental anxiety in young people	Pilot and feasibility studies	2,016	cc-by	7,386	Protocol for a feasibility study of a self-help Cognitive Behavioural Therapy resource for the reduction of dental anxiety in young people MARSHMAN, Zoe, MORGAN, Annie, PORRITT, Jenny <http://orcid.org/0000- 0001-7772-438X>, GUPTA, Ekta, BAKER, Sarah, CRESWELL, Cathy, NEWTON, Tim, STEVENS, Katherine, WILLIAMS, Chris, PRASAD, Suneeta, KIRBY, Jennifer and RODD, Helen This document is the author deposited version. You are advised to consult the publisher's version if you wish to cite from it. Published version MARSHMAN, Zoe, MORGAN, Annie, PORRITT, Jenny, GUPTA, Ekta, BAKER, Sarah, CRESWELL, Cathy, NEWTON, Tim, STEVENS, Katherine, WILLIAMS, Chris, PRASAD, Suneeta, KIRBY, Jennifer and RODD, Helen (2016). Protocol for a feasibility study of a self-help Cognitive Behavioural Therapy resource for the reduction of dental anxiety in young people. Pilot and Feasibility Studies, 2, p. 13. * Correspondence: z.marshman@sheffield.ac.uk 1School of Clinical Dentistry, University of Sheffield, Claremont Crescent, Sheffield S10 2TA, UK Full list of author information is available at the end of the article Copyright and re-use policy See http://shura.shu.ac.uk/information.html See http://shura.shu.ac.uk/information.html Sheffield Hallam University Research Archive http://shura.shu.ac.uk Marshman et al. Pilot and Feasibility Studies (2016) 2:13 DOI 10.1186/s40814-016-0054-2 Open Access © 2016 Marshman et al. 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. Protocol for a feasibility study of a self-help cognitive behavioural therapy resource for the reduction of dental anxiety in young people Zoe Marshman1* , Annie Morgan2, Jenny Porritt3, Ekta Gupta1, Sarah Baker1, Cathy Creswell4, Tim Newton5, Katherine Stevens6, Christopher Williams7, Suneeta Prasad8, Jennifer Kirby2 and Helen Rodd1 Background behave in relation to their problems. It is a type of ther- apy which can be used to teach patients (and often their parents/carers) skills for the self-management of their anxiety. CBT incorporates a variety of different cognitive and behavioural strategies which aim to help the patient modify the unhelpful behaviours or thoughts maintain- ing their anxiety [24]. behave in relation to their problems. It is a type of ther- apy which can be used to teach patients (and often their parents/carers) skills for the self-management of their anxiety. CBT incorporates a variety of different cognitive and behavioural strategies which aim to help the patient modify the unhelpful behaviours or thoughts maintain- ing their anxiety [24]. Dental anxiety is common, affects people of all ages [1], and tends to develop in childhood and adolescence [2, 3]. The prevalence of severe dental anxiety in children ranges from 5 to 20 % in different countries and age groups [4–6]. Dental anxiety impacts on the quality of life of young people with those affected reporting significantly worse oral health-related quality of life, with particular impact on social and emotional well-being such as feeling upset or worried or being teased or left out [7]. Young people with high levels of dental anxiety also have more decayed and extracted teeth [8, 9] and demonstrate increased unmet need for dental care [10, 11] as compared to their peers with low levels of anxiety and who are regu- lar attenders. Up to one in five young people reports not visiting the dentist regularly because of fear [12]. Fear is also a significant barrier to completion of dental treatment [13]. Research has found that children who are most likely to have missed previous dental appoint- ments were those who were rated by their parents as having high levels of dental anxiety [14]. Hallberg and colleagues [15] found that parents experienced difficul- ties trying to persuade their dentally anxious children to go to their dental appointment and felt they lacked strategies to handle their child’s level of fear and effect- ively manage the situation [16]. g y A review of meta-analyses revealed that CBT is highly effective in treating a range of anxiety disorders in both children and adults [25, 26]. Behavioural interventions, such as graded exposure and systematic desensitisation to feared stimuli, have been found to be effective in reducing dental anxiety levels of children [27–29]. Background Studies have also found modelling interventions, which help patients de- velop effective coping skills by observing other people (e.g. other children, parents) successfully receiving dental treat- ment (film, in vivo), are effective in improving the behav- iour of dentally anxious young people [30]. Whilst there has been a paucity of research investigating the effective- ness of cognitive-behavioural interventions in reducing young people’s dental anxiety, research has revealed prom- ising results in the use of such strategies to reduce adults’ dental anxiety [31, 32]. Based on (1) evidence for the effectiveness of self-help CBT for young people with general anxiety; (2) case stud- ies of CBT for children with dental anxiety [33, 34]; and (3) experience from the use of CBT for dental anxiety in adults including CBT delivered by dental nurses in Shef- field, there was sufficient evidence to suggest that a self- help CBT resource for young people with dental anxiety could be highly effective at reducing dental anxiety and its consequences. However, no such resource had been evalu- ated to date. Dental anxiety in young people also has a significant impact on dental services. Providing treatment for anx- ious patients is time consuming, costly, demanding, and a cause of occupational stress for dentists [17]. These factors result in patients being referred to sec- ondary care services, having to wait longer for dental treatment and increased costs to the National Health Service (NHS) [18]. Traditionally, dental anxiety has been managed using pharmacological techniques including inhalational sed- ation and general anaesthetic (GA). However, such ap- proaches only manage rather than reduce children’s dental anxiety [19]. The cost per case for inhalational sedation or GA for dental treatment has been estimated at £273 and £720, respectively [20]. Indeed, dental anxiety remains un- changed in those children who receive dental treatment under GA [21] with anxious children becoming adults with a long-term reliance on expensive interventions in specialist settings. Abstract Background: Childhood dental anxiety is very common, with 10–20 % of children and young people reporting high levels of dental anxiety. It is distressing and has a negative impact on the quality of life of young people and their parents as well as being associated with poor oral health. Affected individuals may develop a lifelong reliance on general anaesthetic or sedation for necessary dental treatment thus requiring the support of specialist dental services. Children and young people with dental anxiety therefore require additional clinical time and can be costly to treat in the long term. The reduction of dental anxiety through the use of effective psychological techniques is, therefore, of high importance. However, there is a lack of high-quality research investigating the impact of cognitive behavioural therapy (CBT) approaches when applied to young people’s dental anxiety. Methods/design: The first part of the study will develop a profile of dentally anxious young people using a prospective questionnaire sent to a consecutive sample of 100 young people referred to the Paediatric Dentistry Department, Charles Clifford Dental Hospital, in Sheffield. The second part will involve interviewing a purposive sample of 15–20 dental team members on their perceptions of a CBT self-help resource for dental anxiety, their opinions on whether they might use such a resource with patients, and their willingness to recruit participants to a future randomised controlled trial (RCT) to evaluate the resource. The third part of the study will investigate the most appropriate outcome measures to include in a trial, the acceptability of the resource, and retention and completion rates of treatment with a sample of 60 dentally anxious young people using the CBT resource. Discussion: This study will provide information on the profile of dentally anxious young people who could potentially be helped by a guided self-help CBT resource. It will gain the perceptions of dental care team members of guided self-help CBT for dental anxiety in young people and their willingness to recruit participants to a trial. Acceptability of the resource to participants and retention and completion rates will also be investigated to inform a future RCT. Keywords: Dental anxiety, CBT, Children, Young people, Feasibility study, CBT self-help, Low intensity, Qualitative research Marshman et al. Pilot and Feasibility Studies (2016) 2:13 Page 2 of 8 Page 2 of 8 Aims of study The study will determine the feasibility of evaluating, in a randomised controlled trial (RCT), a CBT resource for young people (aged 9–16 years) for the reduction of dental anxiety. It will specifically: a) Identify a profile of dentally anxious young people who could potentially be helped by a guided self-help CBT resource b) Explore perceptions of primary dental care team members of guided self-help CBT for dental anxiety in young people and their willingness to recruit participants to a future substantive trial Over recent years, it has been recognised that greater effort should be directed towards behaviour manage- ment and psychological interventions which can reduce the patient’s anxiety in the long term [22, 23]. Advances in the treatment of anxiety in young people have been made in the field of cognitive behavioural therapy (CBT), including self-help approaches. CBT is a goal- orientated talking therapy which aims to help people manage their problems by changing how they think and c) Evaluate the retention and completion rates and acceptability of the guided self-help CBT resource to patients and parents d) Identify potential outcome measures for use in a future RCT e) Determine the sample size required for a future RCT Page 3 of 8 Page 3 of 8 Marshman et al. Pilot and Feasibility Studies (2016) 2:13 Methods/design carers will be invited to help their child complete the questionnaire if necessary. If young people and their parent consent for them to take part, they will be asked to return the completed questionnaire when they attend for their appointment. In order to maximise the response rate, if the participants have not completed their questionnaire when they attend for their appointment, they will be given a copy to complete at the appointment and sufficient time to do this in a private area of the clinic. Based on previous surveys in the department, a response rate of 60 % is anticipated. Participants will continue to be recruited until the sample size of 100 is achieved. Recruitment will be restricted to those aged 11–16 years due to the length of the questionnaire which takes around 15 min to complete and the nature of some of the questions about depression and anxiety. S l i This mixed-method study will be conducted in three phases to allow the objectives to be achieved and to cap- ture the perspectives of all relevant stakeholders. For each phase the design, procedure and approach to data analysis will be described. Ethical approval will be ob- tained from an NHS research ethics committee. Intervention The intervention will be a self-help paper-based CBT resource developed with young people, parents, and dental professionals based on the Five Areas model of CBT [24] which uses a combination of cognitive and behavioural techniques to reduce dental anxiety. The resource will be given to young people by a dentist who will guide them through the use of it during dental ap- pointments. More information about the resource can be found at https://www.sheffield.ac.uk/dentalschool/ research/create/research_projects. Sample size The sample size has been based on a similar questionnaire survey of 100 adults attending a clinic for patients with dental anxiety at a dental hospital in London [39]. The sample size has been based on a similar questionnaire survey of 100 adults attending a clinic for patients with dental anxiety at a dental hospital in London [39]. A) The profile of dentally anxious young people Design of the study This part of the project will develop a profile of dentally anxious young people who could potentially be helped by a self-help CBT resource to reduce dental anxiety. It will include a prospective questionnaire sent to a consecutive sample of 100 young people referred to the Paediatric Dentistry Department in Sheffield for the management of dental anxiety. The questionnaire will include items to establish patient demographic information, quality of life (the Child Health Utility 9D) [35], level of dental anxiety (Modified Child Dental Anxiety Scale) [36], any psychological disorders (Revised Child Depression and Anxiety Scale and the Strengths and Difficulties Questionnaire) [37, 38], and willingness to consider using a CBT resource. The findings will allow an estimate to be made of the number of young people and their parents who could potentially be helped by a guided self-help CBT resource and that would need to be assessed in order to identify sufficient patients who are eligible and willing to take part in a future trial. A) The profile of dentally anxious young people Design of the study clinic for patients with dental anxiety at a dental hospital in London [39]. Type of participants The following inclusion and exclusion criteria will be adopted: p Inclusion criteria p Inclusion criteria p Inclusion criteria Children aged 11–16 years old with mild to moderate dental anxiety Children referred to the Paediatric Dentistry Department by a primary or secondary dental care provider for the management of dental anxiety y Exclusion criteria y Exclusion criteria Children who are unable to complete a questionnaire due to learning or language difficulties Data analysis Data will be entered into an electronic database (Statistical Package for Social Sciences, v20) and analysed using simple descriptive statistics. Setting and recruitment B) The perceptions of dental care team members Design of the study This part of the study involves a qualitative study of the perceptions of primary and secondary dental care team members of the developed self-help CBT resource and their willingness to recruit participants to a trial and potentially offer the resource to their own patients. This will be accomplished through conducting and analysing individual interviews. B) The perceptions of dental care team members Design of the study This part of the study involves a qualitative study of the perceptions of primary and secondary dental care team members of the developed self-help CBT resource and their willingness to recruit participants to a trial and potentially offer the resource to their own patients. This will be accomplished through conducting and analysing individual interviews. Suitable child patients will be identified by a clinician member of the study team by screening of referral letters to the Paediatric Dentistry Department according to the inclusion/exclusion criteria described below. The questionnaire will be posted to potential participants, with covering letters and Participant Information Sheets for both the young person and their parent/carer. Young people will be involved in the design of the covering letter and Participant Information Sheets. Parents/ Marshman et al. Pilot and Feasibility Studies (2016) 2:13 Page 4 of 8 Face-to-face individual or group interviews will be conducted with members of the primary and secondary care dental care teams using a topic guide as the framework for the interview. A combination of individual and group interviews will be employed to gain a breadth and depth of information and at the convenience of the participants. g Data analysis The data will be analysed using framework analysis [43]. The framework will be informed by the theory of planned behaviour which has been used previously to explore dental practitioner’s clinical practice [44]. Two researchers will be involved in the analysis of the qualitative data. 1. Identification: practices will be identified from the list of providers in South Yorkshire. 2. Approaching: potential participants will be sent written information in the post. C), D), and E) Evaluation of retention rates and completion rates, acceptability of the guided self-help CBT resource, evaluation of potential outcome measures, and sample size calculation Design of the study This stage will comprise a pre-post test single group design and will involve asking a consecutive sample of young people and their parents to complete the resource to evaluate retention, completion rates, and acceptability, to evaluate potential outcome measures and provide data to allow a sample size calculation for an RCT. Participants will be asked to complete a battery of measures and will then be given the resource to work through with their parent or carer and clinician. After they have completed three visits for dental treatment, they will then complete the questionnaire a second time. The questionnaire will include patient demographic information, dental anxiety (Modified Child Dental Anxiety C), D), and E) Evaluation of retention rates and completion rates, acceptability of the guided self-help CBT resource, evaluation of potential outcome measures, and sample size calculation Design of the study 3. Recruitment: after the initial approach, the dental care professional will then be contacted by telephone by a researcher, after being given at least 1 week to consider whether or not they want to take part, with the aim of arranging a date for the interview. Written consent will be obtained on the day of the interview. Interviews will be conducted by trained researchers with both dental and social science backgrounds to reduce the risk of influencing the results. Dental care professionals will be given thank-you vouchers in gratitude for their time. Sample size Type of participants Type of participants y The following inclusion and exclusion criteria will be adopted: The following inclusion and exclusion criteria will be adopted: p Inclusion criteria Dental professionals who are primarily employed in general dental practice or the salaried dental service in Sheffield Dental professionals who provide dental treatment to children and young people Dental professionals who have made a referral to a secondary dental care provider for the management of dental anxiety of a child or young person Exclusion criteria Exclusion criteria Exclusion criteria Dental professionals who do not treat children aged 9–16 years Dental professionals who do not provide NHS treatment Data collection g The research participants will be recruited from general dental practices, the salaried dental service, and secondary dental care in South Yorkshire. Participants will be purposively recruited to include dental practices serving varying levels of deprivation and ethnic minorities. The goal of purposive sampling will be to provide a range of experiences and views. Sufficient participants should be involved to achieve saturation of information but not so many to prohibit detailed analysis. It is expected that 15–20 dental care professionals will be interviewed. Interviews will be conducted with individuals or in groups and arranged at a mutually convenient time and place, preferably at the participant’s place of work. The interview will be recorded using a digital sound recorder and will last 30–60 min. A) The profile of dentally anxious young people Design of the study The topic guide will be developed based on the theory of planned behaviour to explore the key barriers and facilitators (e.g. attitudes, perceived behavioural control, subjective norms) which may influence dental team members’ intentions to use a self-help CBT resource with their patients in future [40]. The interviews will be digitally recorded and transcribed verbatim as quickly as possible after the event and the recordings deleted after a short time. All identifying information will be removed from the transcripts to ensure anonymity. Setting and recruitment Sample size As a qualitative approach is being used, no formal sample size calculation has been carried out. Based on previous experience, a maximum of 20 participants is likely to be required to achieve saturation [41, 42]. Recruitment will continue until no new themes emerge. Page 5 of 8 Page 5 of 8 Marshman et al. Pilot and Feasibility Studies (2016) 2:13 Scale [45]), and health-related quality of life (Child Health Utility 9D) [46]. A sample of young people and parents/carers will also be interviewed to explore the acceptability of the resource. The interviews will be digitally recorded and transcribed verbatim as quickly as possible after the event and the recordings deleted after a short time. All identifying information will be removed from the transcripts to ensure anonymity. Scale [45]), and health-related quality of life (Child Health Utility 9D) [46]. A sample of young people and parents/carers will also be interviewed to explore the acceptability of the resource. The interviews will be digitally recorded and transcribed verbatim as quickly as possible after the event and the recordings deleted after a short time. All identifying information will be removed from the transcripts to ensure anonymity. participants before data saturation is reached [47, 48]. In order to evaluate the potential outcome measures and determine the sample size for a future RCT, a total sample size of 60 participants will be sought. A sample size of 50 is recommended by Consensus-based Standards for the selection of health measurement instruments [49]. Allowing for a 40 % drop-out rate between baseline and follow-up, the intent is to recruit 100 participants. Data collection Data will also be recorded by a member of the clinical care team to include: missed or cancelled appointments, completion of the course of treatment, and the level of engagement with the resource. The interviews will take place at a mutually convenient time and place, preferably at the young person’s home. The interview will be recorded using a digital sound recorder and will Type of participants Type of participants The research participants will be recruited purposively from new patients to a general dental practice in South Yorkshire, Derbyshire, salaried dental service and the Paediatric Dentistry Department of the Charles Clifford Dental Hospital in Sheffield. The sample will be recruited to involve young people living in areas with a range of different levels of deprivation and from different black and ethnic minority groups. It is expected that a total of 100 families will be recruited. The following inclusion and exclusion criteria will be adopted: The following inclusion and exclusion criteria will be adopted: Inclusion criteria Inclusion criteria Children aged 9–16 years old with mild to moderate dental anxiety Children requiring a course of dental treatment which requires at least three separate visits Children requiring a course of dental treatment which requires at least three separate visits Exclusion criteria Children diagnosed with an underlying psychological disorder 1. Identification: potential participants will be identified by their dentist based on the inclusion and exclusion criteria. Children with an acute dental problem who require urgent dental treatment 2. Initial approach: potential participants and their parents will be approached by a research nurse and the young person will be asked ‘Do you feel worried or afraid about going to the dentist?’ If they say ‘yes’ then they will be invited to take part in the study. Children with a severe disability where communication is not possible Non-English speaking children and parent/ carers (to avoid the need for translating services where the participant’s responses might be unintentionally altered as a result of being translated) 3. Recruitment: after the initial approach, the children and their parents will be given separate written information sheets to take away and read. Data collection Data collection Data will be collected about the following outcomes: dental anxiety, health-related quality of life, Data will be collected about the following outcomes: dental anxiety, health-related quality of life, Data will be collected about the following outcomes: dental anxiety, health-related quality of life, acceptability, missed or cancelled appointments, completion of the course of treatment, and the level of engagement with the resource. 4. At the next dental appointment, which is part of the child’s normal course of planned treatment and will typically involve a non-anxiety-provoking preventive treatment, child and parental consents will be obtained from those wishing to participate in the study. At this time, the CBT resource will be provided and the participant will also complete the baseline questionnaire (T1). acceptability, missed or cancelled appointments, completion of the course of treatment, and the level of engagement with the resource. A battery of outcome measures including the Modified Child Dental Anxiety Scale [36] and the Child Health Utility 9D [46] will be given to the participants to complete then the CBT resource will be introduced along with the accompanying instructions for young people and their parents. Interviews will be arranged once the participants have completed their course of dental treatment and as much of the resource as they want. Data will also be recorded by a member of the clinical care team to include: missed or cancelled appointments, completion of the course of treatment, and the level of engagement with the resource. A battery of outcome measures including the Modified Child Dental Anxiety Scale [36] and the Child Health Utility 9D [46] will be given to the participants to complete then the CBT resource will be introduced along with the accompanying instructions for young people and their parents. 5. The participant will continue along their normal care pathway for their course of treatment (which could include treatment under inhalation sedation) and will work through the CBT resource at subsequent visits with the same dentist. At their third visit, they will complete the same questionnaire again (T2) and continue with their course of treatment as normal. The time interval between T1 and T2 will vary between participants. Interviews will be arranged once the participants have completed their course of dental treatment and as much of the resource as they want. Sample size For the qualitative aspect, no formal sample size calculation has been carried out although previous studies have required interviews with 10–15 Marshman et al. Pilot and Feasibility Studies (2016) 2:13 Page 6 of 8 Page 6 of 8 last for 45–60 min. Interviews will be conducted with both parents and children. Data analysis unfavourable dental experiences. If through involvement in the study a participant is identified as having an undiag- nosed psychological disorder, this will be dealt with by the clinical dental teams and a referral will be made to the Child Adolescent Mental Health Service. The qualitative data will be analysed using framework analysis [43]. Two researchers will be involved in the analysis of the qualitative data. Quantitative data will be entered into an electronic database (Statistical Package for Social Sciences, v20) and analysed using simple descriptive statistics, internal consistency (Cronbach’s alpha) and validity through correlation coefficients to evaluate the performance of the measures. Our main outcomes of interest are take-up, recruitment rates, use of the resource, completion rates, and our ability to gather data at baseline and follow-up. Practical issues involved in performing the study will be challenges of involving general dental practices in re- search. These include ensuring early and continued in- volvement of clinic staff in the research study, developing the research capacity within the dental practice, e.g. train- ing in research methodology, and maintaining enthusiasm for the research to ensure progress and momentum of the study [53]. y The study findings will be widely disseminated through presentations at national and international dental and psy- chotherapy conferences and publications in peer-reviewed journals. A wider programme of dissemination will involve patients and the public. The findings will be disseminated back to participating young people and their parents in an easy read report. The findings will also be posted on the ‘dental fear in children’ thread of the Dental Fear Central forum, an international online resource for those with dental anxiety and dental professionals. A project steering group will be convened to include two members of the Sheffield Health watch and a parent representative as well as the research team and collaborators. The project steer- ing group will meet every 6 months during the project. A panel of five young people (aged 11–16 years) will also be assembled and will meet twice during the project. Competing interests CW i h f Competing interests CW is an author of a range of CBT-based resources that address anxiety, depression, and other disorders including a book and online resources on back to work. These are available commercially as books, cCBT products, and classes. He receives royalty and is a shareholder and director of a company that commercialises these resources. The other authors have declared no other competing interests. In terms of participant comfort, it is not anticipated that participants will feel distressed during the course of the study. However, discussing dental anxiety could lead a participant to consider a traumatic past dental experience and its impact. If a participant does become upset, the interview will be stopped until the partici- pant feels able to continue. The researcher conducting the interviews will have the appropriate training and experience to support young people who have suffered Sample size The project steering group and young people’s panel will be in- volved in designing participant information leaflets, con- sent forms, questionnaires, and the topic guides for the qualitative interviews. The steering group and panel will also discuss and help interpret the results of the study, ad- vise on dissemination, and make recommendations for the design of the pilot trial to evaluate the effectiveness of the resultant CBT resource. Discussion This study protocol is designed to investigate the feasibil- ity of evaluating, in an RCT, a CBT resource for young people (9–16 years) for the reduction of dental anxiety. The findings will provide valuable information regarding recruitment (including sample size), the most appropriate outcome measures, retention and completion rates, and the acceptability of the guided self-help CBT resource to patients, parents, and clinicians informing the design of a future RCT. If in a subsequent RCT the guided self-help CBT resource is found to be effective at reducing dental anxiety, these findings would be relevant to both users (young people and their parents) and service providers (dental practitioners and NHS). One key issue to consider throughout this study is the involvement of children and young people themselves in the development, modification, and evaluation of the resource. Involving children throughout the research process is important as they have their own perspectives and these should be taken onto account whilst making de- cisions about their care [50]. A recent systematic review suggested the need for greater child-centred oral health research especially for evaluating the effectiveness of clin- ical interventions which could have a positive impact on the health outcomes for children and young people [51]. Additionally, engaging children in research enables age- appropriate measures to be chosen, ensures sustainable recruitment strategies, and assists in better dissemin- ation [41, 52]. Abbreviations CBT iti b Abbreviations CBT: cognitive behavioural therapy; NHS: National Health Service; RCT: randomised control trial. Abbreviations CBT: cognitive behavioural therapy; NHS: National Health Service; RCT: randomised control trial. References 1. McGoldrick P, Levitt J, de Jongh A, Mason A, Evans D. Dental anxiety: referrals to a secondary care dental clinic for anxious adult patients: implications for treatment. Br Dent J. 2001;191(12):686–8. 1. McGoldrick P, Levitt J, de Jongh A, Mason A, Evans D. Dental anxiety: referrals to a secondary care dental clinic for anxious adult patients: implications for treatment. Br Dent J. 2001;191(12):686–8. 25. Hofmann SG, Asnaani A, Vonk IJ, Sawyer AT, Fang A. The efficacy of cognitive behavioral therapy: a review of meta-analyses. Cogn Ther Res. 2012;36(5):427–40. 2. Locker D, Liddell A, Dempster L, Shapiro D. Age of onset of dental anxiety. J Dent Res. 1999;78(3):790–6. doi:10.1177/00220345990780031201. 2. Locker D, Liddell A, Dempster L, Shapiro D. Age of onset of dental anxiety. J Dent Res. 1999;78(3):790–6. doi:10.1177/00220345990780031201. 26. Higa-McMillan CK, Francis SE, Rith-Najarian L, Chorpita BF. Evidence base update: 50 years of research on treatment for child and adolescent anxiety. J Clin Child Adolesc Psychol, 2015:1-23. doi:10.1080/15374416. 2015.1046177. 3. Locker D, Thomson WM, Poulton R. Onset of and patterns of change in dental anxiety in adolescence and early adulthood: a birth cohort study. Community Dent Health. 2001;18(2):99–104. 4. Klingberg G, Broberg AG. Dental fear/anxiety and dental behaviour management problems in children and adolescents: a review of prevalence and concomitant psychological factors. Int J Paediatr Dent. 2007;17(6):391–406. doi:10.1111/j.1365-263X.2007.00872.x. 27. Heaton LJ, Leroux BG, Ruff PA, Coldwell SE. Computerized dental injection fear treatment: a randomized clinical trial. J Dent Res. 2013;92(7 suppl):S37–42. doi:10.1177/0022034513484330. 28. Coldwell SE, Wilhelm FH, Milgrom P, Prall CW, Getz T, Spadafora A, et al. Combining alprazolam with systematic desensitization therapy for dental injection phobia. J Anxiety Disorders. 2007;21(7):871–87. http://dx.doi.org/10. 1016/j.janxdis.2007.01.001. 5. Chhabra N, Chhabra A, Walia G. Prevalence of dental anxiety and fear among five to ten year old children: a behaviour based cross sectional study. Minerva Stomatol. 2012;61(3):83–9. 6. Lee C-Y, Chang Y-Y, Huang S-T. Prevalence of dental anxiety among 5- to 8- year-old Taiwanese children. J Public Health Dent. 2007;67(1):36–41. doi:10.1111/j.1752-7325.2007.00006.x. 29. Dewis LM, Kirkby KC, Martin F, Daniels BA, Gilroy LJ, Menzies RG. Computer-aided vicarious exposure versus live graded exposure for spider phobia in children. J Behav Ther Exp Psychiatry. 2001;32(1):17–27. 7. Luoto A, Lahti S, Nevanpera T, Tolvanen M, Locker D. Oral-health-related quality of life among children with and without dental fear. Int J Paediatr Dent. 2009;19(2):115–20. 30. Melamed BG, Weinstein D, Hawes R, Katin-Borland M. Author details 1 17. Brahm C-O, Lundgren J, Carlsson SG, Nilsson P, Corbeil J, Hägglin C. Dentists’ views on fearful patients. Problems and promises. Swed Dent J. 2012; 36(2):79–89. 1School of Clinical Dentistry, University of Sheffield, Claremont Crescent, Sheffield S10 2TA, UK. 2Paediatric Dentistry Department, Charles Clifford Dental Hospital, Wellesley Road, Sheffield S10 2SZ, UK. 3Department of Psychology, Sociology, and Politics, Sheffield Hallam University, Room 2.05 Heart of the Campus, Collegiate Crescent, Sheffield S10 2BQ, UK. 4School of Psychology and Clinical Language Sciences, University of Reading, Earley Gate, Whiteknights, Reading, Berkshire RG6 6AL, UK. 5Oral Health Services Research and Dental Public Health, King’s College London, Denmark Hill Campus, Caldecot Road, London SE5 9RW, UK. 6School of Health and Related Research (ScHARR), University of Sheffield, Regent Court, 30 Regent Street, Sheffield S1 4DA, UK. 7Institute of Health and Wellbeing, Mental Health and Wellbeing, University of Glasgow, Gartnavel Royal Hospital, Administration Building, 1055 Great Western Road, Glasgow G12 0XH, UK. 8Derbyshire 18. Harris RV, Pender SM, Merry A, Leo A. Unravelling referral paths relating to the dental care of children: a study in Liverpool. Primary Dental Care. 2008; 15(2):45–52. doi:10.1308/135576108784000294. 19. Koroluk LD. Dental anxiety in adolescents with a history of childhood dental sedation. ASDC J Dent Child. 1999;67(3):200–5. 161. 20. NICE. Sedation in children and young people. Costing report: implementing nice guidance. London: National Institute for Health and Care Excellence; 2010. 21. Arch LM, Humphris GM, Lee GTR. Children choosing between general anaesthesia or inhalation sedation for dental extractions: the effect on dental anxiety. Int J Paediatr Dent. 2001;11(1):41–8. doi:10.1046/j.1365-263x. 2001.00238.x. Community Health Services, Long Eaton Dental Clinic, Midland Street, Long Eaton, Nottingham NG10 1RY, UK. 22. Levitt J, McGoldrick P, Evans D. The management of severe dental phobia in an adolescent boy: a case report. Int J Paediatr Dent. 2000;10(4):348–53. doi:10.1046/j.1365-263x.2000.00218.x. Received: 2 December 2015 Accepted: 19 February 2016 Authors’ contributions The design of the study was conceived primarily by Dr Zoe Marshman, Professor Helen Rodd, and Dr Jenny Porritt with Professor Chris Williams, Professor Cathy Creswell, Professor Tim Newton, and Professor Sarah Baker. The clinical aspects of the protocol were designed by Professor Helen Rodd, Miss Annie Morgan, Mrs Suneeta Prasad, and Miss Jennifer Kirby. The health economic aspects of the design of the study were conceived by Dr Katherine Stevens. Dr Zoe Marshman and Miss Annie Morgan wrote the draft, and subsequent revisions of the manuscript were made by Dr Ekta Page 7 of 8 Page 7 of 8 Marshman et al. Pilot and Feasibility Studies (2016) 2:13 Marshman et al. Pilot and Feasibility Studies (2016) 2:13 Gupta. All authors contributed to the critical revision of the manuscript for important intellectual content. All authors read and approved the final manuscript. 13. Gustafsson A. Dental behaviour management problems among children and adolescents—a matter of understanding? Studies on dental fear, personal characteristics and psychosocial concomitants. Swed Dent J Suppl. 2010;202:1–46. 14. Wogelius P, Poulsen S. Associations between dental anxiety, dental treatment due to toothache, and missed dental appointments among six to eight-year- old Danish children: a cross-sectional study. Acta Odontol Scand. 2005;63(3): 179–82. Received: 2 December 2015 Accepted: 19 February 2016 23. Bankole OO, Aderinokun GA, Denloye OO, Jeboda SO. Maternal and child’s anxiety—effect on child’s behaviour at dental appointments and treatments. Afr J Med Med Sci. 2002;31:349–52. 24. Williams C, Garland A. A cognitive–behavioural therapy assessment model for use in everyday clinical practice. Adv Psychiatr Treat. 2002;8(3):172–9. doi:10.1192/apt.8.3.172. Acknowledgements We would like to thank the patients and patient representatives who contributed to the design of the study. We would like to thank the patients and patient representatives who contributed to the design of the study. This paper presents independent research funded by the National Institute for Health Research (NIHR) under its Research for Patient Benefit (RfPB) Programme (Grant Reference Number PB-PG-1111-26029). The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, or the Department of Health. This paper presents independent research funded by the National Institute for Health Research (NIHR) under its Research for Patient Benefit (RfPB) Programme (Grant Reference Number PB-PG-1111-26029). The views 15. Hallberg U, Camling E, Zickert I, Robertson A, Berggren ULF. Dental appointment no-shows: why do some parents fail to take their children to the dentist? Int J Paediatr Dent. 2008;18(1):27–34. doi:10.1111/j.1365-263X.2007.00867.x. g expressed are those of the authors and not necessarily those of the NHS, the NIHR, or the Department of Health. 16. Hirshfeld-Becker DR, Biederman J. Rationale and principles for early intervention with young children at risk for anxiety disorders. Clin Child Fam Psychol Rev. 2002;5(3):161–72. 12. Taani DQ. Dental attendance and anxiety among public and private school children in Jordan. Int Dent J. 2002;52(1):25–9. Marshman et al. Pilot and Feasibility Studies (2016) 2:13 36. Wong HM, Humphris GM, Lee GT. Preliminary validation and reliability of the Modified Child Dental Anxiety Scale. Psychol Rep. 1998;83(3 Pt 2):1179–86. doi:10.2466/pr0.1998.83.3f.1179. 37. Chorpita BF, Moffitt CE, Gray J. Psychometric properties of the Revised Child Anxiety and Depression Scale in a clinical sample. Behav Res Ther. 2005; 43(3):309–22. http://dx.doi.org/10.1016/j.brat.2004.02.004. 38. Goodman R. The Strengths and Difficulties Questionnaire: a research note. J Child Psychol Psychiatry. 1997;38(5):581–6. 38. Goodman R. The Strengths and Difficulties Questionnaire: a research note. J Child Psychol Psychiatry. 1997;38(5):581–6. 39. Boyle CA, Newton T, Milgrom P. Who is referred for sedation for dentistry and why? Br Dent J. 2009;206(6):E12-E. 39. Boyle CA, Newton T, Milgrom P. Who is referred for sedation for dentistry and why? Br Dent J. 2009;206(6):E12-E. 40. Ajzen I. From intentions to actions: a theory of planned behavior. In: Kuhl J, Beckmann J, editors. Action Control. SSSP Springer Series in Social Psychology. Berlin Heidelberg: Springer Berlin Heidelberg; 1985. p. 11-39. 40. Ajzen I. From intentions to actions: a theory of planned behavior. In: Kuhl J, Beckmann J, editors. Action Control. SSSP Springer Series in Social Psychology. Berlin Heidelberg: Springer Berlin Heidelberg; 1985. p. 11-39. 41. Marshman Z, Innes N, Deery C, Hall M, Speed C, Douglas G, et al. The management of dental caries in primary teeth-involving service providers and users in the design of a trial. Trials. 2012;13(1):1–9. 42. Marshall B, Cardon P, Poddar A, Fontenot R. Does sample size matter in qualitative research?: A review of qualitative interviews in IS research. J Computer Information Systems. 2013;54(1):11–22. 43. Ritchie J, Spencer L. Qualitative data analysis for applied policy research. The qualitative researcher’s companion.London: Routledge; 1993. pp. 173–194. 44. Bonetti D, Pitts NB, Eccles M, Grimshaw J, Johnston M, Steen N, et al. Applying psychological theory to evidence-based clinical practice: identifying factors predictive of taking intra-oral radiographs. Soc Sci Med. 2006;63(7):1889–99. 45. Humphris GM, Freeman R, Campbell J, Tuutti H, D’Souza V. Further evidence for the reliability and validity of the Modified Dental Anxiety Scale. Int Dent J. 2000;50(6):367–70. doi:10.1111/j.1875-595X.2000.tb00570.x. 46. Stevens K. Developing a descriptive system for a new preference-based measure of health-related quality of life for children. Qual Life Res. 2009; 18(8):1105–13. doi:10.1007/s11136-009-9524-9. 47. Gilchrist F, Marshman Z, Deery C, Rodd HD. The impact of dental caries on children and young people: what they have to say? Int J Paediatr Dent. 2015;25(5):327–38. doi:10.1111/ipd.12186. 48. O’ Donnell SC, Marshman Z, Zaitoun H. Marshman et al. Pilot and Feasibility Studies (2016) 2:13 Marshman et al. Pilot and Feasibility Studies (2016) 2:13 ‘Surviving the sting’: the use of solicited diaries in children and young people with oral mucosal disease. Int J Paediatr Dent. 2013;23(5):352–8. doi:10.1111/ipd.12028. 49. Mokkink LB, Terwee CB, Patrick DL, Alonso J, Stratford PW, Knol DL, et al. The COSMIN study reached international consensus on taxonomy, terminology, and definitions of measurement properties for health-related patient-reported outcomes. J Clin Epidemiol. 2010;63(7):737–45. http://dx. doi.org/10.1016/j.jclinepi.2010.02.006. 50. RCPCH. Involving children and young people in health services. London: Royal College of Paediatrics and Child Health; 2011.http://www.rcpch.ac.uk/ system/files/protected/page/ Involving%20CAYP%20in%20Health%20Services.pdf. 51. Marshman Z, Gupta E, Baker SR, Robinson PG, Owens J, Rodd HD, et al. Seen and heard: towards child participation in dental research. Int J Paediatr Dent. 2015;25(5):375–82. doi:10.1111/ipd.12179. 51. Marshman Z, Gupta E, Baker SR, Robinson PG, Owens J, Rodd HD, et al. Seen and heard: towards child participation in dental research. Int J Paediatr Dent. 2015;25(5):375–82. doi:10.1111/ipd.12179. 52. Marshman Z, Hall MJ. Oral health research with children. Int J Paediatr Dent. 2008;18(4):235–42. 52. Marshman Z, Hall MJ. Oral health research with children. Int J Paediatr Dent. 2008;18(4):235–42. 53. Martin-Kerry JM, Lamont TJ, Keightley A, Calache H, Martin R, Floate R, et al. Practical considerations for conducting dental clinical trials in primary care. Br Dent J. 2015;218(11):629–34. doi:10.1038/sj.bdj.2015.498. 53. Martin-Kerry JM, Lamont TJ, Keightley A, Calache H, Martin R, Floate R, et al. Practical considerations for conducting dental clinical trials in primary care. Br Dent J. 2015;218(11):629–34. doi:10.1038/sj.bdj.2015.498. References Reduction of fear-related dental management problems with use of filmed modeling. J Am Dent Assoc. 1975;90(4):822–6. 8. Esa R, Ong A, Humphris G, Freeman R. The relationship of dental caries and dental fear in Malaysian adolescents: a latent variable approach. BMC Oral Health. 2014;14(1):19. 8. Esa R, Ong A, Humphris G, Freeman R. The relationship of dental caries and dental fear in Malaysian adolescents: a latent variable approach. BMC Oral Health. 2014;14(1):19. 31. Hofmann SG, Wu JQ, Boettcher H. Effect of cognitive-behavioral therapy for anxiety disorders on quality of life: a meta-analysis. J Consult Clin Psychol. 2014;82(3):375. 9. Nicolas E, Bessadet M, Collado V, Carrasco P, Rogerleroi V, Hennequin M. Factors affecting dental fear in French children aged 5–12 years. Int J Paediatr Dent. 2010;20(5):366–73. doi:10.1111/j.1365-263X.2010.01054.x. 32. Boman UW, Carlsson V, Westin M, Hakeberg M. Psychological treatment of dental anxiety among adults: a systematic review. Eur J Oral Sci. 2013;121(3): 225–34. doi:10.1111/eos.12032. Factors affecting dental fear in French children aged 5–12 years. Int J Paediatr Dent. 2010;20(5):366–73. doi:10.1111/j.1365-263X.2010.01054.x. 10. Colares V, Franca C, Ferreira A, Amorim Filho HA, Oliveira MCA. Dental anxiety and dental pain in 5- to 12-year-old children in Recife, Brazil. Eur Arch Paediatr Dent. 2013;14(1):15–9. doi:10.1007/s40368-012-0001-8. 10. Colares V, Franca C, Ferreira A, Amorim Filho HA, Oliveira MCA. Dental anxiety and dental pain in 5- to 12-year-old children in Recife, Brazil. Eur Arch Paediatr Dent. 2013;14(1):15–9. doi:10.1007/s40368-012-0001-8. 33. Klesges RC, Malott JM, Ugland M. The effects of graded exposure and parental modeling on the dental phobias of a four-year-old girl and her mother. J Behav Ther Exp Psychiatry. 1984;15(2):161–4. http://dx.doi.org/10. 1016/0005-7916(84)90012-0. 11. Skaret E, Berg E, Kvale G, Raadal M. Psychological characteristics of Norwegian adolescents reporting no likelihood of visiting a dentist in a situation with toothache. Int J Paediatr Dent. 2007;17(6):430–8. doi:10.1111/j.1365-263X.2007. 00869.x. 34. Sanders MR, Jones L. Behavioural treatment of injection, dental and medical phobias in adolescents: a case study. Behav Psychother. 1990;18(04):311–6. 34. Sanders MR, Jones L. Behavioural treatment of injection, dental and medical phobias in adolescents: a case study. Behav Psychother. 1990;18(04):311–6. 35. Stevens K. Valuation of the child health utility 9D index. PharmacoEconomics. 2012;30(8):729–47. doi:10.2165/11599120-000000000-00000. 12. Taani DQ. Dental attendance and anxiety among public and private school children in Jordan. Int Dent J. 2002;52(1):25–9. Page 8 of 8 Page 8 of 8 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: • 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 and we will help you at every step: • We accept pre-submission inquiries
https://openalex.org/W4324103076	https://journals.bilpubgroup.com/index.php/jsbe/article/download/5533/4702	English	null	An Analysis of the COVID-19 Consequences on the Uncertainty Socio-economic Indicators of Sub-Saharan Africa	Journal of sustainable business and economics	2,023	cc-by	6,378	Journal of Sustainable Business and Economics \| Volume 06 \| Issue 01 \| January 2023 Journal of Sustainable Business and Economics \| Volume 06 \| Issue 01 \| January 2023 An Analysis of the COVID-19 Consequences on the Uncertainty Socio-economic Indicators of Sub-Saharan Africa Etienne Gatera Pedagogical University of Krakow, ul. Podchorążych 2, 30-084, Kraków, Poland ABSTRACT This study aims to investigate the main sectors of economic development before and the current situation of COVID-19 for Sub-Saharan African countries by demonstrating country experiences, the role of vaccines, and the SSA economy forecast. The study has four main sections, including an introduction, an overview of socioeconomic indicators before the pandemic, methods, results findings, and discussion. The study used mixed methods, including an approach based on secondary data. The quantitative results were analysed using both empirical methods and the researcher’s prior expertise. The analysis of the effects of the COVID-19 pandemic on SSA countries was based on long-term data collected by several international financial institutions. The research findings demonstrated conclusively that COVID-19 is causing the collapse of the SSA economy, the first economic recession in 25 years, $37-79 billion in lost GDP by 2020, and an export decrease of 10.6%. In education, for example, 64% of primary and 50% of secondary students lack ICT training, 89% (216 million) do not have access to a home computer, and 82% (199 million) do not have an Internet connection missed classes during the COVID-19 period. The agricultural sector in SSA is also impacted by over 239 million hungry people. COVID-19 mass vaccinations and public debt amount to over $154 billion in obligations to get the SSA economy back on its feet with zero tolerance for embezzlement of public funds. These results can be used to make the economies of SSA countries resilient to the current crises and to address some thematic issues, such as the implementation of the African Continental Free Trade Area (AfCFTA) in all SSA countries, which will save time and money by getting rid of border taxes. Therefore, policymakers can use the findings to begin formulating plans to address issues like economic development, education, and food insecurity. Keywords: COVID-19; Sub-Saharan Africa (SSA); Economic recession; Vaccines; Economic forecasts D-19; Sub-Saharan Africa (SSA); Economic recession; Vaccines; Economic forecasts CORRESPONDING AUTHOR: Etienne Gatera, Pedagogical University of Krakow, ul. Podchorążych 2, 30-084, Kraków, Poland; Email: etienne.gatera@doktorant.up.krakow.pl ARTICLE INFO Received: 12 April 2022 \| Received in revised form: 3 February 2023 \| Accepted: 13 February 2023 \| Published: 23 February 2023 DOI: https://doi.org/10.30564/jsbe.v6i1.45 CITATION Gatera, E., 2023. An Analysis of the COVID-19 Consequences on the Uncertainty Socio-economic Indicators of Sub-Saharan Africa. Journal of Sustainable Business and Economics. 6(1), 15-25. DOI: https://doi.org/10.30564/jsbe.v6i1.45 COPYRIGHT Published by Bilingual Publishing Group. ABSTRACT This is an open access article under the Creative Commons Attribution (CC BY) license (https://crea tivecommons.org/licenses/by/4.0/). CORRESPONDING AUTHOR: Etienne Gatera, Pedagogical University of Krakow, ul. Podchorążych 2, 30-084, Kraków, Poland; Email: etienne.gatera@doktorant.up.krakow.pl ARTICLE INFO Received: 12 April 2022 \| Received in revised form: 3 February 2023 \| Accepted: 13 February 2023 \| Published: 23 February 2023 DOI: https://doi.org/10.30564/jsbe.v6i1.45 CITATION Gatera, E., 2023. An Analysis of the COVID-19 Consequences on the Uncertainty Socio-economic Indicators of Sub-Saharan Africa. Journal of Sustainable Business and Economics. 6(1), 15-25. DOI: https://doi.org/10.30564/jsbe.v6i1.45 COPYRIGHT Published by Bilingual Publishing Group. This is an open access article under the Creative Commons Attribution (CC BY) license (https://crea tivecommons.org/licenses/by/4.0/). *CORRESPONDING AUTHOR: Etienne Gatera, Pedagogical University of Krakow, ul. Podchorążych 2, 30-084, Kraków, Poland; Email: etienne.gatera@doktorant.up.krakow.pl ARTICLE INFO Received: 12 April 2022 \| Received in revised form: 3 February 2023 \| Accepted: 13 February 2023 \| Published: 23 February 2023 DOI: https://doi.org/10.30564/jsbe.v6i1.45 CITATION Gatera, E., 2023. An Analysis of the COVID-19 Consequences on the Uncertainty Socio-economic Indicators of Sub-Saharan Africa. Journal of Sustainable Business and Economics. 6(1), 15-25. DOI: https://doi.org/10.30564/jsbe.v6i1.45 COPYRIGHT Published by Bilingual Publishing Group. This is an open access article under the Creative Commons Attribution (CC BY) license (https://crea tivecommons.org/licenses/by/4.0/). CITATION Gatera, E., 2023. An Analysis of the COVID-19 Consequences on the Uncertainty Socio-economic Indicators of Sub-Saharan Africa. Journal of Sustainable Business and Economics. 6(1), 15-25. DOI: https://doi.org/10.30564/jsbe.v6i1.45 Published by Bilingual Publishing Group. This is an open access article under the Creative Commons Attribution (CC BY) license (https://crea tivecommons.org/licenses/by/4.0/). 15 Journal of Sustainable Business and Economics \| Volume 06 \| Issue 01 \| January 2023 Economy According to the World Bank forecast, SSA was ex pected to witness a rise in growth rate from 3% in 2018 to 3.5% in 2019. Within 21 countries with per capita growth of 5% or more, 24 countries remain at the same level of growth, including the two largest economies, Nigeria and South Africa [4]. The SSA countries’ public debt was diverse. For some countries, it was sustaina ble, whereas for others, it came with high risks. Coun tries like Cameroon, Cabo Verde, Ethiopia, Ghana, and Zambia were in the high-risk zone [5]. Before the COV ID-19 pandemic, the SSA economy was experiencing a better growth rate. It was an average of 3.6% from 2017 to 2019. But that didn’t stop some countries’ rates of public debt from rising, which handicapped the SSA economy’s autonomy. Originating in Wuhan City in China and later circulating to other continents, COVID-19 was offi cially declared a global pandemic by WHO on 11th March 2020 [1]. It is the fifth pandemic of the coro navirus declared globally after 1918. WHO named it a novel coronavirus (2019-Nov) on 12th January 2020. Later, the International Committee on Taxon omy of Virus named it SARS-CoV-2 [2]. The SARS- CoV-2 spread in different continents, including Asia, Europe, America, and later Africa. Education Education continues to be underfunded and un equal in 25 of the 49 SSA countries. Both affect development plans, create disputes over natural re sources among countries, generate low investment in education, and result in poor education policy [7]. According to the 2018 UNESCO statistical re port, SSA countries have the highest primary out-of- school rate compared to the globe. It stands within the range of 20%-40% [43]. Thus, SSA had the most dropouts in 2019. 32.2 million male and female pri mary school dropouts. The SSA education is the first for children globally who have dropped out due to mismanagement, civil wars, and poverty. Since its recognition as a global pandemic in March 2020 by WHO, no medicines for COVID-19 were available. Only a few vaccines are used at the moment. The WHO has recognised the Pfizer COV ID-19 vaccine (BNT162b2); AstraZeneca/Oxford COVID-19 vaccine, Moderna and Janssen (Johnson & Johnson). WHO approved Sinopharm on 10 May 2021 and Sinovacon on 24 May 2021. Numerous articles have examined and evaluated the impact on the different domains. 1.1 Overview of SSA economy, education and agriculture domains before COVID-19 pandemic In the 21st century, the COVID-19 pandemic is the most talked about, both in developed and devel oping countries. The first official case of COVID-19 was recorded in the city of Wuhan, China in Decem ber 2019. In those difficult times, scientists around the world started research to find the COVID-19 vaccine. Many countries devised different strategies to cope with the effects of the COVID-19 pandemic, including economic, educational, agricultural, and general lifestyle sectors, especially in sub-Saharan Africa. This paper focused on the impact of the COVID-19 pandemic on SSA, country experiences, the role of vaccines, reasons for the delay in the roll out, and economic forecasts. The study was based on a review of various reports from international finan cial institutions (IFIs). 1.2 Hypothesis We hypothesise that there are destructive conse quences in major domains (economy, education, ag riculture, and heather sectors) in SSA caused by the COVID-19 pandemic. According to the WB, in 2019, the SSA economy was annually growing at 2.28%. The WB predicted that the SSA economy would fall to –3.3% in 2020 since it is the first recession in the past 25 years [13]. COVID-19 caused instability in airspace transport as some countries closed borders, leading to uncertainty in industries engaged in international commerce. Agriculture The GDP for SSA is expected to increase from less than 1% in 2005-2007 to 2.3% in 2050 [8]. The SSA and South Asia will need to double agricul tural production to respond to the demand. Hunger and malnutrition remain a challenge. Eradication of hunger is not possible by 2030, not even by 2050; as projected, 8% of the global population will remain The change in human lifestyles, such as the nega tive effect on the world uncertainty economy. It start ed with the US and UK before and after COVID-19; later, global economic fluctuations occurred [3]. 16 Journal of Sustainable Business and Economics \| Volume 06 \| Issue 01 \| January 2023 undernourished [9]. The SSA countries have a higher percentage of malnourished populations, especially in Ethiopia and Chad, due to violent conflict and polit ical instability [10]. Better technologies in the agricul ture sector, its development, and improved govern ance are the fundamental needs of food security [11]. To achieve the goal, they required increased food production and imports; food must be accessible in combination with water accessibility [12]. data is collected and processed. It provides informa tion about the data’s content so that others can use it in future projects and understand the data structure [17]. Furthermore, data are being used to improve trans parency throughout the research process [18]. This study collected data from different official interna tional organisations, such as IFIs, non-governmental organisations, and the WHO. We collected and analysed different official re ports about the effects of COVID-19 on SSA in var ious domains. Most of the data collected regards the impact of COVID-19 on education. (Dropout in pri mary, secondary, and university; early marriage; and teenage girls’ pregnancies). Economically, SSA was in the process of growth. During the discussion, we mainly focus on the role of vaccines and the reasons behind rollout delays, countries’ experiences, and economic forecasts. 2.2 Methodology The methodology is a way of finding procedures for scientific exploration. It provides clarity for understanding many approaches that involve the re search process and design [19]. This study employed quantitative methods as well as secondary data. We use empirical methods and the researcher’s experi ence during data analysis and presentation. Our find ings are based on longitudinal data from various in ternational organisations, like IFIs (WB, IMF, ADB), FAO, UNESCO, and UNCTAD report on the impact of the COVID-19 pandemic on SSA countries. We use these reports from the UN agencies and other official global institutions about how COVID-19 affected the SSA to figure out how horrible things were in the SSA. Therefore, after the WHO declared COVID-19 a pandemic, many SSA countries implemented total lockdowns and self-isolation quarantines [14]. More than 191 schools were closed, with 740 million girls estimated to be out of school until the end of March 2020. Along with a lack of internet, laptops, and electricity. As a result, schools closing the gender gap have played a role in increasing the risks of teen age girls’ pregnancies [15]. The more COVID-19 affected the SSA coun tries, the worse the situation became due to partial lockdowns. It prevented people from going about their daily lives, resulting in population hunger [16]. Women suffer more from hunger compared to men; therefore, the vaccine is the only way to control the COVID-19 pandemic and ensure economic recovery. 2.3 Empirical research and results Empirical research is based on the observation and measurement of phenomena directly experi enced by the researcher [20]. Therefore, empirically, we analysed and interpreted the different reports concerned with the consequences of the COVID-19 pandemic, in which the SSA countries underwent the 2.1 Data description Data description is a method of documenting how 17 Journal of Sustainable Business and Economics \| Volume 06 \| Issue 01 \| January 2023 most crucial phases of socio-economic development, especially in education, economy, and agriculture. Finally, the data analysis helped us confirm the study hypothesis. SSA’s economy has been and continues to be destabilized by COVID-19’s effects in all domains, including agriculture and the exports of the natural resources industry. Following the decline in Nige ria’s revenues in 2020, other major fuel exporters in the region, such as Algeria (–2.5%), Angola (–3.8%), and Congo (–2.4%), have also experienced smaller declines in their sales [23]. Therefore, it led to a re duction in the capacity of imported products for the affected countries. In general, the GDP of SSA coun tries is declining. The trade balance is increasingly deteriorating, domestic supply problems and the growth of the informal sector are leading to reces sion and debt burdens on SSA countries. 3.1 Economy and services The current crisis affects business and govern ment balance sheets, and SSA development gains and growth expectations have been jeopardized for years. According to World Bank, SSA will enter a recession in 2020 for the first time in over 25 years, with the COVID-19 outbreak costing the country be tween $37 billion and $79 billion in lost production in 2020 alone [21]. The IMF, WB, and OECD predict ed a –1.25% fall in world GDP and a 30% fall in fuel prices. The average decline in global GDP predicted was 4%, and fuel prices were expected to fall by 60% in the extreme impact scenario. These predic tions had significant consequences for key exports and trade costs. 3.2 Education and teenage pregnancies Before the COVID-19 pandemic, SSA educa tion ranked last in the global ranking on quality and quantity of education. Many students are at risk of not returning to school in most South Asian and SSA countries. All domains were affected by the COV ID-19 pandemic and the education sector was not spared either [27]. As the virus spread, this weakened the education system worldwide, with about 90% of the 24 million students risked not returning to school. Although the online distance education model has been established in a few SSA countries, only 64% of primary teachers and 50% of secondary teachers have received the necessary training and ICT skills. 89% of students (216 million) do not have comput ers at home for the online learning model; 82% (199 million) do not have access to the Internet; and 26 million students do not have mobile Internet. The COVID-19 pandemic led to the closing of 191 na tional borders, with 1.5 billion pupils estimated to be out of school, with 740 million young girls [15]. The worse impacts were teenage girls’ pregnancies and surviving rape or sexual assault. During the COV ID-19 pandemic, Sierra Leone had to close schools for eight months, and 11,000 teens got pregnant [28]. African exports were expected to fall by 10.6% under the mild impact scenario, primarily due to a decline in fuel exports, followed by food exports. A significant drop in fuel prices was expected to cause substantial losses in government revenue, as ob served in the mild and severe impact scenarios [22]. Between the first and second quarters of 2020, South Africa’s GDP, for example, dropped by 51%. Moreover, the pandemic could cause an additional 26 to 40 million people in SSA to fall into poverty [23]. As a result, the £2.9 billion cut to foreign assistance to SSA countries’ budgets, and the budget still has uncertainty about the COVID-19 response. The WB, remittances are expected to decline in all ma jor regions, with SSA experiencing a 23.1% decline against a global decline of 20% [24]. The travel and tourism industry employs one in 20 people in SSA. Tourism was down since COVID-19 and was a significant source of income for countries such as, Rwanda, Botswana, Seychelles, and Kenya, generat ing billions of dollars and jobs [25]. Role of COVID-19 vaccines and reason behind rollout delay Despite considerable uncertainty about the pan demic’s trajectory, a way out of this health and economic crisis is becoming more apparent. The scientific community worked day and night to find a COVID-19 solution (either a vaccine or medicine). We now have several vaccines that can reduce the severity and frequency of infections. The interna tional community believes that increasing immunisa tion coverage in SSA is imperative for reasons other than local livelihoods and growth. A global public good also has extensive regional treatment. The most long-term recovery for any country, anywhere, ne cessitates a worldwide effort involving everyone [30]. Nigeria Nigeria is economically stronger than SSA. How ever, its growth slowed down as a result of the COV ID-19 effect. Since the 2020 recession entered the Nigerian economy due to the fall of oil prices, failed global demand due to containment measures against the spread of the pandemic. The lockdown measures seriously affected aviation, tourism, manufacturing, and trade. The esteemed GDP shrunk by 3% in 2020. Inflation rose to 12.8% in 2020, pushing the prices of food items higher. The Central Bank of Nigeria cut the policy rate by 100 basis points to 11.5% to con tain soaring food prices. The fiscal deficit stepped up to 5.2% in 2020 from 4.3% in 2019. The public debt increased to $85.9 billion (25% of GDP) in June 2020 [31]. 3.3 Hunger and poverty (agriculture) Thus, according to WFP (2020), it is estimated 18 Journal of Sustainable Business and Economics \| Volume 06 \| Issue 01 \| January 2023 that COVID-19 could double the number of hungry people in the world to 820 million in 2018, of which 239 million will be in SSA countries. COVID-19 will mainly affect rural areas; it will have a signifi cant direct and indirect economic impact on vulner able rural communities, especially women, leading to increased hunger and poverty. Farmers, herders, and foresters are particularly affected. Informality is common in rural areas, especially among women, and is linked to poverty [16]. The decline in remittanc es, compounded by other ongoing crises (such as lo custs, droughts, and conflicts) can impact hunger and poverty [29]. COVID-19 is still present in Africa and can be interpreted differently from country to coun try. However, the loss of small businesses due to the lockdown and social distancing measures leads to poverty, hunger, and even loss of life. The budget deficit was 14% of the GDP. It mainly consisted of government spending for the economy impacted by the pandemic. External debt in South Africa decreased in 2021 compared to 2020. Thus, due to the lockdown measures and the third wave, South Africa is still the most impacted country in the SSA [30]. 3.4 Countries’ experiences Due to the different methods adopted by coun tries, the impact of COVID-19 in all SSA countries is not similar and not at the same level. Since the first case in Africa, some countries have implement ed three waves of total or semi-lockdown, South Africa, Rwanda, and others. Other countries, particu larly Burundi and Tanzania, have not implemented any form of containment strategy. Therefore, the real difference in the impact of this phenomenon is explained. In this section, we explain in detail the experiences of South Africa and Nigeria. 3.5 Role of COVID-19 vaccines, reasons be hind rollout delay, and economic forecasts Role of the COVID-19 vaccines in SSA Indeed, due to the different forms of COVID-19 remarked so far, fewer percent of people vaccinated can be infected with COVID-19. Scientists confirm that vaccination against COVID-19 is a way to re sume normal life, business, schools, tourism, travel, and other human activities. SSA has one of the high est rates of HIV, AIDS, and other diseases. The role of the COVID-19 vaccine is to protect people living with HIV and AIDS from serious health problems or hospitalisation if they have contracted COVID-19 [44]. In the optimistic scenario, we explore the possibility of a much stronger recovery than in the baseline sce nario, as activity in the contact-intensive sector recovers rapidly and improves self-confidence through vaccine rollout. This is because activity in the high-contact sec tor recovers rapidly through vaccine deployment, which lifts the level of confidence [30]. So far, the rollout of vaccines in advanced economies has led to a faster recovery, with factories, restaurants, and tourism re covering, while a slightly less pronounced impact is observed in SSA due to vaccine shortages. 3.6 Economic forecasts According to the IMF, the forecast is contingent not only on the success of the virus-vaccine war but also on how well economic measures implemented amid significant uncertainty can limit the long-term consequences of this unprecedented catastrophe. The IMF also anticipates that growth in SSA will increase to 3.4% in 2022, which is 0.2% higher than its prior prediction. In addition, the IMF expects the global economy to rise by 6% in 2021, up from 5.5% in January. Furthermore, the economic recovery in 2021 will be bolstered by the introduction of the COVID-9 vaccine [30]. Countries’ experiences economic projection a. South Africa: South Africa’s GDP plummeted by a stunning 7% last year. Due to a better-than-ex pected fourth quarter in 2021, there was an upward revision. However, this is likely to be offset by the second wave of COVID-19, which peaked in Janu ary 2021. It resulted in the reintroduction of some contain ment measures in the first quarter. In 2021, the net effect will be a growth rate of 3.1%. The authorities have initiated a large-scale vaccination campaign, hoping that the vaccine might reduce the likelihood of new outbreaks [30]. South Africa South Africa’s real GDP growth was 0.2% in 2019. The pandemic and the lockdown measures have spread further and damaged the national econ omy. The real GDP contracted by 8.2% in 2020. Therefore, a decline in the mining, manufacturing, and communication sectors constituted a significant loss. On the demand side, all components faced a co lossal contraction, with 30.4% in investment. The re serve bank cumulative 300 basis points in 2020 from 6.5% to 3.5% to support businesses and households affected by the pandemic. The inflation rate declined to 3.4% in 2020, with the bank’s reserve at 3% to 6%. Despite these promises of global equity, three-quar 19 Journal of Sustainable Business and Economics \| Volume 06 \| Issue 01 \| January 2023 ters of the one billion doses of COVID-19 vaccine administered globally had been administered in only ten countries by the end of April 2021 [33]. However, in low-income countries, only 1.1% of people have received one dose [34]. Indeed, 2.7 billion doses have been administered globally; only about 1.5% of this total was administered on the African continent. quate refrigeration systems for vaccine storage [35]. Even though SSA is struggling with the doses of the vaccines, the willingness to accept is higher in gen eral in SSA. Reason behind vaccines rollout delay Many advanced economies are focusing on mass vaccinations to rebuild their economies and return to real life. However, in SSA, most countries have found themselves at the back of the queue, with limited purchasing power and few options, and will struggle to meet the basic needs of health workers [30]. Although considerable progress has been made in developing the COVID-19 vaccine, there are many potential barriers to the large-scale rollout of the vaccine in SSA. These include inadequate transport infrastructure and distribution systems, the vulner ability of medical systems to implementing large- scale immunisation programmes, and old or inade b. Nigeria: Despite substantial obstacles, the Nigerian economy is predicted to recover to 1.1% growth in 2021, significantly lower than previous estimates, and will grow to 1.8% in 2022. However, low oil prices are predicted to stifle activity, as are dropping public investment due to insufficient gov ernment income, limited private investment due to corporate failures, and a lack of confidence among investors. Furthermore, the loss of income and in creased precautionary savings of non-poor house holds, as well as the drop in remittances and the depletion of savings of poor and unemployed house 20 Journal of Sustainable Business and Economics \| Volume 06 \| Issue 01 \| January 2023 holds in the setting of insufficient social safety nets, would reduce the prospects for private spending [36]. The recovery of SSA countries will depend on how political leaders manage the rollout of COV ID-19 vaccines to allow for population movements and commodities. SSA needs to strengthen the fight against corruption, reduce inequality and provide countries with security. holds in the setting of insufficient social safety nets, would reduce the prospects for private spending [36]. ments and the private sector to provide immediate liquidity to countries, allowing the ADB to raise approximately $3 billion as a social bond to combat COVID-19, which is quickly becoming the largest bond on the international market [37]. As a result, the EU has set aside 60 million euros to assist the Afri can continent, and $57 billion in loans have been ar ranged for Africa in 2022, including up to $18 billion from the IMF and WB [38]. And Africa’s additional financial needs for an acceptable response to COV ID-19 until 2025 are estimated to be $285 billion during the Paris summit on May 18, 2021 [39]. Reason behind vaccines rollout delay SSA responded to the COVID-19 pandemic by mobilis ing funds from major financial institutions, issuing bonds, and requesting debt suspension. The recovery of SSA countries will depend on how political leaders manage the rollout of COV ID-19 vaccines to allow for population movements and commodities. SSA needs to strengthen the fight against corruption, reduce inequality and provide countries with security. 4.2 Government strategy The first case of COVID-19 in Africa was re ported on February 14, 2020. SSA countries began to behave differently, with some implementing total lockdowns and others semi-lockdowns. Thus, the strategies differ from country to country. Therefore, the common element in SSA countries is to resort to foreign debt and find vaccines for the citizens. 4. Data discussion The most visible consequences on the interna tional scene are concerned with the economic sec tor. However, COVID-19 has weakened the SSA economy and has the highest education rates and teenage pregnancy. In addition, the school dropout rate globally is 97.5%. A larger and more devastating impact existed during and after the school’s lock down and closure, followed by other unemployment effects caused by job loss. The first and most signif icant consequence is that teenage pregnancy leads to complete school dropout and the loss of all hope for the future. In Sierra Leone alone, estimates show that there are approximately 11,000 cases of teenage pregnancy in eight months. Economically, the con sequences of COVID-19 have severely harmed the economies of SSA countries today, with billions of dollars lost, mainly to large companies. However, small businesses are now closed, and it is unlikely that their owners will reopen. The SSA economy still faces borders (taxes). This issue is most difficult to overcome as it blocks free trade for imports and exports between the countries’ zones. Each year, millions of US dollars are lost in border taxes for each country. The implementation of the Africa Continental Free Trade Area (AfCFTA) is the best strategy, but only South Africa, Rwanda, and Botswana are the most prepared for its implementa tion [40]. The AfCFTA combines 54 African countries into a 1.3 billion-person market. This resource might build a $3.4 trillion economic bloc by boosting sustainable markets. Intra-African commerce is predicted to expand by 33%, and Africa’s trade deficit will be eliminated. AfCFTA could create $6.7 trillion in consumer and business expenditure by 2030 according to the Mo Ibrahim Foundation [41]. Nonetheless, COVID-19 has had an economic effect on SSA, but it has also harmed many other industries. Whether it is tourism, manufacturing, mining, or natural resources, it is clear that these industries have been harmed, leading to increased unemployment. Therefore, the SSA countries have suffered significant economic losses, and economic recovery will take longer to be redressed. Nigeria The new legislation seeks to provide businesses registered under the companies and related matters act with a 50% tax break, allowing them to keep their existing employees. The Central Bank of Nige ria’s (CBN) stimulus package provides low-income families impacted by COVID-19 a loan of 3 million Naira. The loan, however, needs collateral and does not come with no interest. Exceptionally, the IMF approved $3.4 billion to tackle and support Nigeria’s economy [31]. 4.1 Responses and recovering strategy Africa could lose between US$157 and US$212 billion, requiring up to US$154 billion in recovery support [26]. The COVID-19 response facility can make up to $10 billion available to African govern The reorganization of some SSA countries re mains a problem, as small businesses are almost 21 Journal of Sustainable Business and Economics \| Volume 06 \| Issue 01 \| January 2023 dead. Therefore, most of the strategies concern big companies. periences it uniquely, there are some common chal lenges that this article outlines for addressing the issue. Lessons from other regions where the virus is more advanced have been successfully extended to this stage. The SSA needs to maintain collaboration to overcome these challenges. Therefore, there are many questions about how SSA countries will re cover from this economic downturn. Can we expect another plan to rebuild the economy of SSA without trading natural resources? Will SSA countries need a special plan to rebuild their economies? For the most corrupt SSA countries, will the debt granted by the WB, IMF, EU, and ADB be used effectively to reconstruct economic activity? As a researcher, one can ask many questions on this subject, but SSA needs a Marshall Plan-styled economic stimulus that rejuvenated Europe after World War II. Instead of demanding more debt from the IMF or the WB, which will encumbrance future generations with 65% of the GDP per year [42], plus 3% for the recent IMF debt in the May 2021 Paris summit, SSA needs strong leadership. However, SSA countries need to work together more, take advantage of AfCFTA to save billions and billions of dollars on taxes and eliminate time-consuming procedures. South Africa With the massive infrastructure rollout, the South African government hopes to unlock over R1 trillion in new infrastructure investments by enabling the private sector and building infrastructure capability. The strategy is planned to be implemented in the following three steps: Recovery strategies differ by country by balancing the total loss for the country’s economy, secondly the common goal for the SSA countries is to find the COVID-19 vaccine doses for all their citizens and Multinational collaboration such as the COVID-19 Vaccine Access Facility (COVAX) outlines the key strategic issues to be addressed in building the national rollout and immunisation plan for COVID-19 vaccines (African Union, CDC, 2020), apart from the joint COVID-19 vaccination programme [45]. Thus, it is necessary to refer to the context of the different economic recovery strategies of SSA countries, as the consequences have not been the same. The uncertainty of the economy in the future, COVID-19 vaccines, which are a solution to revive the tourism industry, foreign trade, including seriously affected airline businesses, public debt, and problems with the import and export of goods and services, are all common problems for SSA countries. There is no conflict of interest. There is no conflict of interest. References [1] United Nations, 2020. Policy Brief: Impact of COVID-19 in Africa [Internet] [cited 2022 Aug 16]. Available from: https://reliefweb.int/ sites/reliefweb.int/files/resources/Policy-brief- Impact-of-COVID-19-in-Africa%20%281%29. pdf. [2] Liu, Y.C., Kuo, R.L., Shih, S.R., 2020. COVID-19: The first documented coronavirus pandemic in history. Biomedical Journal. 43, 328-333. [2] Liu, Y.C., Kuo, R.L., Shih, S.R., 2020. COVID-19: The first documented coronavirus pandemic in history. Biomedical Journal. 43, 328-333. 5. Conclusions [3] Choi, S.Y., 2020. Industry volatility and eco nomic uncertainty due to the COVID-19 pan demic: Evidence from wavelet coherence analy [3] Choi, S.Y., 2020. Industry volatility and eco nomic uncertainty due to the COVID-19 pan demic: Evidence from wavelet coherence analy The African continent has not been spared from the COVID-19 pandemic. While each country ex 22 Journal of Sustainable Business and Economics \| Volume 06 \| Issue 01 \| January 2023 sis. 37, 101783. doi: 10.1016/j.frl.2020.101783. tances-in-recent-history. tances-in-recent-history. [13] Mahuku, E., Yihun, K.L., Deering, K., et al., 2020. Care Rapid Gender Analysis for COVID 19: East, Central and Southern Africa [Inter net]. Care International Report. Available from: https://www.careevaluations.org/evaluation/ care-rapid-gender-analysis-for-covid-19-east- central-and-southern-africa/. [4] IMF, 2019. Regional economic outlook, April 2019, Sub-saharan Africa: Recovery amid ele vated uncertainty (world economic and financial surveys). IMF: Washington. pp. 72. [5] IMF, 2018. World Economic Outlook Database [Internet] [cited 2022 Jul 26]. Available from: https://www.imf.org/en/Publications/WEO/ Issues/2018/01/11/world-economic-outlook-up date-january-2018. [14] Albrectsen, A.B., Giannini, S., 2020. COVID-19 School Closures around the World will Hit Girls Hardest [Internet] [cited 2022 Jun 15]. Available from: https://www.unesco.org/en/articles/covid- 19-school-closures-around-world-will-hit-girls- hardest. [6] Chikoko, V., Mthembu, P., 2020. Financing pri mary and secondary education in Sub-Saharan Africa: A systematic review of literature. South African Journal of Education. 40(4), 1-9. doi: 10.15700/saje.v40n4a2046. [15] FAO, 2020. COVID-19 and malnutrition: Situa tion analysis and options in Africa. FAO: Accra. pp. 6. doi: 10.4060/ca9896en. [7] Alexandratos, N., Bruinsma, J., 2012. World ag riculture towards 2030/2050: The 2012 revision. ESA Working Paper No. 12-03. Rome: FAO. Available from: https://www.fao.org/3/ap106e/ ap106e.pdf. [16] Arzberger, P., Schroeder, P., Beaulieu, A., et al., 2004. Promoting access to public research data for scientific, economic, and social develop ment. Data Science Journal. 3, 135-152. [8] FAO IFAD & WFP, 2015. Achieving zero hun ger: The critical role of investment in social pro tection and agriculture. FAO: Rome, Italy. [17] Hossain, M.A., Dwivedi, Y.K., Rana, N.P., 2016. State-of-the-art in open data research: Insights from existing literature and a research agenda. Journal of Organizational Computing and Elec tronic Commerce. 26(1-2), 14-40. [9] FAO, 2017. The Future of Food and Agricul ture—Trends and Challenges [Internet] [cited 2022 Jul 28]. Available from: http://www.fao. org/publications/card/en/c/d24d2507-41d9- 4ec2-a3f8-88a489bfe1ad. [18] Babbie, E., 2010. The practice of social research (12th edition). Wadsworth, Cengage Learning: Boston. [10] Bjornlund, V., Bjornlund, H., Van Rooyen, A.F., 2020. Why agricultural production in sub-Sa haran Africa remains low compared to the rest of the world—a historical perspective. 5. Conclusions Interna tional Journal of Water Resources Development. 36(sup1), S20-S53. [19] Saunders, M., Lewis, P., Thornhill, A., 2004. Research methods for business students (3re edition). Prentice-Hall: USA. [20] World Bank, IFC, 2020. COVID-19 Economic Impact; Sub-Saharan Africa [Internet] [cited 2022 Aug 26]. Available from: https://www.ifc. org/wps/wcm/connect/publications_ext_content/ ifc_external_publication_site/publications_list ing_page/covid-19-response-brief-ssa. [11] Ehui, S.K., Benin, S.E., Williams, T., et al., 2002. Food security in sub-Saharan Africa to 2020. Socio-economics and Policy Research Working Paper 49. ILRI (International Livestock Research Institute): Nairobi, Kenya. pp. 60. [21] UNCTAD Report, 2020. Assessing the Impact of COVID-19 on Africa’s Economic Develop ment [Internet] [cited 2022 Sep 27]. Available from: https://unctad.org/webflyer/assessing-im pact-covid-19-africas-economic-development. [12] World Bank, 2020. Predicts sharpest decline of remittances in recent history [Internet] [cited 2022 Aug 28]. Available from: https://www.world bank.org/en/news/press-release/2020/04/22/ world-bank-predicts-sharpest-decline-of-remit [22] FAO, 2021. Economic inclusion and social pro 23 Journal of Sustainable Business and Economics \| Volume 06 \| Issue 01 \| January 2023 tection to reduce poverty: Rural social protec tion and climate change after COVID-19. FAO COVID-19 response and recovery programme. doi: 10.4060/cb3625en. Articles/2020/04/28/pr20191-nigeria-imf-exec utive-board-approves-emergency-support-to-ad dress-covid-19. [31] Kreier, F., 2021. Unprecedented achievement: Who received the first billion COVID vaccina tions? Nature. (Ahead of print). doi: 10.1038/ d41586-021-01136-2. [23] Lakemann, T., Lay, J., Tafese, T., 2020. Af rica after the COVID-19 Lockdowns: Eco nomic impacts and Prospects [Internet] [cited 2022 Sep 27]. Available from: https://www. giga-hamburg.de/en/publications/21606562-af rica-after-covid-19-lockdowns-economic-im pacts-prospects/. [32] Mathieu, E., Ritchie, H., Ortiz-Ospina, E., et al., 2021. A global database of COVID-19 vaccina tions. Nat Hum Behav. 5, 947-953. doi: 10.1038/ s41562-021-01122-8. [24] World Bank, 2020. Predicts Sharpest Decline of Remittances in Recent History [Internet] [cited 2022 Aug 28]. Available from: https:// www.worldbank.org/en/news/press-re lease/2020/04/22/world-bank-predicts-sharp est-decline-of-remittances-in-recent-history. [33] Akwataghibe, N.N., Ogunsola, E.A., Broerse, J.E.W., et al., 2019. Exploring factors influ encing immunization utilization in Nigeria— A mixed methods study. Frontiers in Public Health. 7, 392. [34] World Bank, 2020. Nigeria in times of COVID-19: Laying foundations for a strong recovery—Nigeria development update. World Bank: Washington, DC. [25] Mishra, A., 2020. Africa and COVID-19: Im pact, response, and challenges to recovery. ORF Occasional Paper No. 275, September. Observer Research Foundation: Delhi. [35] African Development Bank Group, 2021. Af rican Development Bank Group Unveils $10 Billion Response Facility to Curb COVID-19 [Internet]. Available from: https://www.ecofina gency.com/public-management/0804-41230-af rican-development-bank-group-unveils-10-bil lion-response-facility-to-curb-covid-19. [26] UNESCO, 2020. COVID-19 Education Re sponse: How Many Students are at Risk of not Returning to School? [Internet] [cited 2022 Aug 15]. Available from: https://unesdoc.unesco.org/ ark:/48223/pf0000373992. 5. Conclusions [27] UN, OCHA, 2020. COVID-19 Aftershocks: Access denied: Teenage Pregnancy Threatens to Block a Million Girls Across Sub-Saharan Af rica from Returning to School [Internet]. World Vision, USA. [cited 2022 Sep 24]. Available from: https://reliefweb.int/report/world/covid- 19-aftershocks-access-denied-teenage-pregnan cy-threatens-block-million-girls. [36] World Bank Group, 2020. World Bank Group and IMF Mobilize Partners in the Fight Against COVID-19 in Africa [Internet]. Available from: https://www.worldbank.org/en/news/press-re lease/2020/04/17/world-bank-group-and-imf- mobilize-partners-in-the-fight-against-covid-19- in-africa. [37] IMF, 2021. Summit on the Financing of Afri can Economies, Paris, 18 May 2021: Decla ration. [Internet] [cited 2022 Sep 20]. Avail able from: https://reliefweb.int/report/world/ summit-financing-african-economies-par is-18-may-2021-declaration. [28] UN, FAO, 2020. Migrant workers and remit tances in the context of COVID-19 in sub-Saha ran Africa. FAO: Africa. doi: 10.4060/cb0184en. [29] IMF, 2021. Regional economic outlook. Sub-Sa haran Africa: Navigating a long pandemic. IMF: Washington, DC. [38] Petersen, H.G., 2010. Tax systems and tax harmonisation in the East African Community (EAC). Finanzwissenschaftliche Diskussionsbe iträge 60, Universität Potsdam, Wirtschafts-und [30] IMF, 2021. IMF Executive Board Approves US$ 3.4 Billion in Emergency Support to Nigeria to Address the COVID-19 Pandemic [Internet]. Available from: https://www.imf.org/en/News/ 24 Journal of Sustainable Business and Economics \| Volume 06 \| Issue 01 \| January 2023 Sozialwissenschaftliche Fakultät. Sozialwissenschaftliche Fakultät. Regional Statistics on the Status of the Aids Ep idemic [Internet]. Available from: https://www. unaids.org/en/resources/documents/2022/UN AIDS_FactSheet. [39] Boateng, G., Dankyi, B.O., 2020. AFCFTA: How Africa’s Free Trade Agreement Would Re duce COVID-19’s Impact [Internet]. Available from: https://www.brinknews.com/how-an-afri can-free-trade-agreement-would-reduce-covid- 19s-impact/. [43] UNESCO, 2021. Out-of-School Children and Youth [Internet] [cited 2022 Apr 15]. Available from: https://uis.unesco.org/en/topic/out-school- children-and-youth. [40] IMF, 2020. Regional Economic Outlook for Sub-Saharan Africa October 2020 [Internet] [cited 2022 Aug 27]. Available from: https:// www.imf.org/en/Publications/REO/SSA/ Issues/2020/10/22/regional-economic-out look-sub-saharan-africa. [44] World Health Organization, 2022. Coronavirus Disease (Covid-19) and People Living with HIV [Internet] [cited 2022 Aug 8]. Available from: https://www.who.int/news-room/ques tions-and-answers/item/coronavirus-disease- (covid-19)-covid-19-and-people-living-with-hiv. [41] UNESCO Report, 2019. New Methodology Shows that 258 Million Children, Adolescents and Youth are out of School [Internet] [cited 2022 Aug 15]. Available from: http://uis.unesco. org/en/topic/out-school-children-and-youth. [45] Africa Union, CDC., 2020. Implementation Guide for COVID-19 Vaccines in Africa [Inter net]. Available from: https://africacdc.org/down load/implementation-guide-for-covid-19-vac cines-in-africa/. [42] UNAIDS, 2021. Fact Sheet: Latest Global and 25
https://openalex.org/W4396557658	https://www.frontiersin.org/journals/endocrinology/articles/10.3389/fendo.2024.1385756/pdf	English	null	Patterns of distant metastasis and survival outcomes in de novo metastatic breast cancer according to age groups	Frontiers in endocrinology	2,024	cc-by	9,215	TYPE Original Research PUBLISHED 01 May 2024 DOI 10.3389/fendo.2024.1385756 TYPE Original Research PUBLISHED 01 May 2024 DOI 10.3389/fendo.2024.1385756 TYPE Original Research PUBLISHED 01 May 2024 DOI 10.3389/fendo.2024.1385756 Ke Liu 1†, An-Le Huang 2†, Xue-Qin Chen 1* and San-Gang Wu 3* Ke Liu 1†, An-Le Huang 2†, Xue-Qin Chen 1* and San-Gang Wu 3* 1Xiamen Key Laboratory of Clinical Efﬁcacy and Evidence Studies of Traditional Chinese Medicine, The First Afﬁliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China, 2Department of Gastrointestinal Oncology Surgery, The First Afﬁliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China, 3Department of Radiation Oncology, Xiamen Cancer Quality Control Center, Xiamen Cancer Center, Xiamen Key Laboratory of Radiation Oncology, The First Afﬁliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China Background: Is de novo metastatic breast cancer (dnMBC) the same disease in the elderly as in younger breast cancer remains unclear. This study aimed to determine the metastatic patterns and survival outcomes in dnMBC according to age groups. CITATION Liu K, Huang A-L, Chen X-Q and Wu S-G (2024) Patterns of distant metastasis and survival outcomes in de novo metastatic breast cancer according to age groups. Front. Endocrinol. 15:1385756. doi: 10.3389/fendo.2024.1385756 Methods: We included patients from the Surveillance Epidemiology and End Results program. Chi-square test, multivariate logistic regression analyses, and multivariate Cox regression models were used for statistical analyses. Results: A total of 17719 patients were included. There were 3.6% (n=638), 18.6% (n=3290), 38.0% (n=6725), and 39.9% (n=7066) of patients aged <35, 35-49, 50- 64, and ≥65 years, respectively. Older patients had a signiﬁcantly higher risk of lung metastasis and a signiﬁcantly lower risk of liver metastasis. There were 19.1%, 25.6%, 30.9%, and 35.7% of patients with lung metastasis in those aged <35, 35- 49, 50-64, and ≥65 years, respectively. Moreover, the proportion of liver metastasis was 37.6%, 29.5%, 26.3%, and 19.2%, respectively. Age was the independent prognostic factor associated with breast cancer-speciﬁc survival (BCSS) and overall survival (OS). Those aged 50-64 years had signiﬁcantly inferior BCSS (P<0.001) and OS (P<0.001) than those aged <35 years. Patients aged ≥65 years also had signiﬁcantly lower BCSS (P<0.001) and OS (P<0.001) than those aged <35 years. However, similar outcomes were found between those aged 35- 49 and <35 years. COPYRIGHT © 2024 Liu, Huang, Chen and Wu. 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. breast cancer, age, prognosis, metastatic patterns, SEER OPEN ACCESS OPEN ACCESS EDITED BY Guangliang Chen, Fudan University, China REVIEWED BY Xudong Zhu, University of Kentucky, United States Eswari Dodagatta-Marri, University of California, San Francisco, United States y, REVIEWED BY Xudong Zhu, University of Kentucky, United States Eswari Dodagatta-Marri, University of California, San Francisco, United States Ke Liu 1†, An-Le Huang 2†, Xue-Qin Chen 1* and San-Gang Wu 3* No use, distribution or reproduction is permitted which does not comply with these terms. Conclusion: Our study suggests that different age groups may affect the metastatic patterns among patients with dnMBC and the survival of younger patients is more favorable than those of older patients. KEYWORDS breast cancer, age, prognosis, metastatic patterns, SEER Frontiers in Endocrinology 01 frontiersin.org 10.3389/fendo.2024.1385756 Liu et al. Introduction However, the age thresholds were inconsistent in the above studies. The median age in those with dnMBC was 61 years, which was similar to those with non-metastatic patients (16, 17). Young BC patients typically display more aggressive tumor characteristics, whereas older patients often experience a poorer prognosis (18, 19). However, the extent to which age is closely associated with metastasis in BC patients remains largely uncertain. Is dnMBC the same disease in the elderly as in younger BC? In light of this, our study aimed to investigate the patterns of DM and survival outcomes among the age groups in this population. Breast cancer (BC) is the most common type of malignant neoplasm in women (1). Due to the initiation of BC screening, approximately 65% of patients were diagnosed with early-stage BC and the 5-year overall survival (OS) has exceeded 90% in this population (2, 3). However, 5% of patients still present with distant metastasis (DM) disease at BC diagnosis (de novo metastatic breast cancer, dnMBC). Bone metastasis was the most common site of DM in BC, followed by lung, liver, and brain (4). With a deep understanding of BC, it is found that BC is a highly heterogeneous disease (5). According to the speciﬁc biological characteristics of different BC subtypes (BCS), corresponding therapeutic strategies such as radiotherapy, chemotherapy, targeted therapy, endocrine therapy, or immunotherapy have been developed in recent decades (6). According to the data from the United States (US) between 2012 and 2018, patients with dnMBC had the lowest 5-year OS rate (29%), which was much lower than stage I (>99%), stage II (93%), and stage III (75%) (7). A study from the US found that the 5-year disease-speciﬁc survival rate of the dnMBC increased from 28% to 55% in those diagnosed in 1990 and 2010, respectively (8). However, a cohort study from Germany found that the survival of this population hardly changed between 1978 and 2013 (9). Identifying clinical risk factors closely correlated with DM can offer insights into the underlying mechanism of advanced BC and inform the development of treatment strategies. Patient demographics We included 17719 patients in this study. The patient characteristics have listed in Table 1. The median age was 61 years (range, 15-99 years), and 3.6% (n=638), 18.6% (n=3290), 38.0% (n=6725), and 39.9% (n=7066) were aged <35, 35-49, 50-64, and ≥65 years, respectively. There were 74.4% (n=13198) of patients were white, 77.8% (n=13777) were invasive ductal carcinoma subtype, 51.7% (n=9166) were stage T3-4 disease, and 66.9% (n=13619) were nodal positive diseases. Regarding BCS, 60.1% Metastasis patterns A total of 28155 metastatic sites were identiﬁed in this study (Table 2). The SEER database only records distant organ metastases and does not include information on the number of metastatic lesions within speciﬁc metastatic organs. Therefore, the speciﬁc metastatic sites indicated the speciﬁc metastatic organs in this study. Bone was the most common metastatic site (n=11977, 42.5%), followed by lung (n=5566, 19.8%), distant lymph nodes (n=5176, 18.4%), liver (n=4331, 15.4%), and brain (n=1105, 3.9%). There were 10683 (60.3%), 4385 (24.7%), 1986 (11.2%), 581 (3.3%), and 84 (0.5%) patients who had one, two, three, four, and ﬁve metastatic sites, respectively. Patients with HoR+/HER2- were more likely to have bone metastasis (76.1% vs. 46.1-65.7%, P<0.001), those with HoR+/HER2+ and HoR-/HER2+ subtypes were more likely to have liver metastasis (35.2-44.5% vs. 17.8-27.0%, P<0.001), and those with HoR-/HER2- disease were more likely to have brain metastasis (10.2% vs. 4.8-9.1%, P<0.001), lung metastasis (42.2% vs. 28.2- 36.0%, P<0.001), and distant lymph nodes metastasis (39.0% vs. 25.6-34.8%, P<0.001) (Figure 2). There was a small difference in the proportion of bone (Figure 3A), brain (Figure 3D), and distant lymph node metastasis (Figure 3E) among the four age subgroups. However, the risk of lung metastasis was signiﬁcantly higher and the risk of liver metastasis was signiﬁcantly lower in the older patients. There were 19.1%, 25.6%, 30.9%, and 35.7% of patients with lung metastasis in those aged <35, 35-49, 50-64, and ≥65 years, respectively (Figure 3B). Moreover, 37.6%, 29.5%, 26.3%, and 19.2% of patients had liver metastasis in those aged <35, 35-49, 50-64, and ≥65 years, respectively (Figure 3C). We found similar distributions of the sites of DM among the four age subgroups in patients with single-site metastasis. Moreover, similar distributions of the sites of DM among the four age subgroups were found after stratiﬁcation by the BCS. Measures The following patient and tumor characteristics were included: age at diagnosis, race, histological subtype, grade, T stage, N stage, hormone receptor (HoR) status, HER2 status, BCS, and treatment. All the patients were divided into four age groups: <35 years, 35-49 years, 50-64 years, and ≥65 years, these cut-offs having been selected based on previously reported studies (21–24). BCS was classiﬁed into four subtypes: HoR+/HER2-, HoR+/HER2+, HoR-/HER2+, and HoR-/HER2-. The main endpoints of our study were breast cancer-speciﬁc survival (BCSS) and OS. BCSS was calculated as the time from the initial diagnosis of BC to the date of BC-speciﬁc death or last follow-up. OS was deﬁned as the time from the initial diagnosis of BC to the date of death due to any cause. We used the variable names COD_ to_ site_record in the SEER database to analyze the cause of death in patients. This record was introduced to account for several newly valid International Classiﬁcation of Diseases-10 codes and includes both cancer and non-cancer causes of death. Patient selection The study data was retrieved from the Surveillance Epidemiology and End Results (SEER) database from 2010 to 2019 which was released in April 2022 (available at: https:// seer.cancer.gov/), using the SEERStat software (version 8.3.9) (20). The SEER is a population-based dataset that covers approximately 30% population of the US, including demographic, clinicopathologic, diagnostic, ﬁrst course of treatment, and survival information. The diagnosis of BC was identiﬁed using the International Classiﬁcation of Disease for Oncology, Third Edition, which were all pathologically conﬁrmed. The following inclusion criteria were used: 1) female with dnMBC; 2) available sites of DM included bone, lung, liver, brain, or distant lymph nodes; 3) available information included tumor grade, estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) status. The patient selection ﬂowchart in this study has listed in Figure 1. Patients with an unknown tumor (T) stage, unknown nodal (N) stage, or unknown surgical procedure were excluded. This study did not require BC is an age-related disease. Age, as a prominent risk factor of tumorigenesis in BC, contributes greatly to the development of metastasis, possibly due to age-related changes in patient homeostasis and the tumor microenvironment (10, 11). The median age of those with non-metastatic patients was 62 years (12). Disproportionately higher rates of BC-related death have been found in those with younger BC as well as elderly BC (13–15). FIGURE 1 The patient selection ﬂowchart. 02 Frontiers in Endocrinology frontiersin.org 10.3389/fendo.2024.1385756 Liu et al. (n=10646), 16.8% (n=2976), 9.0% (n=1588), and 14.2% (n=2509) of patients had HoR+/HER2-, HoR+/HER2+, HoR-/HER2+, and HoR-/HER2- subtypes, respectively. Those with older age were more likely to be white race, invasive lobular carcinoma subtype, T4 diseases, ER+ diseases, and HER2- diseases (all P<0.001). However, those with older age were less likely to have N3 disease and poorly/ undifferentiated disease (all P<0.001). Regarding treatment, patients of older age were less likely to receive surgery, chemotherapy as well as radiotherapy (all P<0.001). In those receiving surgical treatment for the primary breast tumors (n=6120), there were 1701 (27.8%) patients treated with breast-conserving surgery and 4419 (72.2%) treated with mastectomy. Patients with older age were more likely to receive breast-conserving surgery compared to those with younger age (P<0.001). approval from the institutional review board due to the de- identiﬁed information in the SEER program. Statistical analysis The patient and tumor characteristics were compared using the Chi-square test. Multivariate logistic regression analyses were used to determine the relationship between different predictive factors and the metastatic patterns. The BCSS and OS curves were described using the Kaplan-Meier methods and compared by the log-rank test. A Cox proportional hazard regression model was based on the assumption that hazard rates were proportional over time. Variables with a P value <0.1 in the univariate Cox regression model were incorporated into the multivariate Cox proportional analysis to determine the prognostic factors that were signiﬁcantly related to the survival outcomes. For the multivariable analyses, we used time-dependent variable approaches to check the proportional hazards (PH) assumption. Statistical analyses were conducted by the IBM SPSS 22.0 package (IBM Corp., Armonk, NY, USA) and a two-sided P-value <0.05 was considered statistically signiﬁcant. Frontiers in Endocrinology The association between age groups and metastatic patterns Moreover, the risk of liver metastasis decreased with age, those aged 35-49, 50-64, and ≥65 years had 0.786 (P<0.001), 0.677 (P<0.001), and 0.494 (P<0.001) time of liver metastasis compared to those aged <35 years. Age was also the independent predictive factor for bone and distant lymph node metastases, but there was no signiﬁcant change with age. Age was not an independent risk factor for brain metastasis. The association between age groups and metastatic patterns 10.3389/fendo.2024.1385756 Variables n <35 years (%) 35-49 years (%) 50-64 years (%) ≥65 years (%) P Radiotherapy No 11474 354 (55.5) 1912 (58.1) 4179 (62.1) 5029 (71.2) <0.001 Yes 5835 262 (41.1) 272 (38.7) 2379 (35.4) 1922 (27.2) Unknown 410 22 (3.4) 106 (3.2) 167 (2.5) 115 (1.6) IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma; T, tumor; N, nodal; G1, well differentiated; G2, moderately differentiated; G3, poorly/undifferentiated; ER, estrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor 2. IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma; T, tumor; N, nodal; G1, well differentiated; G2, moderately differentiated; G3, poorly/undifferentiated; ER, estrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor 2. TABLE 2 Continued Number of metastatic sites Patterns of distant metastasis N % Bone+brain+lung+distant lymph nodes 85 14.6 Bone+brain+liver+distant lymph nodes 40 6.9 Brain+liver+lung+distant lymph nodes 16 2.8 Five metastatic sites (n=84) Bone+brain+lung+liver+distant lymph nodes 84 100 TABLE 2 The patterns of distant metastasis in de novo metastatic breast cancer patients (n=28155). TABLE 2 The patterns of distant metastasis in de novo metastatic breast cancer patients (n=28155). cancer patients (n 28155). Number of metastatic sites Patterns of distant metastasis N % Single metastatic site (n=10683) Bone 6318 59.1 Distant lymph nodes 1589 14.9 Lung 1486 13.9 Liver 1150 10.8 Brain 140 1.3 Second metastatic sites (n=4385) Bone+lung 1090 24.9 Bone+liver 1074 24.5 Bone+distant lymph nodes 890 20.3 Lung+distant lymph nodes 552 12.6 Liver+lung 260 5.9 Bone+brain 208 4.7 Liver+distant lymph nodes 175 4.0 Brain+lung 82 1.9 Brainl+distant lymph nodes 35 0.8 Brain+liver 19 0.4 Three metastatic sites (n=1986) Bone+lung+distant lymph nodes 710 35.8 Bone+lung+liver 438 22.1 Bong+liver+distant lymph nodes 374 18.8 Liver+lung+distant lymph nodes 156 7.9 Bone+brain+lung 98 4.9 Bone+brain+liver 68 3.4 Bong+brain+distant lymph nodes 60 3.0 Brain+lung+distant lymph nodes 45 2.3 Brain+liver+lung 24 1.2 Brain+liver+distant lymph nodes 13 0.7 Four metastatic sites (n=581) Bone+lung+liver+distant lymph nodes 352 60.6 Bone+brain+liver+lung 88 15.1 (Continued) patterns (Table 3). The following variables were included in the multivariate logistic regression model: speciﬁc site of DM, race, age, histology, tumor stage, nodal stage, ER status, PR status as well as HER2 status. The results indicated that age was the independent risk factor for lung and liver metastases. The risk of lung metastasis increased with age, those aged 35-49, 50-64, and ≥65 years had 1.631 (P<0.001), 2.177 (P<0.001), and 2.963 (P<0.001) times of lung metastasis compared to those aged <35 years. The association between age groups and metastatic patterns The multivariate logistic regression analysis was conducted to analyze the association between the age groups and the metastatic Frontiers in Endocrinology 03 frontiersin.org Liu et al. Liu et al. 10.3389/fendo.2024.1385756 TABLE 1 Patient baseline characteristics according to age groups (n=17719). TABLE 1 Patient baseline characteristics according to age groups (n=17719). TABLE 1 Patient baseline characteristics according to age groups (n=17719). Variables n <35 years (%) 35-49 years (%) 50-64 years (%) ≥65 years (%) P Race White 13198 414 (64.9) 2257 (68.6) 4842 (72.0) 5685 (80.5) <0.001 Black 2843 153 (24.0) 615 (18.7) 1197 (17.8) 878 (12.4) Other 1678 71 (11.1) 418 (12.7) 686 (10.2) 506 (7.1) Histology IDC 13777 568 (89.0) 2676 (81.3) 5238 (77.9) 5295 (74.9) <0.001 ILC 1705 12 (1.9) 214 (6.5) 626 (9.3) 853 (12.1) Other 2237 58 (9.1) 400 (12.2) 861 (12.8) 918 (13.0) Tumor stage T1 2448 73 (11.4) 428 (13.0) 914 (13.6) 1033 (14.6) <0.001 T2 6105 215 (33.7) 1222 (37.1) 2202 (32.7) 2466 (34.9) T3 3247 175 (27.4) 681 (20.7) 1241 (18.5) 1150 (16.3) T4 5919 175 (27.4) 959 (29.1) 236 (35.2) 2417 (34.2) Nodal stage N0 4100 103 (16.1) 588 (17.9) 1386 (20.6) 2023 (28.6) <0.001 N1 8368 324 (50.8) 1684 (51.2) 3155 (46.9) 3205 (45.4) N2 2168 77 (12.1) 407 (12.4) 904 (13.4) 780 (11.0) N3 3083 134 (21.0) 611 (18.6) 1280 (19.0) 1058 (15.0) Tumor grade G1 1452 20 (3.1) 208 (6.3) 489 (7.3) 735 (10.4) <0.001 G2 7648 204 (32.0) 1325 (40.3) 2815 (41.9) 3304 (46.8) G3 8619 414 (64.9) 1757 (53.4) 3421 (50.9) 3027 (42.8) ER status Negative 4355 208 (32.6) 847 (25.7) 1752 (26.1) 1548 (21.9) <0.001 Positive 13364 430 (67.4) 2443 (74.3) 4973 (73.9) 5518 (78.1) PR status Negative 6812 274 (42.9) 1186 (36.0) 2798 (41.6) 2554 (36.1) <0.001 Positive 10907 364 (57.1) 2104 (64.0) 3927 (58.4) 4512 (63.9) HER2 status Negative 13155 382 (59.9) 2293 (69.7) 4842 (72.0) 5638 (79.8) <0.001 Positive 4564 256 (40.1) 997 (30.3) 1883 (28.0) 1428 (20.2) Surgery No 11599 346 (54.2) 1932 (58.7) 4332 (64.4) 4989 (70.6) <0.001 Yes 6120 292 (45.8) 1358 (41.3) 2393 (35.6) 2077 (29.4) Chemotherapy No 6219 70 (11.0) 662 (20.1) 1854 (27.6) 3633 (51.4) <0.001 Yes 11500 568 (89.0) 2628 (79.9) 4871 (72.4) 3433 (48.6) (Continued) Variables n <35 years (%) 35-49 years (%) 50-64 years (%) ≥65 04 Frontiers in Endocrinology frontiersin.org Liu et al. Prognostic analysis We conducted the prognostic analysis on patients with HoR +/HER- and patients who received chemotherapy in the HoR +/HER2+, HoR−/HER2+, and HoR−/HER2−subtypes (n=16218). All variables in the univariate Cox regression model were P value <0.1 and were incorporated into the multivariate Cox proportional analysis. In addition, all variables in the multivariate Cox regression analyses satisﬁed the PH hypothesis with its hazard ratio [HR] value changes with time. The results of the multivariate Cox regression analyses indicated that age was the independent prognostic factor related to survival outcomes (Table 3, Supplementary Table 1). Those aged 50-64 years had signiﬁcantly lower BCSS (HR 1.258, P<0.001) and OS (HR 1.227, P<0.001) compared to those aged <35 Frontiers in Endocrinology 05 frontiersin.org Liu et al. Liu et al. 10.3389/fendo.2024.1385756 A B C D E FIGURE 2 The patterns of distant metastases by different breast cancer subtypes in de novo metastatic breast cancer patients (A, bone metastasis; B, lung metastasis; C, liver metastasis; D, brain metastasis; E, distant lymph node metastasis). A B C D E FIGURE 3 The patterns of distant metastases by different age groups in de novo metastatic breast cancer patients (A, bone metastasis; B, lung metastasis; C, liver metastasis; D, brain metastasis; E, distant lymph node metastasis). A B C D E FIGURE 2 The patterns of distant metastases by different breast cancer subtypes in de novo metastatic breast cancer patients (A, bone metastasis; B, lung metastasis; C, liver metastasis; D, brain metastasis; E, distant lymph node metastasis). D A A E B B E C FIGURE 2 The patterns of distant metastases by different breast cancer subtypes in de novo metastatic breast cancer patients (A, bone metastasis; B, lung metastasis; C, liver metastasis; D, brain metastasis; E, distant lymph node metastasis). A B C D E FIGURE 3 The patterns of distant metastases by different age groups in de novo metastatic breast cancer patients (A, bone metastasis; B, lung metastasis; C, liver metastasis; D, brain metastasis; E, distant lymph node metastasis). D E B E C FIGURE 3 The patterns of distant metastases by different age groups in de novo metastatic breast cancer patients (A, bone metastasis; B, lung metastasis; C, liver metastasis; D, brain metastasis; E, distant lymph node metastasis). 06 Frontiers in Endocrinology frontiersin.org 10.3389/fendo.2024.1385756 Liu et al. years. Prognostic analysis Patients aged ≥65 years also had signiﬁcantly lower BCSS (HR 1.648, P<0.001) and OS (HR 1.722, P<0.001) compared to those aged <35 years. However, similar BCSS (P=0.262) and OS (P=0.681) were found between those aged 35-49 and <35 years. The survival curves of different age groups have shown in Figure 4. Patients with bone (BCSS, HR 1.290, P<0.001; OS, HR 1.238, P<0.001), lung (BCSS, HR 1.164, P<0.001; OS, HR 1.156, P<0.001), liver (BCSS, HR 1.580, P<0.001; OS, HR 1.517, P<0.001), and brain metastasis (BCSS, HR 1.882, P<0.001; OS, HR 1.881, P<0.001), and ≥2 metastatic sites (BCSS, HR 1.180, TABLE 3 Multivariate logistic regression analysis of the association between age at diagnosis and the patterns of distant metastasis in de novo metastatic breast cancer patients. Variables Years OR 95%CI P Bone metastasis <35 1 35-49 1.027 0.852-1.237 0.781 50-64 0.996 0.833-1.191 0.966 ≥65 0.763 0.638-0.913 0.003 Lung metastasis <35 1 35-49 1.631 1.314-2.023 <0.001 50-64 2.177 1.769-2.679 <0.001 ≥65 2.963 2.408-3.647 <0.001 Liver metastasis <35 1 35-49 0.786 0.655-0.943 0.010 50-64 0.677 0.568-0.806 <0.001 ≥65 0.494 0.414-0.589 <0.001 Brain metastasis <35 1 35-49 1.071 0.754-1.523 0.702 50-64 1.188 0.849-1.663 0.314 ≥65 0.920 0.655-1.293 0.631 Distant lymph node metastasis <35 1 35-49 1.171 0.960-1.428 0.118 50-64 1.359 1.124-1.644 0.002 ≥65 1.393 1.150-1.685 0.001 CI, conﬁdence interval; OR, odds ratio. A B FIGURE 4 Comparison of survival outcomes by different age groups in de novo metastatic breast cancer patients (A, breast cancer-speciﬁc survival; B, overall survival). Frontiers in Endocrinology frontiersin.org 07 tivariate logistic regression analysis of the association between age at diagnosis and the patterns of distant metastasis in de novo east cancer patients. Variables Years OR 95%CI P Bone metastasis <35 1 35-49 1.027 0.852-1.237 0.781 50-64 0.996 0.833-1.191 0.966 ≥65 0.763 0.638-0.913 0.003 Lung metastasis <35 1 35-49 1.631 1.314-2.023 <0.001 50-64 2.177 1.769-2.679 <0.001 ≥65 2.963 2.408-3.647 <0.001 Liver metastasis <35 1 35-49 0.786 0.655-0.943 0.010 50-64 0.677 0.568-0.806 <0.001 ≥65 0.494 0.414-0.589 <0.001 Brain metastasis <35 1 35-49 1.071 0.754-1.523 0.702 50-64 1.188 0.849-1.663 0.314 ≥65 0.920 0.655-1.293 0.631 Distant lymph node metastasis <35 1 35-49 1.171 0.960-1.428 0.118 50-64 1.359 1.124-1.644 0.002 ≥65 1.393 1.150-1.685 0.001 CI, conﬁdence interval; OR, odds ratio. CI, conﬁdence interval; OR, odds ratio. Prognostic analysis We conducted sensitivity analyses to determine the effect of age on survival after stratiﬁcation by the metastatic sites (Table 4). The following variables were included in the multivariate Cox regression models: race, age, histology, tumor stage, nodal stage, ER status, PR status, HER2 status, surgery, radiotherapy as well as the speciﬁc site of DM. The results also showed that age was the independent prognostic factor for BCSS and OS regardless of the sites of DM. When stratiﬁed by age groups (Table 6), the HoR-/HER2- subtype was consistently associated with an inferior BCSS (HR for aged <35 years: 2.584, 95% CI 1.828-3.651; 35-49 years: 3.415, 95% CI 2.942-3.964; 50-64 years: 2.373, 95% CI 2.143-2.627; ≥65 years: 2.188, 95% CI 1.956-2.446) and OS (HR for aged <35 years: 2.539, 95% CI 1.832-3.521; 35-49 years: 3.284, 95% CI 2.846-3.791; 50-64 years: 2.291, 95% CI 2.080-2.524; ≥65 years: 1.925, 95% CI 1.733-2.139) regardless of age groups. In addition, those with HoR When stratiﬁed by the sites of DM (Table 5), HoR-/HER2- subtype was consistently associated with an inferior BCSS (HR for any site: 2.469, 95% CI 2.318-2.630; bone: 2.870, 95% CI 2.634- 3.126; lung: 2.109, 95% CI 1.902-2.339; liver: 2.092, 95% CI 1.854- 2.360; brain: 2.055, 95%CI 1.669-2.530; distant lymph node: 2.339, 95%CI 2.102-2.602) and OS (HR for any site: 2.329, 95% CI 2.190- TABLE 4 Multivariate Cox regression analyses of the association between age at diagnosis and the breast cancer-speciﬁc surviva n de novo metastatic breast cancer patients (n=16218). Prognostic analysis Patients with bone (BCSS, HR 1.290, P<0.001; OS, HR 1.238, P<0.001), lung (BCSS, HR 1.164, P<0.001; OS, HR 1.156, P<0.001), liver (BCSS, HR 1.580, P<0.001; OS, HR 1.517, P<0.001), and brain metastasis (BCSS, HR 1.882, P<0.001; OS, HR 1.881, P<0.001), and ≥2 metastatic sites (BCSS, HR 1.180, Patients with bone (BCSS, HR 1.290, P<0.001; OS, HR 1.238, P<0.001), lung (BCSS, HR 1.164, P<0.001; OS, HR 1.156, P<0.001), liver (BCSS, HR 1.580, P<0.001; OS, HR 1.517, P<0.001), and brain metastasis (BCSS, HR 1.882, P<0.001; OS, HR 1.881, P<0.001), and ≥2 metastatic sites (BCSS, HR 1.180, Patients with bone (BCSS, HR 1.290, P<0.001; OS, HR 1.238, P<0.001), lung (BCSS, HR 1.164, P<0.001; OS, HR 1.156, P<0.001), liver (BCSS, HR 1.580, P<0.001; OS, HR 1.517, P<0.001), and brain metastasis (BCSS, HR 1.882, P<0.001; OS, HR 1.881, P<0.001), and ≥2 metastatic sites (BCSS, HR 1.180, years. Patients aged ≥65 years also had signiﬁcantly lower BCSS (HR 1.648, P<0.001) and OS (HR 1.722, P<0.001) compared to those aged <35 years. However, similar BCSS (P=0.262) and OS (P=0.681) were found between those aged 35-49 and <35 years. The survival curves of different age groups have shown in Figure 4. A B FIGURE 4 Comparison of survival outcomes by different age groups in de novo metastatic breast cancer patients (A, breast cancer-speciﬁc survival; B, overall survival). B A B FIGURE 4 Comparison of survival outcomes by different age groups in de novo metastatic breast cancer patients (A, breast cancer-speciﬁc survival; B, overall survival). 07 07 Frontiers in Endocrinology frontiersin.org 10.3389/fendo.2024.1385756 Liu et al. Liu et al. 2.471; bone: 2.720, 95% CI 2.508-2.951; lung: 1.969, 95% CI 1.786- 2.172; liver: 2.020, 95% CI 1.779-2.267; brain: 1.795, 95%CI 1.469- 2.194; distant lymph node: 2.202, 95%CI 1.989-2.437) regardless of DM site. The BCSS and OS curves among the four subtypes have listed in Figure 5. Those with HoR+/HER2+ disease had better BCSS and OS compared to those with HoR+/HER2- disease regardless of DM site. However, comparable BCSS and OS were found between HoR+/HER2- and HoR-/HER2+ subtypes in those with bone, lung, brain, and distant lymph node metastases. P<0.001; OS, HR 1.155, P<0.001) also had inferior BCSS and OS, while those with distant lymph node metastasis (BCSS, HR 0.994, P=0.863; OS, HR 1.104, P=0.648) had no signiﬁcant effect on survival outcomes (Supplementary Table 1). terval; HR, hazard ratio; BCSS, breast cancer-speciﬁc survival; OS, overall survival. Prognostic analysis Variables Years BCSS OS HR 95%CI P HR 95%CI P Entire cohort <35 1 1 35-49 1.081 0.944-1.238 0.262 1.027 0.904-1.167 0.681 50-64 1.258 1.104-1.433 <0.001 1.227 1.086-1.387 <0.001 ≥65 1.648 1.444-1.880 <0.001 1.722 1.523-1.948 <0.001 Bone metastasis <35 1 1 35-49 1.041 0.883-1.227 0.631 1.007 0.862-1.177 0.930 50-64 1.242 1.061-1.455 0.007 1.862 1.059-1.426 0.007 ≥65 1.652 1.408-1.937 <0.001 1.484 1.484-2.004 <0.001 Lung metastasis <35 1 1 35-49 0.923 0.709-1.201 0.551 0.896 0.698-1.105 0.392 50-64 1.035 0.803-1.333 0.792 1.043 0.820-1.326 0.732 ≥65 1.309 1.015-1.689 0.038 1.407 1.105-1.790 0.006 Liver metastasis <35 1 1 35-49 1.132 0.912-1.405 0.263 1.095 0.892-1.345 0.384 50-64 1.398 1.137-1.719 0.001 1.361 1.119-1.656 0.002 ≥65 1.957 1.581-2.422 <0.001 1.986 1.624-2.430 <0.001 Brain metastasis <35 1 1 35-49 0.983 0.629-1.535 0.938 1.054 0.677-1.640 0.817 50-64 1.117 0.729-1.713 0.611 1.246 0.816-1.905 0.309 ≥65 1.672 1.074-2.602 0.023 1.881 1.216-2.909 0.005 Distant lymph node metastasis <35 1 1 35-49 1.186 0.921-1.528 0.185 1.162 0.917-1.473 0.215 50-64 1.357 1.066-1.727 0.013 1.309 1.044-1.642 0.020 ≥65 1.540 1.204-1.968 <0.001 1.602 1.273-2.015 <0.001 CI conﬁdence interval; HR hazard ratio; BCSS breast cancer-speciﬁc survival; OS overall survival Frontiers in Endocrinology 08 frontiersin.org Liu et al. 10.3389/fendo.2024.1385756 Liu et al. TABLE 5 Multivariate Cox regression analyses of the association between breast cancer subtypes and the breast cancer-speciﬁc survival and overall survival according to the site of distant metastasis in de novo metastatic breast cancer patients (n=16218). ; HR, hazard ratio; BCSS, breast cancer-speciﬁc survival; OS, overall survival; HoR, hormone receptor; HER2, human epidermal growth factor receptor 2. Prognostic analysis Variables Subtypes BCSS OS HR 95%CI P HR 95%CI P Entire cohort HoR+/HER2- 1 1 HoR+/HER2+ 0.632 0.586-0.681 <0.001 0.640 0.597-0.684 <0.001 HoR-/HER2+ 0.823 0.752-0.900 <0.001 0.829 0.762-0.902 <0.001 HoR-/HER2- 2.469 2.318-2.630 <0.001 2.326 2.190-2.471 <0.001 Bone metastasis HoR+/HER2- 1 1 HoR+/HER2+ 0.654 0.598-0.716 <0.001 0.655 0.602-0.713 <0.001 HoR-/HER2+ 0.887 0.784-1.004 0.057 0.898 0.800-1.007 0.067 HoR-/HER2- 2.870 2.634-3.126 <0.001 2.720 2.508-2.951 <0.001 Lung metastasis HoR+/HER2- 1 1 HoR+/HER2+ 0.665 0.586-0.754 <0.001 0.658 0.585-0.741 <0.001 HoR-/HER2+ 0.913 0.790-1.055 0.216 0.914 0.799-1.046 0.190 HoR-/HER2- 2.109 1.902-2.339 <0.001 1.969 1.786-2.172 <0.001 Liver metastasis HoR+/HER2- 1 1 HoR+/HER2+ 0.552 0.489-0.624 <0.001 0.566 0.504-0.635 <0.001 HoR-/HER2+ 0.646 0.564-0.740 <0.001 0.640 0.562-0.728 <0.001 HoR-/HER2- 2.092 1.854-2.360 <0.001 2.020 1.779-2.267 <0.001 Brain metastasis HoR+/HER2- 1 1 HoR+/HER2+ 0.712 0.557-0.911 0.007 0.707 0.562-0.891 0.003 HoR-/HER2+ 1.271 0.981-1.647 0.069 1.179 0.920-1.512 0.193 HoR-/HER2- 2.055 1.669-2.530 <0.001 1.795 1.469-2.194 <0.001 Distant lymph node metastasis HoR+/HER2- 1 1 HoR+/HER2+ 0.648 0.567-0.740 <0.001 0.654 0.578-0.740 <0.001 HoR-/HER2+ 0.924 0.796-1.072 0.297 0.905 0.787-1.041 0.164 HoR-/HER2- 2.339 2.102-2.602 <0.001 2.202 1.989-2.437 <0.001 CI conﬁdence interval; HR hazard ratio; BCSS breast cancer-speciﬁc survival; OS overall survival; HoR hormone receptor; HER2 human epidermal growth factor receptor 2 B A B FIGURE 5 Comparison of survival outcomes by different breast cancer subtypes in de novo metastatic breast cancer patients (A, breast cancer-speciﬁc survival; B, overall survival). A B FIGURE 5 Comparison of survival outcomes by different breast cancer subtypes in de novo metastatic breast cancer patients (A, breast cancer-speciﬁc survival; B, overall survival). 09 09 Frontiers in Endocrinology frontiersin.org Liu et al. 10.3389/fendo.2024.1385756 Liu et al. TABLE 6 Multivariate Cox regression analyses of the association between breast cancer subtypes and the breast cancer-speciﬁc survival and overall survival according to the age groups in de novo metastatic breast cancer patients. Prognostic analysis Age (years) Subtypes BCSS OS HR 95%CI P HR 95%CI P <35 HoR+/HER2- 1 1 HoR+/HER2+ 0.385 0.267-0.555 <0.001 0.449 0.321-0.628 <0.001 HoR-/HER2+ 0.482 0.318-0.729 <0.001 0.565 0.387-0.824 0.003 HoR-/HER2- 2.584 1.828-3.651 <0.001 2.539 1.832-3.521 <0.001 35-49 HoR+/HER2- 1 1 HoR+/HER2+ 0.463 0.389-0.552 <0.001 0.461 0.390-0.545 <0.001 HoR-/HER2+ 0.752 0.611-0.926 0.007 0.774 0.636-0.942 0.010 HoR-/HER2- 3.415 2.942-3.964 <0.001 3.284 2.846-3.791 <0.001 50-64 HoR+/HER2- 1 1 HoR+/HER2+ 0.585 0.521-0.656 <0.001 0.597 0.536-0.665 <0.001 HoR-/HER2+ 0.715 0.623-0.820 <0.001 0.712 0.625-0.811 <0.001 HoR-/HER2- 2.373 2.143-2.627 <0.001 2.291 2.080-2.524 <0.001 ≥65 HoR+/HER2- 1 1 HoR+/HER2+ 0.631 0.549-0.724 <0.001 0.632 0.558-0.716 <0.001 HoR-/HER2+ 0.840 0.709-0.996 0.044 0.827 0.709-0.964 0.015 HoR-/HER2- 2.188 1.956-2.446 <0.001 1.925 1.733-2.139 <0.001 CI, conﬁdence interval; HR, hazard ratio; BCSS, breast cancer-speciﬁc survival; OS, overall survival; HoR, hormone receptor; HER2, human epidermal growth factor receptor 2. studies, 63.9-68% of patients had a single site of DM, and 60.3% were in our study. This suggests that the early diagnosis of advanced BC may not be further improved under the contemporary screening modes. The number of sites of DM was associated with survival outcomes in our study. Therefore, it is still necessary to explore strategies that can detect the disease in time. +/HER2+ and HoR-/HER2+ subtypes had signiﬁcantly better BCSS and OS compared to HoR+/HER2- subtype regardless of age groups. Discussion There was a small difference in the proportion of bone, brain, and distant lymph node metastases among the four age groups. However, the risk of lung metastasis was higher and liver metastasis was lower in the older patients. A previous SEER study from Chen et al. included 4932 patients, they found that patients with older age had signiﬁcantly higher rates of lung metastasis and a lower rate of distant lymph node metastasis. However, no signiﬁcant differences in bone, brain, and liver metastasis were found among the age groups (27). We should notice that there were 5.0%, 63.7%, and 31.2% of patients were aged <50 years, 50-69 years, and ≥70 years in the above study. However, there were 22.2% of patients were aged <50 years in our study. A recent SEER study, which included 24155 patients diagnosed with dnMBC, categorized the age of these patients into four groups: ≤40 years, 41-60 years, 61-80 years, and >80 years (28). However, this analysis did not account for distant lymph node metastasis, limiting the ability to fully assess the patterns of DM within this cohort. Furthermore, the study observed a similar distribution of lung metastasis among the age groups of 61-80 and >80 years (32.4% vs. 33.6%, respectively). The age distribution of patients in our study is more highly represented in the literature (22–24, 29), providing a more comprehensive perspective. Therefore, the distribution of different age groups may affect the assessment of the metastatic patterns. However, Purushotham et al. found a decreased risk of bone and viscera metastases with the increase of age at diagnosis in those who developed DM during follow-up (30). Moreover, Hung et al. showed that younger patients were particularly prone to brain metastasis regardless of BCS (31). In our study, older patients presented more often with larger tumor sizes. However, patients of older age were less likely to have N3 disease and have higher tumor grade. Therefore, older BC patients have special tumor biological behavior. The inferior outcomes in older patients might be related to lower administration of surgery, chemotherapy, radiotherapy, and endocrine therapy in older BC. Several studies also supported undertreatment in older BC (36, 37). Although most patients included in our multivariate survival analysis were treated with chemotherapy, the completion of chemotherapy, endocrine therapy, and targeted therapy were not recorded in the SEER database. Therefore, we could assume that elderly patients may have lower compliance with multimodal treatment. Discussion We also found that those who did not undergo surgery had worse survival. Although the results of the prospective studies did not ﬁnd the survival beneﬁt of additional surgery in this population (38, 39), many real-world studies including ours have found that surgical treatment could improve the survival of these patients (40, 41). Therefore, the local treatment strategy is still worth further exploration for this population, especially for the elderly. Moreover, some older women with BC may have biological invasive diseases, but their characteristics have not been fully determined (42, 43). There were 19.8% of patients had lung metastasis in our study and the risk of lung metastasis increased with age. However, we found that age was not a prognostic factor affecting the survival of those with lung metastasis. The reason for this difference remains unclear. It should be noted that patients with HoR-/HER2- were more likely to have lung metastasis (42.2% vs. 28.2-36.0%, P<0.001) compared to other subtypes, which was similar to a previous study (44). Similarly, Ahn et al. found inferior survival for young patients with HoR+ disease, but not for those with HoR-/HER2- BC (45). In particular, 60-70% of metastatic patients who eventually died were diagnosed with lung metastasis (46). Therefore, the probability of HoR-/HER2- BC was higher among patients with lung metastasis, which may compensate for the differences in survival caused by age. The reasons behind the observed diverse patterns of DM among different age groups remain unclear. In this study, older patients were found to be more prone to lung metastases. This phenomenon may be attributed to chronic lung inﬂammation, such as chronic obstructive pulmonary disease, which is common in the elderly (32). Several studies have shown that potential mechanisms involving inﬂammatory cell neutrophils speciﬁcally support metastatic initiation and promote the awakening of dormant cancer cells in the lungs using mouse models (33, 34). Additionally, we observed a higher prevalence of liver metastases among younger patients. The increased risk of liver metastases in this demographic may be linked to a higher percentage of HER2+ disease in younger patients, consistent with previous studies including our own (4, 35). Given the high proportions of HER2+ subtypes in young patients, further research focusing on potential mechanisms of liver metastases in HER2+ BC may help in developing novel strategies for metastasis prevention. Discussion There were speciﬁc metastatic patterns of different malignant tumors. In small-cell lung cancer, the liver was the most common metastatic site (44.8%), followed by bone (35.0%), brain (25.8%), and lung (20.6%) (25). In those with stage IV esophageal cancer, the lung was the most common metastatic site, followed by liver, bone, and brain (26). BC is a highly heterogeneous tumor (5). In our previous study, we found that patients with different BCS have various metastatic patterns. Patients with HoR+/HER2- and HoR +/HER2+ subtypes were more prone to bone metastases. Lung and brain metastases were common in HoR-/HER2+ and HoR-/HER2- subtypes and patients with HoR+/HER2+ and HoR-/HER2+ subtypes were more likely to have liver metastases (4). Similar results were found in the present study using a larger cohort. Therefore, patients with different tumor locations should have different priorities in surveillance, and individualized follow-up strategies should be formulated for different malignant tumors. In the present study, we investigated the effect of age on metastatic patterns and survival outcomes in dnMBC. Our results showed that there were certain speciﬁcities in dissemination to different distant organs by different age groups. In addition, patients of older age were associated with inferior prognoses in this population. In our previous SEER study included 7575 patients diagnosed between 2010 and 2013, a total of 11140 sites of DM were found and bone (51.2%) was the most common metastatic site, followed by lung (23.2%), liver (20.1%), and brain (5.5%) (4). However, the distant lymph nodes were not analyzed in the study. In this study, we included 17719 patients with 28155 sites of DM, we found that bone was also the most common metastatic site (42.5%), followed by lung (19.8%), distant lymph nodes (18.4%), liver (15.4%), and brain (3.9%). Another study from Malmgren et al. also found that bone metastasis was the most common metastatic site (58%), followed by distant lymph nodes (33%), lung (21%), liver (21%), and brain (1%) (8). However, we should note that in the previous In this study, we further analyzed the association among the age groups and the metastatic patterns. We found that age could also affect the patterns of DM in BC, and these metastatic patterns by age Frontiers in Endocrinology frontiersin.org 10 Liu et al. Liu et al. 10.3389/fendo.2024.1385756 10.3389/fendo.2024.1385756 groups were not affected by the BCS. Acknowledgments The authors acknowledge the efforts of the Surveillance, Epidemiology, and End Results (SEER) Program tumor registries in the creation of the SEER database. Discussion Moreover, it appears that tumor cell subsets interact differently with the tumor microenvironments in various distant metastatic organs, providing favorable conditions for the invasion and proliferation of tumor cells. We hypothesized that the immune system may also inﬂuence the metastatic patterns of patients. Younger patients tend to have stronger immune responses, resulting in differences in the tumor microenvironment. Therefore, it is essential to employ different strategies to treat different tumor cell subsets. Nonetheless, further investigation into the underlying molecular mechanisms is still needed, particularly concerning those with bone, brain, and distant lymph node metastases across different age groups. y p y g Only 3.6% of patients were diagnosed with dnMBC at <35 years of age in our study. However, BC in younger women is often diagnosed at more advanced stages of the disease. The main reason for this is the lack of screening, which is not recommended in this age group, as well as the longer delay in diagnosis. In addition, young women often have dense breast parenchyma, which could reduce the sensitivity and accuracy of digital mammography (47). There is no evidence of a mortality beneﬁt from mammographic screening in women under the age of 35 years (48). Our results showed that younger patients were more likely to have N3 disease and poorly/undifferentiated disease. Therefore, screening is also needed for high-risk young women, and new screening methods should be explored. A previous study has shown worse 5-year survival in young women with non-metastatic BC at diagnosis compared with older, premenopausal patients (49). However, in our study, patients aged <35 years had comparable BCSS and OS compared to those with aged 35-49 years, while they had signiﬁcantly better BCSS and OS than those aged ≥50 years. Moreover, those with HER2+ subtypes had signiﬁcantly better BCSS and OS, while those with HoR-/HER2- had signiﬁcantly Frontiers in Endocrinology 11 frontiersin.org Liu et al. Liu et al. 10.3389/fendo.2024.1385756 inferior BCSS and OS in patients aged <35 years. Therefore, advances in systemic therapy have contributed to improving survival outcomes for young patients with dnMBC by tailoring treatments to their disease biology. effect of age in predicting metastatic patterns and prognosis in this population. Author contributions KL: Conceptualization, Data curation, Formal analysis, Visualization, Writing – original draft. A-LH: Conceptualization, Data curation, Formal analysis, Funding acquisition, Resources, Writing – original draft. X-QC: Conceptualization, Funding acquisition, Methodology, Resources, Supervision, Validation, Visualization, Writing – review & editing. S-GW: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – review & editing. Our study had several limitations. First, miscoding and missing information inevitably existed in any retrospective studies. Second, there was no information concerning systemic treatment in different age groups, including the chemotherapy regimen, the completion of chemotherapy, endocrine therapy, targeted therapy, and immunotherapy. Third, although these are the common sites of DM in BC, metastases to other distant sites may inﬂuence the survival of BC patients. Fourth, information regarding patient comorbidities was inaccessible in the SEER database, and we could not adjust for patient comorbidities in the multivariable analysis. This limitation may have inﬂuenced estimations of survival and treatment selection. Moreover, older BC patients are frequently excluded from ongoing clinical trials, leading to inadequate treatment and inferior survival rates. Therefore, the correlation between age and BC mortality is far more complex, with potential confounding factors possibly contributing to better survival outcomes in younger women. Finally, only 3.6% of patients were under 35 years old in our study, which could limit the representativeness and generalizability of our ﬁndings. However, the incidence of dnMBC among young women has been steadily increasing over the last decades, while remaining stable in other age groups (52, 53). Data availability statement The median age at the time of diagnosis was 61 years, with approximately 40% of patients diagnosed at the age of 65 or older in our study. Despite the high prevalence of cancer in older patients, their participation in oncology clinical trials has traditionally been inadequate. Recent data indicated that only 1% of trials have enrolled solely patients aged 65 or 70 and older (50). In general, using upper age limits in clinical trials presents challenges due to the considerable heterogeneity of aging. This means that there is often no direct correlation between an older woman’s chronological age and her biological age. This is particularly signiﬁcant for patients who may undergo chemotherapy or newer biological therapies, where published trials have typically included a few older patients, limiting information on both short-term and long-term toxicity among this demographic. This underrepresentation is often attributed to age bias among clinicians (51). However, studies have demonstrated that this healthy older age group tolerates treatment as well as younger patients, including breast and axillary surgery, chemotherapy, and radiotherapy. Therefore, it is important that older patients are actively recruited into studies evaluating novel agents and therapeutic approaches whenever possible and appropriate, as is recommended for all patients. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Ethics statement The studies were conducted in accordance with the local legislation and institutional requirements. This study did not require approval from the institutional review board due to the deidentiﬁed information in the SEER program. Informed consent is not required because the data were extracted from the SEER database after obtaining the permission of the administrator. In addition, the privacy of the patients was well protected due to the anonymization and de-identiﬁcation of the patient information. Funding The author(s) declare that no ﬁnancial support was received for the research, authorship, and/or publication of this article. Frontiers in Endocrinology Supplementary material 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 The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fendo.2024.1385756/ full#supplementary-material References 20. Surveillance, Epidemiology, and End Results (SEER) Program. SEERStat Database: Incidence - SEER Research Data, 17 Registries, Nov 2021 Sub (2000-2019) - Linked To County Attributes - Time Dependent (1990-2019) Income/Rurality, 1969- 2020 Counties. Bethesda, Maryland: National Cancer Institute, DCCPS, Surveillance Research Program (2022). Available at: www.seer.cancer.gov. 1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. (2021) 71:209–49. doi: 10.3322/caac.21660 2. Berry DA, Cronin KA, Plevritis SK, Fryback DG, Clarke L, Zelen M, et al. Effect of screening and adjuvant therapy on mortality from breast cancer. N Engl J Med. (2005) 353:1784–92. doi: 10.1056/NEJMoa050518 2. Berry DA, Cronin KA, Plevritis SK, Fryback DG, Clarke L, Zelen M, et al. Effect of screening and adjuvant therapy on mortality from breast cancer. N Engl J Med. (2005) 353:1784–92. doi: 10.1056/NEJMoa050518 21. Sparano JA, Gray RJ, Makower DF, Pritchard KI, Albain KS, Hayes DF, et al. Adjuvant chemotherapy guided by a 21-gene expression assay in breast cancer. N Engl J Med. (2018) 379:111–21. doi: 10.1056/NEJMoa1804710 3. Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer statistics, 2022. CA Cancer J Clin. (2022) 72:7–33. doi: 10.3322/caac.21708 22. Puigpinos-Riera R, Continente X, Serral G, Bargalló X, Doménech M, Espinosa- Bravo M, et al. Inﬂuence of social determinants, lifestyle, emotional well-being and the use of unconventional therapies in breast cancer progression in a cohort of women in Barcelona: protocol for the DAMA cohort. JMIR Res Protoc. (2017) 6:e249. doi: 10.2196/resprot.7653 4. Wu SG, Li H, Tang LY, Sun JY, Zhang WW, Li FY, et al. The effect of distant metastases sites on survival in de novo stage-IV breast cancer: A SEER database analysis. Tumour Biol. (2017) 39:1010428317705082. doi: 10.1177/1010428317705082 5. Joseph C, Papadaki A, Althobiti M, Alsaleem M, Aleskandarany MA, Rakha EA. Breast cancer intratumour heterogeneity: current status and clinical implications. Histopathology. (2018) 73:717–31. doi: 10.1111/his.13642 23. Darwish AD, Helal AM, Aly El-Din NH, Solaiman LL, Amin A. Breast cancer in women aging 35 years old and younger: The Egyptian National Cancer Institute (NCI) experience. Breast. (2017) 31:1–8. doi: 10.1016/j.breast.2016.09.018 6. Wang J, Wu SG. Breast cancer: an overview of current therapeutic strategies, challenge, and perspectives. Breast Cancer (Dove Med Press). (2023) 15:721–30. doi: 10.2147/BCTT.S432526 24. References Blouet A, Zinger M, Capitain O, Landry S, Bourgeois H, Seegers VT, et al. Sexual quality of life evaluation after treatment among women with breast cancer under 35 years old. Support Care Cancer. (2019) 27:879–85. doi: 10.1007/s00520-018-4374-z 7. Giaquinto AN, Sung H, Miller KD, Kramer JL, Newman LA, Minihan A, et al. Breast cancer statistics, 2022. CA Cancer J Clin. (2022) 72:524–41. doi: 10.3322/caac.21754 25. Li J, Liu F, Yu H, Zhao C, Li Z, Wang H, et al. Different distant metastasis patterns based on tumor size could be found in extensive-stage small cell lung cancer patients: a large, population-based SEER study. PeerJ. (2019) 7:e8163. doi: 10.7717/ peerj.8163 8. Malmgren JA, Mayer M, Atwood MK, Kaplan HG. Differential presentation and survival of de novo and recurrent metastatic breast cancer over time: 1990-2010. Breast Cancer Res Treat. (2018) 167:579–90. doi: 10.1007/s10549-017-4529-5 9. Hölzel D, Eckel R, Bauerfeind I, Baier B, Beck T, Braun M, et al. Survival of de novo stage IV breast cancer patients over three decades. J Cancer Res Clin Oncol. (2017) 143:509–19. doi: 10.1007/s00432-016-2306-1 26. Qiu G, Zhang H, Wang F, Zheng Y, Wang Y. Patterns of metastasis and prognosis of elderly esophageal squamous cell carcinoma patients in stage IVB: a population-based study. Transl Cancer Res. (2021) 10:4591–600. doi: 10.21037/tcr 10. Gu X, Wang B, Zhu H, Zhou Y, Horning AM, Huang TH, et al. Age-associated genes in human mammary gland drive human breast cancer progression. Breast Cancer Res. (2020) 22:64. doi: 10.1186/s13058-020-01299-2 27. Chen MT, Sun HF, Zhao Y, Fu WY, Yang LP, Gao SP, et al. Comparison of patterns and prognosis among distant metastatic breast cancer patients by age groups: a SEER population-based analysis. Sci Rep. (2017) 7:9254. doi: 10.1038/s41598-017-10166-8 11. Wu AML, Gossa S, Samala R, Chung MA, Gril B, Yang HH, et al. Aging and CNS myeloid cell depletion attenuate breast cancer brain metastasis. Clin Cancer Res. (2021) 27:4422–34. doi: 10.1158/1078-0432.CCR-21-1549 28. Xiao Q, Zhang W, Jing J, Zhong T, Li D, Zhou J, et al. Patterns of de novo metastasis and survival outcomes by age in breast cancer patients: a SEER population-based study. Front Endocrinol (Lausanne). (2023) 14:1184895. doi: 10.3389/fendo.2023.1184895 12. DeSantis CE, Ma J, Goding Sauer A, Newman LA, Jemal A. Breast cancer statistics, 2017, racial disparity in mortality by state. CA Cancer J Clin. (2017) 67:439– 48. doi: 10.3322/caac.21412 29. Schreiber J, Ignatov A, Burger E, Meinecke AM, Eggemann H. Conﬂict of interest In conclusion, our study suggests that different age groups may affect the metastasis pattern of patients with dnMBC, and the survival of younger patients is relatively more favorable than those with older age. More studies are needed to fully verify the 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. Frontiers in Endocrinology frontiersin.org 12 Liu et al. Liu et al. 10.3389/fendo.2024.1385756 The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the ﬁnal decision. 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. References Breast cancer therapy in women under 35 years and between 50 and 69 years: inﬂuence of the observation period. J Cancer Res Clin Oncol. (2023) 149:5665–76. doi: 10.1007/s00432-022-04520-1 13. Zhang X, Yang J, Cai H, Ye Y. Young age is an independent adverse prognostic factor in early stage breast cancer: a population-based study. Cancer Manag Res. (2018) 10:4005–18. doi: 10.2147/CMAR 30. Purushotham A, Shamil E, Cariati M, Agbaje O, Muhidin A, Gillett C, et al. Age at diagnosis and distant metastasis in breast cancer–a surprising inverse relationship. Eur J Cancer. (2014) 50:1697–705. doi: 10.1016/j.ejca.2014.04.002 14. Gnerlich JL, Deshpande AD, Jeffe DB, Sweet A, White N, Margenthaler JA. Elevated breast cancer mortality in women younger than age 40 years compared with older women is attributed to poorer survival in early-stage disease. J Am Coll Surg. (2009) 208:341–7. doi: 10.1016/j.jamcollsurg.2008.12.001 31. Hung MH, Liu CY, Shiau CY, Hsu CY, Tsai YF, Wang YL, et al. Effect of age and biological subtype on the risk and timing of brain metastasis in breast cancer patients. PloS One. (2014) 9:e89389. doi: 10.1371/journal.pone.0089389 32. Cosio MG, CazzufﬁR, Saetta M. Is chronic obstructive pulmonary disease a disease of aging? Respiration. (2014) 87:508–12. doi: 10.1159/000360770 15. de la Rochefordiere A, Asselain B, Campana F, Scholl SM, Fenton J, Vilcoq JR, et al. Age as prognostic factor in premenopausal breast carcinoma. Lancet. (1993) 341:1039–43. doi: 10.1016/0140-6736(93)92407-K 33. Albrengues J, Shields MA, Ng D, Park CG, Ambrico A, Poindexter ME, et al. Neutrophil extracellular traps produced during inﬂammation awaken dormant cancer cells in mice. Science. (2018) 361:eaao4227. doi: 10.1126/science.aao4227 16. Roder DM, de Silva P, Zorbas HM, Kollias J, Malycha PL, Pyke CM, et al. Age effects on survival from early breast cancer in clinical settings in Australia. ANZ J Surg. (2012) 82:524–8. doi: 10.1111/j.1445-2197.2012.06114.x 34. Wculek SK, Malanchi I. Neutrophils support lung colonization of metastasis- initiating breast cancer cells. Nature. (2015) 528:413–7. doi: 10.1038/nature16140 17. Brandt J, Garne JP, Tengrup I, Manjer J. Age at diagnosis in relation to survival following breast cancer: a cohort study. World J Surg Oncol. (2015) 13:33. doi: 10.1186/ s12957-014-0429-x 35. Kennecke H, Yerushalmi R, Woods R, Cheang MC, Voduc D, Speers CH, et al. Metastatic behavior of breast cancer subtypes. J Clin Oncol. (2010) 28:3271–7. doi: 10.1200/JCO.2009.25.9820 18. Johnson HM, Irish W, Muzaffar M, Vohra NA, Wong JH. Quantifying the relationship between age at diagnosis and breast cancer-speciﬁc mortality. Breast Cancer Res Treat. References (2019) 177:713–22. doi: 10.1007/s10549-019-05353-2 36. Hughes KS, Schnaper LA, Bellon JR, Cirrincione CT, Berry DA, McCormick B, et al. Lumpectomy plus tamoxifen with or without irradiation in women age 70 years or older with early breast cancer: long-term follow-up of CALGB 9343. J Clin Oncol. (2013) 31:2382–7. doi: 10.1200/JCO.2012.45.2615 19. Nasrazadani A, Marti JLG, Kip KE, Marroquin OC, Lemon L, Shapiro SD, et al. Breast cancer mortality as a function of age. Aging (Albany NY). (2022) 14:1186–99. doi: 10.18632/aging.v14i3 37. Kunkler IH, Williams LJ, Jack WJ, Cameron DA, Dixon JMPRIME II investigators. Breast-conserving surgery with or without irradiation in women aged Frontiers in Endocrinology 13 frontiersin.org Liu et al. 10.3389/fendo.2024.1385756 65 years or older with early breast cancer (PRIME II): a randomised controlled trial. Lancet Oncol. (2015) 16:266–73. doi: 10.1016/S1470-2045(14)71221-5 resistance: nationwide survival data in Korea–a report from the Korean Breast Cancer Society. J Clin Oncol. (2007) 25:2360–8. doi: 10.1200/JCO.2006.10.3754 38. Khan SA, Zhao F, Goldstein LJ, Cella D, Basik M, Golshan M, et al. Early local therapy for the primary site in de novo stage IV breast cancer: results of a randomized clinical trial (EA2108). J Clin Oncol. (2022) 40:978–87. doi: 10.1200/JCO.21.02006 46. Dan Z, Cao H, He X, Zhang Z, Zou L, Zeng L, et al. A pH-responsive host-guest nanosystem loading succinobucol suppresses lung metastasis of breast cancer. Theranostics. (2016) 6:435–45. doi: 10.7150/thno.13896 39. Badwe R, Hawaldar R, Nair N, Kaushik R, Parmar V, Siddique S, et al. Locoregional treatment versus no treatment of the primary tumour in metastatic breast cancer: an open-label randomised controlled trial. Lancet Oncol. (2015) 16:1380– 8. doi: 10.1016/S1470-2045(15)00135-7 47. Huang J, Lin Q, Cui C, Fei J, Su X, Li L, et al. Correlation between imaging features and molecular subtypes of breast cancer in young women (≤30 years old). Jpn J Radiol. (2020) 38:1062–74. doi: 10.1007/s11604-020-01001-8 48. Gentilini O, Partridge AH, Pagani O. Breast Cancer in Young Women. Switzerland: Springer International Publishing (2020) p. 1–33. 40. Reinhorn D, Mutai R, Yerushalmi R, Moore A, Amir E, Goldvaser H. Locoregional therapy in de novo metastatic breast cancer: systemic review and meta- analysis. Breast. (2021) 58:173–81. doi: 10.1016/j.breast.2021.05.003 49. Guo F, Kuo YF, Shih YCT, Giordano SH, Berenson AB. Trends in breast cancer mortality by stage at diagnosis among young women in the United States. Cancer. (2018) 124:3500–9. doi: 10.1002/cncr.31638 41. Yu Y, Hong H, Wang Y, Fu T, Chen Y, Zhao J, et al. 51. Karuturi M, VanderWalde N, Muss H. Approach and management of breast cancer in the elderly. Clin Geriatr Med. (2016) 32:133–53. doi: 10.1016/j.cger.2015.08.011 References Clinical evidence for locoregional surgery of the primary tumor in patients with de novo stage IV breast cancer. Ann Surg Oncol. (2021) 28:5059–70. doi: 10.1245/s10434-021-09650-3 50. Szlezinger K, Pogoda K, Jagiełło-Gruszfeld A, Kłosowska D, Górski A, Borysowski J. Eligibility criteria in clinical trials in breast cancer: a cohort study. BMC Med. (2023) 21:240. doi: 10.1186/s12916-023-02947-y 42. Wang MX, Ren JT, Tang LY, Ren ZF. Molecular features in young vs elderly breast cancer patients and the impacts on survival disparities by age at diagnosis. Cancer Med. (2018) 7:3269–77. doi: 10.1002/cam4.1544 51. Karuturi M, VanderWalde N, Muss H. Approach and management of breast cancer in the elderly. Clin Geriatr Med. (2016) 32:133–53. doi: 10.1016/j.cger.2015.08.011 43. Azim HAJr, Nguyen B, Brohée S, Zoppoli G, Sotiriou C. Genomic aberrations in young and elderly breast cancer patients. BMC Med. (2015) 13:266. doi: 10.1186/ s12916-015-0504-3 52. Johnson RH, Chien FL, Bleyer A. Incidence of breast cancer with distant involvement among women in the United States, 1976 to 2009. JAMA. (2013) 309:800–5. doi: 10.1001/jama.2013.776 44. Foulkes WD, Smith IE, Reis-Filho JS. Triple-negative breast cancer. N Engl J Med. (2010) 363:1938–48. doi: 10.1056/NEJMra1001389 53. Tripathy D, Brufsky A, Cobleigh M, Jahanzeb M, Kaufman PA, Mason G, et al. De novo versus recurrent HER2-positive metastatic breast cancer: patient characteristics, treatment, and survival from the systHERs registry. Oncologist. (2020) 25:e214–22. doi: 10.1634/theoncologist.2019-0446 45. Ahn SH, Son BH, Kim SW, Kim SI, Jeong J, Ko SS, et al. Poor outcome of hormone receptor-positive breast cancer at very young age is due to tamoxifen Frontiers in Endocrinology 14 frontiersin.org
https://openalex.org/W2985041512	https://scindeks-clanci.ceon.rs/data/pdf/0042-8426/2019/0042-84261905005S.pdf	Latin	null	The role of war games in the operational planning process	Vojno delo	2,019	cc-by	11,473	DOI: 10.5937/vojdelo1905005S DOI: 10.5937/vojdelo1905005S Introduction t the beginning of this century, many countries experienced rapid economic growth which was driven by numerous achievements made in science and technology, and first of all, in the field of information and technology. The economic growth was mainly driven by achievements made in the military industry, especially at the time of the division into blocs, and the arms race of the world’s most developed countries. The situation like this resulted in a need to strengthen the function of strategic planning, and to permanently analyze the oppo- nent’s capabilities in order to come up with warfare models which would give commands and units the initial advantage and provide better conditions for operational development. The actual testing of models by implementing real exercise-related activities required the employ- ment of considerable human and material resources along with enormous financial expenses, which resulted in a need to develop simulation and create war games. A w A g War games were made on the grounds of the game theory and were primarily de- veloped within military sciences in accordance with the scenarios made by possible bel- ligerents. Most research on basic concepts of the game theory and structural elements of war games is based either on looking for answers to some military and security-related questions or finding solutions to specific simulated conflicts (for example: the Battle of the Bismarck Sea, the Game of Intimidation, the Game of Deterrence (Mukić, 2014). In the Serbian Armed Forces, war games are generally applied in the decision- making process while preparing and conducting tasks from the first Mission, which refers to the defense of the Republic of Serbia from the armed threats. Depending on the pur- pose and type of commands, the decision-making process follows the standard operating procedures where all phases and steps of the operational planning process have been implemented in accordance with the relevant instructions and standards and criteria that have already been set up (the General Staff of the Serbian Armed Forces, 2017). The experiences gained by commands and HQs of the Serbian Armed Forces in ap- plying war games in the operational planning process indicate that war games have been insufficiently, inadequately and very often superficially applied, especially by the army branches, which do not take them into consideration at all. Radiša Saković and Vlada Mitić Republic of Serbia, Ministry of Defence, Defense Policy Sector, Strategic Planning Department Mitar Kovač Edukons University, Faculty of Project and Innovation Management, Belgrade Radiša Saković and Vlada Mitić Republic of Serbia, Ministry of Defence, Defense Policy Sector, Strategic Planning Department Mitar Kovač Edukons University, Faculty of Project and Innovation Management, Belgrade ver the past several decades, most regional and local conflicts have merely been the scenarios prepared in advance in the “kitchens” of great powers. The aforesaid scenarios are practically tested on simulation-based models that are formed as war games, which con- siderably save resources and improve the strategic and operational- tactical planning process. O v h O Information technology has had the decisive impact on the development of war games resulting in the fact that various software packages have been developed, as well as the systems which provide support to commands and HQs in a decision-making process at all command and control levels. The findings showing that resources can be rationally exploited and financial funds considerably saved indicate the efficiency of applying war games in the operational planning process and cause specific simulation-based mod- els to be developed in commands and HQs of modern armed forces. The aim of this paper is to point out the role and importance of war games in the operational planning process. To that end, various models of war games have been analyzed, along with the possibility of applying them in the operational planning process and activities conducted by the commands and HQs of the Serbian Armed Forces. The paper examines the disadvantages of models that have already been applied in the drill of the Serbian Armed Forces units, at the same time indicating the modern models of war games. The topic has been addressed through four items: 1) the term and characteristics of war games; 2) the elements of war games; 3) the structure of war games and 4) the application of war games in the operational planning process. It has been concluded by the research results that war games are an integral and inseparable part of the operational planning process in mod- ern armed forces and that they are implemented as various models for making decisions during the preparation and execution of combat actions and armed conflicts. Radiša Saković and Vlada Mitić Republic of Serbia, Ministry of Defence, Defense Policy Sector, Strategic Planning Department Mitar Kovač Edukons University, Faculty of Project and Innovation Management, Belgrade The collected findings have shown that war games are an integral part of the operational planning process in the Serbian Armed Forces too, as well as that the level they have reached is not sat- isfactory and should be improved by being more comprehensively imple- 5 VOJNO DELO, 5/2019 mented in commands and HQs, provided that automated command-information systems have previously been established. Ensuring that the above-mentioned conditions are met at all command and control levels would contribute to introducing a decision-making support system in an organized manner and improving the efficiency of the operational planning process, and thus making timely decisions while conducting missions and tasks of the Serbian Armed Forces. mented in commands and HQs, provided that automated command-information systems have previously been established. Ensuring that the above-mentioned conditions are met at all command and control levels would contribute to introducing a decision-making support system in an organized manner and improving the efficiency of the operational planning process, and thus making timely decisions while conducting missions and tasks of the Serbian Armed Forces. Key Words: War Games, Commands and HQs, Operational Planning, Conflicts Concept and Characteristics of War Games Over the course of history, military sciences have constantly advanced, striving to be fully developed and following, in doing so, the other social and liberal arts, technical- technological and other types of sciences, including all their constituents. 1 The greatest problems in defining military sciences and their scientific disciplines have arisen while selecting the methods of military sciences and laying their foundations, where war games have very often been, as a matter of priority, discussed to be the only possible and the most eligible solution. The selection of the accredited methods of military sciences has been addressed by a lot of foreign and national authors, where the greatest achieve- ments have been made with reference to theoretical development of tactics and opera- tional art (Liptai, 1996). ( ) War games were derived from games, which people find entertaining and which con- tributed to introducing people to each other, making them closer and outsmarting each other, with no intention of inflicting losses, injuries, etc. to the opposing party, but with the aim to win by making higher-quality decisions. The situations in which interests of two or more parties are entwined, confronted or conflicted are called conflicts. That is why we often say that the parties which make decisions are in a conflict. For example, in the game of bridge, the result of the game does not depend only on a move of one player, but of the other, as well. Their interests are in a conflict because both parties want to win. This concept of a game is impossible to apply when various governmental and non- governmental stakeholders have the same interests, and are ready to exercise not only political and military, but also regular and irregular forms of strength and power. Bearing in mind that in each conflict losses are inevitable due to diametrically opposite goals of adver- saries, a war game is a mechanism for testing the strength and in doing so its duration and intensity directly depend on the quality of the decision made. In some cases, the conse- quences of the decision being made depend only on one party, as it is the case when a commander makes a decision which route should be used by a unit to move in the opera- tion of preventive deployment of forces. Introduction In order to successfully elimi- nate shortcomings, it is necessary to observe the role and importance of war games, as well as the possibilities for procurement of new software solutions and introduction of the already existing ones all the way down to HQs of the lowest tactical units, which will make it possible for command staff to be trained during preparatory activities and execu- tion of all actions and counteractions. 6 The Role of War Games in the Operational Planning Process If the systematic approach to the development of war games is applied in order to enhance the operational planning process in the Serbian Armed Forces, the conditions will be made for applying new operational methods in commands and HQs and more successful standardized procedures. By attributing greater importance to war games in the Serbian Armed Forces, a comprehensive approach to the operational planning proc- ess would be made possible, and thereby timely decisions would be made at all com- mand and control levels. 1 Science constituents: subjects, theories, methods and language. Concept and Characteristics of War Games However, the most frequent decisions are the ones whose the consequences do not depend on one party only, but also on the decisions made by the other party, as well, which is particularly typical for armed conflicts. By experience, it is well-known that the bloc-divided countries and the leading world powers have different approaches to the interpretation of war and the definition of the concept and categories of armed conflicts, which is particularly characteristic for the be- 7 VOJNO DELO, 5/2019 ginning of this century with the emergence of hybrid threats and asymmetric forms of conflicts. Such a situation resulted in having different approaches to the perception of war games, particularly with reference to their concept. The available facts have shown that a great number of authors think that the equipment makes a key element in war games, while not so many authors think that a human element is a decisive factor. ginning of this century with the emergence of hybrid threats and asymmetric forms of conflicts. Such a situation resulted in having different approaches to the perception of war games, particularly with reference to their concept. The available facts have shown that a great number of authors think that the equipment makes a key element in war games, while not so many authors think that a human element is a decisive factor. Generally, a war game is a model trying to present an armed conflict as real as pos- sible through a descriptive and prognostic character (Radinović, 1983). In this respect, the previous, already waged conflicts can be modelled by war games, which, in that case, mainly have a descriptive character, or some future conflicts which, generally, have a prognostic character. The descriptive character of war games is used to confirm the long-time practical experience or to explain the knowledge gained in the previous period. The prognostic character of war games emphasizes that war games are focused on the prediction of outcomes in the modelled conflicts by introducing various variables, as well by evaluating their effects, so that scientific conclusions can be made. War games are characterized by specific attributes or properties which are based on the attitudes gained by experience and the results obtained in the process of confronting capabilities between the opposing parties (players). Concept and Characteristics of War Games These characteristics are based on the impartiality of a war game and avoidance of the subjectivity by commands and HQs, because the information collected should not reflect the commander’s wishes, but the facts which reflect the real situation and create a clear operational picture of the battle- field. War games are most often characterized by a lack of relevant intelligence informa- tion, uncertainty in estimating the situation and the risk in predicting our own and the opponent’s future steps. Apart from the above - mentioned characteristics, there are a lot of others, as well, which primarily refer to the insufficiency of the strength and assets, i.e. the limitations in human and material and, quite often, spatial and time resources. Bearing in mind the technological progress, as well as the rapid development of in- formation technologies and their growing presence in modern armies, war games are gaining more and more importance in the operational planning process, despite the vari- ous attitudes with reference to their characteristics and concept. The above - mentioned trend will lead to the conditions in which war games will not be applied only for solving operational combat tasks, but also for solving tasks of civil defense, emergency man- agement and other security-related fields. Elements of War Games War games have evolved through history and they are widely used in other fields, as well, not only the military one. Nowadays, there are war games developed for entertain- ment (hobby war games), as well as professional war games, which are used solely in the military field (Perla, 1990). There is a large number of examples of war games, which have been developed for entertainment (a hobby), and which imitate historical conflicts with great accuracy of the final outcome (Figure 1). Thus, a lot of hobby war games have a descriptive character with the aim to bring armed conflicts from the past closer to a huge audience of players. 8 The Role of War Games in the Operational Planning Process Figure 1 – Examples of hobby war games: left – the strategic war game of World War II World in flames; right – the war game Flames of War: the Battle of Normandy in World War II (Salute 2014 – World War II) Figure 1 – Examples of hobby war games: left – the strategic war game of World War II World in flames right – the war game Flames of War: the Battle of Normandy in World War II (Salute 2014 – World War II) The expansion of hobby war games has brought about the development of profes- sional war games, but the tools and rules of hobby games are adjusted and used for the improvement of professional war games (Perla, 1990). The expansion of hobby war games has brought about the development of profes- sional war games, but the tools and rules of hobby games are adjusted and used for the improvement of professional war games (Perla, 1990). p p g ( ) The necessary elements of a war game, generally, are: goals, scenario, database, players, rules, models (simulation) and analysis (Figure 2). Figure 2 – Elements of war games (taken, adapted and designed (Perla, 1990: United Kingdom Ministry of Defense, 2017) Figure 2 – Elements of war games (taken, adapted and designed (Perla, 1990: United Kingdom Ministry of Defense, 2017) Goals of war games should be focused on collecting experiences and information re- quired for conducting various processes related to security of countries, so that the de- sired situation is achieved. 2 Red player(s) will have their goals and blue player(s) will have different goals. Moreover, the goals of other actors can appear in a war game. g gy 4 The break-up of conflicts by types (intensity) can be different, for example: by forms of combat operations, such as a fight, battle or operation (the Federal Secretariat of People’s Defense, 1981); or by levels of objec- tives which have to be reached, at tactical, operational and strategic level (Djordjevic, 2012; General Staff of the Serbian Armed Forces, 2012). Elements of War Games For example, during the operational planning process, the goals of a war game are set up in the phase of identifying required strength and assets, by means of which all conditions are provided for conducting the assigned missions and tasks. That is how the goals become guidelines for creating the game, and particularly for guiding players in the game. 2 9 9 VOJNO DELO, 5/2019 A scenario of a war game is a specific situation in which the players are placed and which has a decisive impact on all war game factors (Perla, 1990). The scenario can result from previous steps of the environment analysis and scenario development, i.e. classified and grouped factors from the environment, which have a decisive impact on a war game and their possible values. Furthermore, the scenario can be a product of the future steps of the environment analysis and scenario development, i.e. selected, inter- preted and analyzed scenarios, which have been developed at a higher level 3. In terms of hierarchy, high-level scenarios are the product of the morphological analysis-based methods and high-level war games. This is how the integration of the morphological analysis-based methods and war games has been established, being two techniques of the scenario development which complement each other. p p A database comprises the information which players need to know, so they can make de- cisions while playing a war game (Perla, 1990). The database needs to have information on key factors and their values, as well as on some planning process products, and especially on analyses of adversaries and interested parties, analyses of strategy, concept and legal frame- work, as well as the identification of required capabilities and option development for achieving the required capabilities. In regard to key factors and their possible values, the selection of factors will, to the greatest extent, depend on the level of operations execution (Тable 1). 3 Since the design of scenarios by using war games is, to the greatest degree, created for the purpose of th conflict scenario, higher level scenarios can be scenarios of the environment, threats and strategy. 4 Elements of War Games Тable 1 – Types of factors found in the database with reference to the level of war games (Weiner, 1959) Type (intensity) of a conflict4 Types of factors involved in a war game Duel Battle Campaign War Resources X X X X Objectives X X X X Military intelligence information X X X X Environment characteristics X X X Background information X X X Logistics X X Economy X X Psychology X X Politics X Other parties X able 1 – Types of factors found in the database with reference to the level of war games (Weiner, 1959) In the low-intensity conflicts based on war games, such as duels, which consider a plat- form-to-platform situation, the factors describing the platforms themselves are important (for example: a number, tactical-technical characteristics, performances, etc.), as well as the objectives of each of those platforms and the basic intelligence information, which play- ers should have about each other (Weiner, 1959) (for example: possible locations, the time of opponent’s actions, the battle order, organization, support, etc.). The battles, which are 10 The Role of War Games in the Operational Planning Process high-intensity conflicts in war games, should, along with the aforesaid information, involve the background information, as well as the environment characteristics (Weiner, 1959). Campaigns, which are reviewed much longer than battles, should include the important information referring to logistics, economy and politics. War, as the highest type of a conflict, must include the information on political factors and other parties involved in war. Models of war games are designed to transform the database data and players’ deci- sions into events in a war game (Perla, 1990). While making a model, the following rec- ommendations should be taken into consideration (Perla, 1990): the model flexibility, accuracy and uncertainty. Due to the complexity of war games, design of war games requires a combination of various models in order to increase the impartiality and reality. The selection of models, to the greatest extent, affects the establishment of war games rules. The theory of war games includes the following models (Weiner, 1959): mathe- matical, mechanical (computer-aided simulation), board games and umpired models. , ( p ), g p Mathematical models imply that various mathematical models are applied and combined for the needs of war games. Elements of War Games The application of mathematical models begins, in principle, with the methods of game theories (Weiner, 1959; Kahn & Mann, 1957), which later build on the mathematical methods for calculating the outcome. Mathematical models are based on de- veloping and linking possible player’s actions, as well as on using the data on the value fac- tors from the database related to the characteristics of the strategy applied by both players, so the conflict outcomes can be calculated by applying the mathematical tools (Figure 3). Figure 3 – Mathematical model of war games – taken and supplemented (Weiner, 1959) 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1. Circle Actions (strategy) player A Actions (strategy) player B Factors from the database Outcome Factors from the database Game theory Game theory Game theory Outcome Factors from the database 2. Circle Outcome 3. Circle Actions (strategy) player B Actions (strategy) player A Factors from the database Factors from the database Figure 3 – Mathematical model of war games – taken and supplemented (Weiner, 1959) 11 11 VOJNO DELO, 5/2019 By applying the methods which are based on the theory of games, the actions of one side are related to the reactions of the other side, so the form matrix of “the strat- egy of the player A” and “the strategy of the player B” is established, which presents a circle of the game (Weiner, 1959). Various combinations of strategies, i.e. actions taken by both players in one move will result in different outcomes 5. The outcomes obtained should be in accordance with the required effects determined in the phase of identifying the required capabilities, so the war games are used to determine if some activities being implemented result in the required effects. The outcomes from the first circle have an impact on forming new strategies for both sides, the outcomes from the second circle affect the strategy for the third circle and it continues until one of the sides achieves the objectives. Professional war games are most often played by mak- ing sequential moves, such as action-reaction-counteraction 6 (General Staff of the Serbian Armed Forces, 217), i.e. 5 The outcome provides the following information: if the player A plays strategy 1, and the player B plays the strategy 2, it will happen that… for example the player A makes x and player B makes y losses, etc. 6 Actions of a player (actions-reactions-counteractions) are identified in the phase of identifying the required capabilities, but war gaming itself helps them to be identified, so a required parallel bond is obvious between the phase of identifying the required capabilities and the phase of analyzing the environment and scenario development, particularly in the domain of war games. 7 Taking the objectives of war games into consideration, the player A can be either red or blue, which de- pends on the scenario, rules and objectives of a war game, but the red player is most often in the role of the player A, and the blue player in the role of the player B. Moreover, the sequence of players defining who of them makes the first move can be changed from one move to other, so one move can begin with the blue player, the second one with the red player and the third one with the blue player again. 5 The outcome provides the following information: if the player A plays strategy 1, and the player B plays th strategy 2, it will happen that… for example the player A makes x and player B makes y losses, etc. 6 Elements of War Games the circle is made up of three moves made by players (the actions made by players A – the reactions made by player B – the counteractions made by player C) 7 (Figure 4). Figure 4 – Possible movements made by players in one circle of a war game – taken and supplemented (Morgan, McLeod, Nixon, Lynch, & Hura, 2018) Figure 4 – Possible movements made by players in one circle of a war game – taken and supplemente (Morgan, McLeod, Nixon, Lynch, & Hura, 2018) Every possible action of player A should be related to every reaction of player B, while every reaction of player B should be related to every counteraction of player A. Each of possible action-reaction-counteraction combinations in one circle needs to be 12 The Role of War Games in the Operational Planning Process evaluated from the point of factors which have the key impact. It can be seen that such a structure of actions taken by both players makes it possible for the morphological analy- sis to be used, so relevant morphological fields are here complemented with possible actions-reactions-counteractions. The morphological analysis can be used to test the possible intercompatibility (consistency) of the actions taken by both players, as well as the compatibility of the actions with key factors from the environment. The results of the morphological analysis can complement war games. That is how a parallel bond can be established between the methods based on the morphological analysis and war games. Numerous mathematical methods have been developed and applied so far for calcu- lating the outcomes. Since actions taken by both parties in one move put their resources in the interrelationship of conflicts, the mathematical methods based on the combat dy- namics can be applied, i.e. Lanchester’s equations, as well as more simple and practical models of calculating combat possibilities (Kovač, Dulanović, & Stojković, 2006; General Staff of the Serbian Armed Forces, 2015). ) The mathematical models based on the combat dynamics take into consideration tactical-technical characteristics of platforms 8, the environment, as well as the informa- tion and assumptions about the conflict expressed by military intelligence information, on whose basis, they, by certain mathematical equations, calculate the outcome as the degradation of a specific platform, i.e. 8 Hit probability, rate of fire, radius of action, penetrability, range, rate of movement, kinetic protection, etc. 9 For example, modifiers that reduce or increase the likelihood of destruction due to the effects of morale, ca- pability level, terrain, the use of chemical and biological weapons, degree of preparedness, degree of land development and fortification and the like (General Staff of the Serbian Armed Forces, 2015) 8 Hit probability, rate of fire, radius of action, penetrability, range, rate of movement, kinetic protection, etc. 9 if ([action A] Ʌ [reaction B] Ʌ [counteraction A])  [outcome] (1) Elements of War Games losses made by a party in a game (Przemieniecki, 2000), as in the case of the conflict of infantry units (Mitić & Petrović, 2015), unmanned anti-armor aircraft and armored personnel carrier (Petrović, Mitić, & Mudri, 2015), etc. The above - mentioned methods require a great number of experts from the field of mathematics and various military sciences to be involved. The mathematical models are not applied sufficiently in the Serbian Armed Forces because the methods based on the combat dynamics are not adjusted to be used by com- mands and HQs during the operational planning process, but they can be the good ground for the development of methods which are based on combat capabilities. On the other hand, the methods based on the calculation of combat capabilities take into account the same input data as the methods based on the combat dynamics, with the only difference of having simpler mathematical tools, which are based on the possibil- ity of destroying the opponent, as well as on modifiers, i.e. coefficients of probability of decrease and increase, which depend on the environment where the conflict takes place 9. The introduction of modifiers makes the calculations easier and enables the models based on combat capabilities to be used by commands and staff during the operational planning process, but the accuracy of the final outcome is lower than with the methods based on combat dynamics. It can be concluded concerning the mathematical models that the measurement of outcomes is based on deduction, which can be presented as the if-then function: (1) if ([action A] Ʌ [reaction B] Ʌ [counteraction A])  [outcome] if ([action A] Ʌ [reaction B] Ʌ [counteraction A])  [outcome] 13 VOJNO DELO, 5/2019 Machine war games or computer-assisted simulation are based on the use of in- formation technology and a game. Machine games have a database and maps rele- vant for war games and they use mathematical models in order to calculate the game outcome. All mathematical operations required for a game are processed by a com- puter according to previously set algorithms. With reference to machine war games, the following types of games can be distinguished according to the criterion of players participating in the war game (Weiner, 1959): machine-machine, machine-man and man-man war games. 10 Information on the exact situation in the field can be entered in the computer war game either manually or automatically by using the telecommunication-information devices together with the devices for navigation, locators, fire and hit simulators and the like. Elements of War Games A large number of computer-assisted war games have been developed so far, both for entertainment and professional purposes related to planning operations and conduct- ing military exercises. Along with the computer simulation, a live one-sided or two-sided exercise can also be conducted by completing the war game database with the informa- tion 10 gathered in the field during the actual exercise (Sennersten, 2010), thus achieving the greater objectivity in the execution of the war game. In the Ministry of Defense and the Serbian Armed Forces, the machine- (com- puter-) assisted war game of JCATS type is applied (Lawrence Livermore National Laboratory, 2018) – Figure 5. The JCATS software runs on the Linux operating sys- tem and represents a multi-sided, stochastic and interactive high-resolution simula- tion designed to model the interaction of forces, from joint forces to individual plat- forms. In JCATS software, players-users have different visual displays of JCATS dur- ing the simulation. Figure 5 – Examples of possible JCATS war games (Lawrence Livermore National Laboratory, 2018) Figure 5 – Examples of possible JCATS war games (Lawrence Livermore National Laboratory, 2018) 10 Information on the exact situation in the field can be entered in the computer war game either manually or automatically by using the telecommunication-information devices together with the devices for navigation, locators, fire and hit simulators and the like. 14 The Role of War Games in the Operational Planning Process JCATS visual presentation is a physical model that includes all the parameters of the digital military topographic map (Military Geographical Institute, 2016) 11, as well as the database related to the characteristics of the combat platforms available to the players. The connectivity with the database ensures the accuracy in simulating reconnaissance and observation, as well as the accuracy of data collecting sensors, weapons systems, ammunition, vehicles, not only on the ground, but also at sea and in the air, as well as the connectivity with the real command-information systems (Lawrence Livermore National Laboratory, 2018). Based on the software algorithms, which are founded on mathematical models, the effects of the conflict are calculated and presented, which al- lows the examination of different decisions in the planning process. Board war games are the most common model of war games that is implemented in military practice, especially because of its price and ease of production and use. 11 For example: digital maps of the Military Geographical Institute (Military Geographical Institute, 2016). 12 Unlike the quadrangular grid that allows forward-to-left-to-right directions. Elements of War Games The elements of board war games are most often a map (a board) or a topographic map of a certain scale, unit markers (Weiner, 1959), and often a grid that is placed over the map (Figure 6). Figure 6 – Example of RAND war game elements with the map, grid and unit markers – taken (Shlapak & Johnson, 2016) Figure 6 – Example of RAND war game elements with the map, grid and unit markers – taken (Shlapak & Johnson, 2016) The maps used for playing war games can be topographic or relief maps of various scales (Military Geographical Institute, 1971-2001), which correspond to the area, where the combat operations are planned to be conducted, while the scale corresponds to the level of performance of the operations. Unit markers are most often two-dimensional cards made on cardboard, paper, plastic or other materials, and different types and lev- els of units are marked in accordance with the Instructions for Operational Planning and Work of Commands (General Staff of the Serbian Armed Forces, 2017). The grid is a transparent layer placed over the map, and it is recommended that it has a hexagonal form to allow the establishment of motion rules and actions of units in all directions, as well as the simulation of support to adjacent units (Figure 7) 12. 15 VOJNO DELO, 5/2019 Figure 7 – Example of the use of the hexagonal grid: left – possible directions for the movement of the blue player; right – possible directions of action and cooperation – support of the blue player Figure 7 – Example of the use of the hexagonal grid: left – possible directions for the movement of the blue player; right – possible directions of action and cooperation – support of the blue player The players in board war games are people, where there might be one man, teams or units at each side. Unlike machine games, players have countless possible actions they can take at their disposal (Weiner, 1959). Playing board war games is not possible without previously applied mathematical models, which establish the rules of board war games, and the calculations of combat capabilities are of particular importance. g p p p Umpired war games are based on the assessment made by an umpire on the out- come of a player's actions. Umpires are usually the oldest and most experienced officers in commands-HQs. Elements of War Games This type of games is applied when clear rules of the game are not elaborated, when there are no mathematical models that are used and when there is a lack of time as a resource (Weiner, 1959).The shortcoming of these games is subjectivity that can influence the assessment of outcomes, and give wrong information. Taking into account the practical experience gained on war games in the Serbian Armed Forces, which are described in the Instructions for Operational Planning and Work of Commands (General Staff of the Serbian Armed Forces, 2017) it can be concluded that although they are played on the map, these war games belong to a group of umpired war games due to the vaguely defined rules, as well as due to the assessment of the outcome made by umpires. Rules are the next element of a war game that needs to be determined during the design of the war game. The choice of different models of war games will imply different rules, so the development of rules goes on concurrently with the selection and develop- ment of war game models. The purpose of rules in war games is to: dictate which models will be used and when; establish rules of connectivity between action, reaction and coun- teraction; provide the delivery of necessary information to players, as well as possible mistakes in information in relation to their reliability; determine the roles of players in a war game and the like (Weiner, 1959). 14 For example, range, seclusion (shelters and revetments), morals, effects in case chemical and biological weapons are used, the target movement, the unit (platform) delivering fire and the like. When it comes to the rules of hits, the rules for direct and indirect fire, as well as close combat, have to be clearly specified. 13 Modifiers increase the detection radius if some electronic and optical devices are used, i.e. they decreas the radius due to the impacts of terrain, part of a day, meteorological conditions and the like. Elements of War Games 16 The Role of War Games in the Operational Planning Process By analyzing a large number of sources dealing with rules, both professional and hobby war games (Exercise Aldershot Skirmish, 2016; Martin, 1982; KDV Technology & Consulting, 2004-2018; United Kingdom Ministry of Defense, 2017; McHugh, 1966; War games research group, 1972; First Battle Basic Rules, 1989), it can be concluded that some of the main rules of war games are based on the identification of the following ele- ments: – the flow of time, which regulates how long the cycle lasts and what the ratio of pas time and movement by the coordinate system is; – the flow of time, which regulates how long the cycle lasts and what the ratio of past time and movement by the coordinate system is; – game sequences that regulate which player plays first and when, how many moves there is in one circle, and when the outcome of the circle is calculated (either after each move or when the circle ends); – the size of the base because it specifies how much space on the map in relation to the grid elements a certain unit type and size occupies; 13 – detection radius and detection radius modifiers; 13 – movement and movement modifiers, which define the rules of movement and regu – movement and movement modifiers, which define the rules of movement and regu- late how many elements of a coordinate system a certain type of unit can pass in a unit of time, while modifiers change the number of possible elements of the coordinate sys- tem that can be crossed depending on the terrain, as well as the impact of various ob- stacles as movement modifiers and channeling of units; – rules of direct and indirect fire and close combat, as well as fire delivering modifi- ers, which regulate the distances for each type of units when a direct or indirect fire oc- curs, as well as the distances when a close battle occurs because the modifiers deter- mine whether and under which conditions the unit can achieve direct or indirect fire or be in close combat; – rules of direct and indirect fire and close combat, as well as fire delivering modifi- ers, which regulate the distances for each type of units when a direct or indirect fire oc- curs, as well as the distances when a close battle occurs because the modifiers deter- mine whether and under which conditions the unit can achieve direct or indirect fire or be in close combat; – the way in which the likelihood is carried out makes it possible to determine more precisely the probability of certain events and the way of their inclusion in the game (for example, the use of many-sided dice for playing is one of the ways of determining the likelihood of events in game theory - Figure 8); – the way in which the likelihood is carried out makes it possible to determine more precisely the probability of certain events and the way of their inclusion in the game (for example, the use of many-sided dice for playing is one of the ways of determining the likelihood of events in game theory - Figure 8); – the hits and modifiers of the hits refer to the rules that specify when the goal is to be hit, and the different conditions that may affect the hit such as its modifiers 14; – the hits and modifiers of the hits refer to the rules that specify when the goal is t be hit, and the different conditions that may affect the hit such as its modifiers 14; – fire radii and radius modifiers refer to the effects that can be achieved at certain dis- tances around the target center; – ranges and range modifiers indicate conditions that can reduce or increase radius effects, and specify the range of certain types of weapons, as well as the different condi- tions that may affect the increase or decrease in range; – target effects and effect modifiers are related to the scale of effects that can be achieved on a specific target depending on the weapon used for firing a target, as well as different modifiers that can reduce or increase the effects, etc. 13 Modifiers increase the detection radius if some electronic and optical devices are used, i.e. they decrease the radius due to the impacts of terrain, part of a day, meteorological conditions and the like. 14 For example, range, seclusion (shelters and revetments), morals, effects in case chemical and biological weapons are used, the target movement, the unit (platform) delivering fire and the like. When it comes to the rules of hits, the rules for direct and indirect fire, as well as close combat, have to be clearly specified. Elements of War Games 18 The Role of War Games in the Operational Planning Process The analysis of the collected data after completion of a war game includes the study of collected facts, data classification and categorization at a lot of levels of generality, data inter- pretation and data description (War Gaming Department, U.S. Naval War College, 2015). The analysis of the collected data after completion of a war game includes the study of collected facts, data classification and categorization at a lot of levels of generality, data inter- pretation and data description (War Gaming Department, U.S. Naval War College, 2015). 15 Based on the results of the morphological analysis, where the scenarios developed by morphological analysis can be further developed in war games, or based on the results of the high-level war games. Elements of War Games – target effects and effect modifiers are related to the scale of effects that can be achieved on a specific target depending on the weapon used for firing a target, as well as different modifiers that can reduce or increase the effects, etc. 17 VOJNO DELO, 5/2019 Figure 8 – Example of multi-sided dice that can include the likelihood in war games – taken (Memories from FRP, 2013) Figure 8 – Example of multi-sided dice that can include the likelihood in war games – taken (Memories from FRP, 2013) Players are one of the required elements of a game, regardless of a model being used. Even when it comes to machine games, i.e. computer simulation, the role of a man as a player making decisions about actions to be taken is inevitable. With regard to the number of players, war games can be with one player – most often computer simulation, with two players, which is the most common war game in the military with red-blue type and with multiplayers – red, blue, allies, neutral and the like. p y The analysis is an element of a war game that is inevitable for professional games, and war games should focus on the analysis itself (Perla, 1990). The analysis of war games can focus on two aspects: the decision-making process, where the analysis focus is on actions made by players; and gathering evidence of certain events, where the analysis focus is on the outcomes, i.e. the effects (Perla, 1990). For the development planning process, both analyses are important and are related to the phase of identifying the required capabilities. q p The analysis needs to be timely planned and human resources have to be pro- vided, so that it can be conducted. The analysis process consists of two parts: the data collection, which is carried out during playing war games and the collected data analy- sis, which is carried out after the completion of games (Perla, 1990; War Gaming De- partment, U.S. Naval War College, 2015). When it comes to data collection, various methods can be used, particularly observation, content analysis, testing, and opera- tional research methods (Radinović, 1983). For the purpose of collecting data, it is necessary to develop adequate instruments to collect data on players' actions in war games and outcomes of actions taken, which are relevant for the operational planning process. Construction of War Games The construction of war games is the most innovative and creative phase in the de- velopment of war games. At this stage, a large number of iterations are performed while the observed shortcomings are constantly removed in order to improve all elements of the war game. The models used during the construction of war games most often result from the requests submitted by a client, that is, by the entity that finances the project and directs the development of the war game scenario. Taking into account the elements of war games, as well as the recommendations made in some books and related to the break-down of war game process into stages, one can conclude on the activities of the scenario construction steps when war games are used (Figure 9). Figure 9 – Construction of scenarios using war games – taken, adapted and supplemented (United Kingdom Ministry of Defense, 2017, War Gaming Department, U.S. Naval War College, 2015) Figure 9 – Construction of scenarios using war games – taken, adapted and supplemented United Kingdom Ministry of Defense, 2017, War Gaming Department, U.S. Naval War College, 2015) Tasking is the first activity that determines the objectives of a war game for all players and the purpose of war games in the context of the development planning, and formulates the problem for which a war game is created. In the context of the development planning process, tasking is carried out on the basis of the phase of identifying the required capabili- ties, and particularly the analysis of key factors and the formulation of objectives. The next activity is the design of a war game that primarily specifies the level of con- flict, the number of players and the scenario. In this regard, the design is made on the results of the previous steps of the environment analysis and the scenario development phase, as well as during the scenario transfer. 15 The development of war games refers to the activity that selects and develops mod- els, specifies the rules of a war game, selects the methods of data collection and creates data collection instruments, determines preliminary data classification and communica- tion in war games. 19 VOJNO DELO, 5/2019 The war game testing is an activity that integrates different elements of a game and checks their compatibility, and also the design or development itself is revised in case of a need. Construction of War Games These forms of testing are more and more represented in open sources, and first of all between players on social networks (Saković & Terzić, 2018). p y ( ) Testing refers to playing a game with real players in order to identify possible shor comings and make the players familiar with the game. Execution is the stage in which the game is played directly by the players followin the rules of the game and making moves that produce certain outcomes. War game analysis is the last activity of the scenario construction by war game appli- cation, by means of which the data is collected on the players’ moves and outcomes of the moves and the reports are made. It is necessary to distinguish between the war game analysis and the stage of the scenario analysis, selection and interpretation. The stage of the scenario analysis, selection and interpretation requires players to try out more than one possible action in each move in order to get different scenarios based on different variants of use by both blue and red player. On the other hand, the war game analysis focuses on each individual variant of use, considering various actions taken by players and outcomes, so that the required skills can be identified. In this regard, the war games analysis needs to be made for each variant of use separately, as each will give different combinations of actions and outcomes (effects). ( ) In addition to the scenario analysis, selection and interpretation, war game analysis data are also used to construct scenarios by using morphological analysis. The integra- tion of the methods, which are based on war games and morphological analysis are con- ducted in the scenario employment (transfer) stage, when, based on the war game analysis, the parameters of the morphological analysis are formulated, and then the pa- rameters are used in the scenario construction stage by using the morphological analy- sis. 16 In this way, a parallel bond is established between the methods of developing sce- narios by using the morphological analysis and the methods of developing scenarios by using war games. 16 For example, a parameter of morphological analysis can be: action, reaction, counteraction, effect, target and the like, which has been identified by war games. Application of War Games in the Operational Planning Process Due to intensive changes in the strategic environment, many countries, insisting on the collective security system, have followed the trend of reducing financial expendi- tures for the development of their armed forces. In contrast, investment in modern telecommunications and information technologies has significantly increased in order to develop early warning systems for potential security challenges, risks and threats. This approach has determined the development of certain software solutions in the field of defense systems in the developed countries along with the prediction of poten- tial scenarios in case of crisis and conflict situations worldwide. Regardless of security, economic, military and other forms of confrontation between states and military alli- 20 The Role of War Games in the Operational Planning Process ances, the scenarios are most often developed in the form of war games. The Republic of Serbia has the concept of total defense and military neutrality, and war games are exclusively within the framework of the employment of the armed forces, that is, in the field of engagement of the Serbian Armed Forces in order to conduct the assigned missions and tasks. In the Serbian Armed Forces, war games are implemented in the fourth phase of the operational planning process – the analysis of employment variants. 17 In principle, war games identify which employment variants can conduct tasks with minimum losses and at the same time ensure that the forces maintain the initiative and supremacy in the planned operation. According to the Instructions for Operational Planning and Work of Commands in the Serbian Armed Forces (on temporary basis), war games are con- ducted in the HQs of the maneuver, special, river and reconnaissance units, as well as in the HQs of joint forces in the Armed Forces, while they are not conducted in other units, where the third stage in the operational planning (the development of employment vari- ants) is directly followed by the fifth one (the comparison of employment variants). The process of analyzing employment variants (war games) is applied at all planning levels with the aim to help the commander and headquarters – the command to come up with a method of optimum employment of the unit combat power and, at the same time, to pro- tect its own forces with the fewest losses. In the above-mentioned correlations of operational planning, the role of war games is seen in selecting an optimum employment variant. 17 The same. 18 The Instructions for Operational Planning and Work of Commands in the Serbian Armed Forces (on tempo- rary basis), defines eight steps of war games: 1) resources raising; 2) making the list of effective forces; 3) making assumption lists; 4) making the list of important events and decision items; 5) selection of war games methods; 6) selection of methods for marking and presenting results; 7) playing out military operations and results assessment and 8) briefing on war games (not necessary). 17 The same. Application of War Games in the Operational Planning Process The war games process provides an outline of the operation course and takes place according to certain rules and clearly defined steps, with full consideration of all key factors of actions and counteractions (hu- man, material, special and time factors). While conducting analyses, commands and HQs inevitably rely on doctrinal attitudes, intelligence-security and other assessments, as well as previous training and war experience. The command conducts the analysis of employment variants through the continuous action, reaction and counteraction process, with which it encourages ideas and gives the picture of the state in the operations area and the wider environment. In this way, the command, by applying war games methods, ensures that major tasks are accomplished by connecting them to the combat capabili- ties of its own forces. War games test the already-developed employment variants or improve them accord- ing to the analyses performed and the latest changes made in the operational situation. This means that the commander and the command staff can change the existing em- ployment variant or develop a new one if unforeseen critical events, tasks, demands or problems are identified. For the sake of a proper and comprehensive approach to the implementation of the fourth stage of the operational planning i.e. the analysis of em- ployment variants, the general rules, responsibilities of the staff officers and steps during the execution of war games are defined in advance. 18 21 VOJNO DELO, 5/2019 According to standard operational procedures, the operational planning process has been implemented at all command and control levels in accordance with the purpose and specifics of the Serbian Armed Forces commands and units, and thus the fourth phase of the operational planning process. When it comes to the steps of war games, the follow- ing steps deserve the special attention: work of the command during the fifth step (selec- tion of war games techniques), the sixth step (selection of ways to record and display results) and the seventh step (the operation war-gaming and results assessment). Within the fifth step, which refers to the choice of war games techniques, the operations officer in the command, i.e. HQ, selects the technique to be used in the further process of operational planning in accordance with the assigned time and mission specifics. Application of War Games in the Operational Planning Process At the moment, three techniques of war games – the belt technique, the in-depth technique and the technique of critical areas (boxing technique) are used at the operational-tactical level in the Serbian Armed Forces, each of them taking into account the area of interest and all enemy forces that affect the operation outcome. These techniques can be used individually or they can be combined (General Staff of the Serbian Armed Forces, 2017). In the sixth step of a war game referring to the selection of methods to record and display results, the HQ, i.e. the command, writes and interprets data on the course of implementing the selected war game technique, on the basis of which it sets up the combat formation, coordinates the activities and, subsequently, makes plans or orders for the employment of units. The data are recorded through the synchronization matrix and a war games worksheet and, at the same time the employment variants are ana- lyzed on the basis of the identified advantages and disadvantages. Within the seventh step of a war game that refers to the operation war-gaming and results assessment, a commander and the HQ or the command try to predict the dynam- ics of combat actions, the action and reaction, and they analyze each selected event, identifying a task that the forces have to conduct at a lower level, and if necessary at two lower levels. The dynamics of war games does not decline in importance at this stage either, because the "action-reaction-counteraction" cycle continues until the important event ends or until the commander concludes that he has to change the employment variant, so that the mission can be conducted. During a war game, the command staff members analyze the risks for each employment variant, in order to obtain as realistic and objective indicators as possible. The results of the war game should provide all the elements necessary for making an appropriate decision, that is, to give answers to all essential issues and remove the majority of the commander’s dilemmas. One of key shortcomings of the existing instructions refers to the inadequate under- standing and insufficient consideration of the employment variants in support units. Conclusion The phenomenon of war games has begun to develop since the first conflicts in the human community. Throughout history, the everlasting aspiration of a nation to dominate the other one has caused the emergence of the first strategies and tactics of warfare. These forms of con- flicts have mainly been fought over the territories rich in food and water, and later in other natu- ral resources. Human nature has remained unchanged over the centuries, so the battle for domination still goes on, and the only difference is in the mechanisms that are applied to achieve projected goals. The greatest contribution to meeting strategic objectives is provided by countries through the strength and power, which they exercise in international relations in all areas of social life, and above all through exercising political, economic and military power. g g p y p In the field of standard armed conflicts, and according to the previously established set objectives, war games are conducted at strategic, operational and tactical levels. Irrespec- tive of the implementation level and due to intensive development of information technolo- gies, war games start to increasingly gain importance in the field of non-conventional con- flicts. They do not only have the armed character, but also the hybrid one, because they represent the combined use of state regular and irregular forces in various forms of conflict. By this approach and partial relocation of war games from the standard battlefield into the sphere of civilian environment, that is, outside the operation zone, in the premises of cer- tain power centers, the operational picture of the battlefield becomes increasingly complex under the conditions of dynamic changes in security challenges, risks and threats, and the defense of countries becomes more and more vulnerable, especially during asymmetric conflicts. The aforementioned changes in the strategic environment are one of the main reasons for the different approaches of the majority of authors to the conceptual determina- tion and identification of key characteristics of war games. The elements of war games are constantly improved, first of all when it comes to technology. It often happens that software games (hobby war games) that have been developed for entertainment quickly turn into war games, which, with certain modifica- tions, are short-listed for professional war games. The current forms of war games are more widely applied than the conventional forms of warfare, which were characteristic for the conflicts in the past century. Application of War Games in the Operational Planning Process By this approach, that is, by excluding support units from the analyses of employment vari- ants at all planning levels, a complete operational picture of the battlefield is blurred and the conditions that can lead to making wrong decisions are created. The role of war games in commands’ operational planning process is of great importance because it represents a mechanism for checking the achieved level of the planning process and allows for the work of commands to be guided in preparing the elements, so that a com- mander can make a decision on timely basis. Basically, war games help the unit's com- mander use combat power optimally and at the same time protect the unit’s strength with the fewest losses possible. The ultimate goal of war games is to select an optimal military em- 22 The Role of War Games in the Operational Planning Process ployment variant. A special contribution of war games is made while analyzing the employ- ment variants of maneuver units, for whose purpose numerous software solutions have been developed. War games represent an indispensable phase in the decision-making process of the developed militaries in the world, and therefore in the Serbian Armed Forces, as well. ployment variant. A special contribution of war games is made while analyzing the employ- ment variants of maneuver units, for whose purpose numerous software solutions have been developed. War games represent an indispensable phase in the decision-making process of the developed militaries in the world, and therefore in the Serbian Armed Forces, as well. Conclusion Conclusion A special contribution to the development of war games, in addition to the development of weapons and military equipment, is made by the infor- mation technology, because, through social networks, it includes all social classes, even the whole population, into asymmetric forms of conflicts. War games are a salient phase in the decision-making process in commands and headquarters of the Serbian Armed Forces, and especially in evolution units and special units. Insufficient development of the war game elements is a key problem in the process of implementing the operational planning process. The major shortcomings can be found in tactical units and are reflected in the lack of adequate models and the failure to establish clear rules for conducting a war game. In commands and HQs of arms and service units, 23 VOJNO DELO, 5/2019 discrepancies during the implementation of the operational planning process increase, which is especially characteristic for the support units, where, along with the material- related, there are problems with the incomplete competence of junior commanding officers. discrepancies during the implementation of the operational planning process increase, which is especially characteristic for the support units, where, along with the material- related, there are problems with the incomplete competence of junior commanding officers. p p p j g With regard to the development and construction of war games elements in commands and HQs of the Serbian Armed Forces, especially at the tactical level, the models showing how they should be used are not developed to the full extent because the economic base of training is slightly poor. This, to some extent, means that the ability of commands and HQs to analyze employment variants as real as possible is reduced in the operational planning process. In contrast, the conditions for the implementation of the operational planning proc- ess at the operational and strategic level are much better, primarily due to the introduction of some computer simulation, such as JCATS software, which is under the authority of the Training and Doctrine Department (J-7) of the Serbian Armed Forces General Staff. g p ( ) The role of war games in the operational planning process is very important, although they are not sufficiently implemented in commands and HQs of the Serbian Armed Forces. 19 The operational planning process at the operational-tactical level in the Serbian Armed Forces is carried out through three methods: 1) a complete work method; 2) an abridged work method with headquarters and 3) an abridged work method without headquarters. Conclusion Under these conditions, commanders of tactical units are forced to resort to improvisation and implementation of shortened work methods in the decision-making process due to the lack of adequate software solutions and models for the implementation of war games. 19 q p g By fully understanding the role of war games in the operational planning process, by train- ing the commanding staff to implement the modern methods and management techniques, and by introducing adequate software solutions to the Serbian Armed Forces commands and HQs, the decision-making process can be significantly improved. By this approach, the ra- tionalization of resources and risk reduction are ensured, and units’ commanders and tempo- rary formations are provided with necessary conditions for making timely decisions. Literature Naval War College, (2015), War gamers handbook-a guide for professional wargamers, Retrieved Јун 9, 2018, from Defense Technical Information Center: http://www.dtic.mil/dtic/tr/fulltext/u2/1001766.pdf [16] Weiner, M. (1959), An Introduction to War Games, Santa Monica: RAND corporation. [17] Војногеографски институт, (1971-2001), Аналогни картографски производи, Retrieved Јун 13, 2018, from Војногеографски институт: http://www.vgi.mod.gov.rs/proizvodi/ analogni/analogni.html [16] Weiner, M. (1959), An Introduction to War Games, Santa Monica: RAND corporation. [17] Војногеографски институт, (1971-2001), Аналогни картографски производи, Retrieved Јун 13, 2018, from Војногеографски институт: http://www.vgi.mod.gov.rs/proizvodi/ analogni/analogni.html [18] Војногеографски институт, (2016), Дигитални производи, Retrieved Јун 10, 2018, from Војногеографски институт: http://www.vgi.mod.gov.rs/cirilica/proizvodi_cir/digitalni_cir/ digitalni_cir.html [18] Војногеографски институт, (2016), Дигитални производи, Retrieved Јун 10, 2018, from Војногеографски институт: http://www.vgi.mod.gov.rs/cirilica/proizvodi_cir/digitalni_cir/ digitalni_cir.html [19] Генералштаб Војске Србије, (2012), Доктрина операција Војске Србије, Београд: Медија центар "Одбрана". [20] Генералштаб Војске Србије, (2015), Упутство за одређивање борбених могућности Војске Србије, Београд. [20] Генералштаб Војске Србије, (2015), Упутство за одређивање борбених могућности Војске Србије, Београд. [21] Генералштаб Војске Србије, (2017), Упутство за оперативно планирање и рад ко- манди у Војсци Србије, Београд. [21] Генералштаб Војске Србије, (2017), Упутство за оперативно планирање и рад ко- манди у Војсци Србије, Београд. [22] Ђорђевић, В. (2012, зима), Операције Војске Србије - објекат пројектног менаџмен- та, Војно дело, Београд. [22] Ђорђевић, В. (2012, зима), Операције Војске Србије - објекат пројектног менаџмен- та, Војно дело, Београд. [23] Ковач, М., Дулановић, Ж., & Стојковић, Д. (2006), Одређивање ефикасности војно- организационих система, Београд: Војноиздавачки завод. [23] Ковач, М., Дулановић, Ж., & Стојковић, Д. (2006), Одређивање ефикасности војно организационих система, Београд: Војноиздавачки завод. [24] Липтаи, С. (1996). Теоријска изграђеност тактике, Београд: Центар високих војнх школа. [25] Митић, В., & Петровић, Д. (2015), Модел развоја ватрене способности тактичких јединица копнене војске у извршавању задатака пробоја, SYM-OP-IS 2015 , 454-457. [24] Липтаи, С. (1996). Теоријска изграђеност тактике, Београд: Центар високих војнх школа. [25] Митић, В., & Петровић, Д. (2015), Модел развоја ватрене способности тактичких јединица копнене војске у извршавању задатака пробоја, SYM-OP-IS 2015 , 454-457. [24] Липтаи, С. (1996). Теоријска изграђеност тактике, Београд: Центар високих војнх школа [ ] , ( ) р ј р ђ , р д Ц р ј [25] Митић, В., & Петровић, Д. (2015), Модел развоја ватрене способности тактичких диница копнене војске у извршавању задатака пробоја, SYM-OP-IS 2015 , 454-457. [26] Мукић, Н. (2014), Теорија игара-математичке основе митова и парадокса, Нови ад: Природно-математички факултет. [27] Петровић, Д., Митић, В., & Мудри, И. Literature [1] Exercise Aldershot Skirmish, (2016), Retrieved Јун 9, 2018, from Connections UK: http://professionalwargaming.co.uk/RMASExerciseAldershotSkirmish.pdf [2] First Battle Basic Rules, (1989), Retrieved Јун 23, 2018, from Connections UK: http://professionalwargaming.co.uk/FirstBattleRules.pdf [1] Exercise Aldershot Skirmish, (2016), Retrieved Јун 9, 2018, from Connections UK: ttp://professionalwargaming.co.uk/RMASExerciseAldershotSkirmish.pdf http://professionalwargaming.co.uk/RMASExerciseAldershotSkirmish.pdf [2] First Battle Basic Rules, (1989), Retrieved Јун 23, 2018, from Connections UK: http://professionalwargaming.co.uk/FirstBattleRules.pdf [2] First Battle Basic Rules, (1989), Retrieved Јун 23, 2018, from Connections UK http://professionalwargaming.co.uk/FirstBattleRules.pdf [3] Kahn, H., & Mann, I. (1957), War gaming, Santa Monica: RAND corporation. [4] KDV Technology & Consulting, (2004-2018), Combat Table Card, Retrieved Јун 18, 2018, from GHQ: http://www.ghqmodels.com/pdf/CombatTableCard.pdf [3] Kahn, H., & Mann, I. (1957), War gaming, Santa Monica: RAND corporation. [4] KDV Technology & Consulting, (2004-2018), Combat Table Card, Retrieved Јун 18, 2018, from GHQ: http://www.ghqmodels.com/pdf/CombatTableCard.pdf [5] Lawrence Livermore National Laboratory, (2018), JCATS, Retrieved Јун 10, 2018, from Conflict Simulation Laboratory: https://csl.llnl.gov/content/assets/docs/JCATS-LLNL-Brochure-2015.pdf [6] Martin, J. (1982), RECON- Roleplaying Game of the Viet nam war. Retrieved from Free Wargames Rules: g tp://web.archive.org/web/20031117000119/http://www.palladiumbooks.com/stuff/recon/originalrecon.pdf [7] McHugh, F. (1966), Fundamentals of war gaming, The United States War College. [7] McHugh, F. (1966), Fundamentals of war gaming, The United States War College. [8] Morgan, F., McLeod, G., Nixon, M., Lynch, C., & Hura, M. (2018), Gaming space, Santa Monica: RAND Corporation. [8] Morgan, F., McLeod, G., Nixon, M., Lynch, C., & Hura, M. (2018), Gaming space, Santa onica: RAND Corporation. [9] Perla, P. (1990), The art of wargaming, Annapolis: Naval institute press. 24 The Role of War Games in the Operational Planning Process [10] Przemieniecki, J. (2000), Mathematical methods in defense analyses (3rd ed.), Redston: IAA Education Series. [11] Sennersten, C. (2010), Model-based Simulation Training Supporting Military Operational Processes-Doctoral Dissertation Series No. 2010:05, Blekinge: Blekinge Institute of Technology, School of Computing. [12] Shlapak, D., & Johnson, M. (2016), Reinforcing Deterrence on NATO's, Santa Monica: RAND corporation. [13] United Kingdom Ministry of Defense, (2017, August), Wargaming Handbook, Retrieved Јун 9, 2018, from Connections UK: http://professionalwargaming.co.uk/DCDC_Doctrine_UK_ Wargaming.pdf [14] War games research group, (1972), War games rules-Infantry action, Retrieved Јун 19, 2018, from Wargames Research Group Ltd: http://www.wrg.me.uk/WRG.net/History/ OLDWRG/InfantryAction.pdf [14] War games research group, (1972), War games rules-Infantry action, Retrieved Јун 19, 2018, from Wargames Research Group Ltd: http://www.wrg.me.uk/WRG.net/History/ OLDWRG/InfantryAction.pdf [15] War Gaming Department, U.S. Naval War College, (2015), War gamers handbook-a guide for professional wargamers, Retrieved Јун 9, 2018, from Defense Technical Information Center: http://www.dtic.mil/dtic/tr/fulltext/u2/1001766.pdf [15] War Gaming Department, U.S. Literature (2015), Вартена способност беспилотног хеликопте- ра наоружаног невођеним ракетним зрнима у противоклопној борби, SYM-OP-IS 2015 , 450-453. [28] Радиновић, Р. (1983), Метода ратне вештине, Београд: Војноиздавачки завод. [28] Радиновић, Р. (1983), Метода ратне вештине, Београд: Војноиздавачки завод. [29] Савезни секретаријат за народну одбрану, (1981), Тактика борбених дејстава та [28] Радиновић, Р. (1983), Метода ратне вештине, Београд: Војноиздавачки завод. [29] Савезни секретаријат за народну одбрану, (1981), Тактика борбених дејстава так- тичких јединица КоВ и територијалне одбране, Београд: Војноиздавачки завод. [29] Савезни секретаријат за народну одбрану, (1981), Тактика борбених дејстава та тичких јединица КоВ и територијалне одбране, Београд: Војноиздавачки завод. [30] Саковић, Р., & Терзић, М. (2018), Употреба друштвених мрежа у хибридном рато- вању, Београд: Војноиздавачки завод, Војно дело бр. 7. [31] Сећања са FRP, (2013), Retrieved Јун 19, 2018, from Dimmypiano wordpress: https://dimmypiano.wordpress.com/2013/04/27/25/ [32] Стојковић, Д. (2017), Хибридне претње безбедности Републике Србије, Београд: Војноиздавачки завод, Војно дело бр. 6. 25
https://openalex.org/W3048670646	https://www.frontiersin.org/articles/10.3389/fphar.2020.01239/pdf	English	null	Editorial: Big Data, Pharmacogenomics and Real-World Research in Pharmacology	Frontiers in pharmacology	2,020	cc-by	1,372	EDITORIAL published: 13 August 2020 doi: 10.3389/fphar.2020.01239 Editorial on the Research Topic Big Data, Pharmacogenomics and Real-World Research in Pharmacology Citation: Wei JC-C, Chang W-C and Mushiroda T (2020) Editorial: Big Data, Pharmacogenomics and Real-World Research in Pharmacology. Front. Pharmacol. 11:1239. doi: 10.3389/fphar.2020.01239 Keywords: big data, database, real-world study, pharmacogenomics, cohort Keywords: big data, database, real-world study, pharmacogenomics, cohort Editorial: Big Data, Pharmacogenomics and Real-World Research in Pharmacology James Cheng-Chung Wei 1,2,3,4,5, Wei-Chiao Chang 6,7 and Taisei Mushiroda 8 1 Department of Rheumatology, BenQ Medical Center, The Afﬁliated BenQ Hospital of Nanjing Medical University, Nanjing, China, 2 School of Clinical Medicine, Beijing Tsinghua Changgung Hospital, Tsinghua University, Beijing, China, 3 Department of Allergy, Immunology & Rheumatology, Chung Shan Medical University Hospital, Taichung, Taiwan, 4 Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan, 5 Graduate Institute of Integrated Medicine, China Medical University, Taichung, Taiwan, 6 Department of Clinical Pharmacy, School of Pharmacy, Taipei Medical University, Taipei, Taiwan, 7 Integrative Research Center for Critical Care, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan, 8 RIKEN Center for Integrative Medical Sciences (IMS), Yokohama, Japan Big data is characterized by large volume, velocity of volume increase, and variety of information that requires speciﬁc technology and analytical methods to derive useful knowledge for clinical applications. Big data research in biomedical science has the potential to directly affect personalized and precision medical care, reduce costs of treatment, predict out breaks of epidemics, avoid preventable diseases, and improve the quality of life and clinical practice. Through advances in bioinformatics and medical information systems, big data research is now a hot topic in omics approaches and epidemiological studies. Edited and reviewed by: Jose´ A. G. Agu´ ndez, University of Extremadura, Spain Correspondence: James Cheng-Chung Wei jccwei@gmail.com Wei-Chiao Chang wcc@tmu.edu.tw Edited and reviewed by: Jose´ A. G. Agu´ ndez, University of Extremadura, Spain Correspondence: James Cheng-Chung Wei jccwei@gmail.com Wei-Chiao Chang wcc@tmu.edu.tw A parallel trend with big data and clinical researches is the development of real-world studies (RWS). The health authorities had increasingly recognized the critical role of high-volume, real-world data, including electronic medical data, post-marketing surveillance, and claim-based databases, as an important reference of drug approval and pharmacovigilance. With the increasing volume, velocity, and variety of information, the trend of RWS is reaching the big data level. Specialty section: This article was submitted to Pharmacogenetics and Pharmacogenomics, a section of the journal Frontiers in Pharmacology Received: 09 July 2020 Accepted: 29 July 2020 Published: 13 August 2020 Citation: Wei JC-C, Chang W-C and Mushiroda T (2020) Editorial: Big Data, Pharmacogenomics and Real-World Research in Pharmacology. Front. Pharmacol. 11:1239. doi: 10.3389/fphar.2020.01239 Specialty section: This article was submitted to Pharmacogenetics and Pharmacogenomics, a section of the journal Frontiers in Pharmacology Specialty section: This article was submitted to Pharmacogenetics and Pharmacogenomics, a section of the journal Frontiers in Pharmacology In this topic “Big Data, Pharmacogenomics and Real-World Research in Pharmacology”, we aimed for studies of pharmacogenomics and pharmacogenetics using big data approaches, studies of real-world registry or cohort studies in therapeutics, claim-based health database, and omics-level big data studies. We received 29 manuscripts globally from March 2019 to January 2020. Finally, 16 manuscripts were accepted for publication and 13 were rejected. The acceptance rate was 55%. We herein thank all authors and reviewers’ great contributions to this important topic. Some manuscripts are highlighted below. Received: 09 July 2020 Accepted: 29 July 2020 Published: 13 August 2020 REAL-WORLD STUDIES Real-world evidence on a big data level can compensate the deﬁcit of clinical trials, especially on the long-term effectiveness and safety of drugs. In this issue, Wessie et al. described the use of opioids August 2020 \| Volume 11 \| Article 1239 Frontiers in Pharmacology \| www.frontiersin.org Editorial: Big Data, Pharmacogenomics and RWS Wei et al. BIG DATA AND ARTIFICIAL INTELLIGENCE STUDIES increases with age in older adults from the Nivel Primary Care Database, which includes 283,600 patients in the Netherlands from 2005 to 2017. Fernandez et al. reported the off-label use of antineoplastic in oncology is limited but has notable scientiﬁc support in a university hospital setting. Tsai T-L et al. disclosed the association between the usage of colchicine and pneumonia in a nationwide, population-based cohort study. Yeh et al. reported the relationship of the usage of statin and vital organ failure in patients with asthma-chronic obstructive pulmonary disease overlap in a time-dependent population-based study. They nicely demonstrated that statin use was associated with vital organ failure, including the heart, lung, and renal failures in patients with asthma-chronic obstructive pulmonary disease overlap. We appreciated Ji et al. who demonstrated the effectiveness of subcutaneous tumor necrosis factor inhibitors in patients with ankylosing spondylitis (AS) from 804 patients with AS in China. Merging big data and artiﬁcial intelligence would be an even more powerful tool in biomedical research. For example, Mo et al. successfully used machine learning technique to predict clinical response of methotrexate treatment in juvenile idiopathic arthritis patients. Noguchi et al. did a nice review of statistical methodologies for detecting drug–drug interactions by using spontaneous reporting systems. Lee et al. developed a proteotranscriptomic-based computational drug-repositioning method for Alzheimer’s disease (AD) that might be a shortcut to discover new efﬁcacy of drugs for AD. Zamami et al. searched for therapeutic agents for cardiac arrests by using “TargetMine”, a drug discovery tool and large-scale medical information database. They extracted data from the Japan Medical Data Center (JMDC) claims database and found that isosorbide dinitrate, nitroglycerin, and nicardipine may be novel therapeutic agents to improve prognosis of cardiac arrest patients. BIG DATA AND REGISTRY STUDIES Overall, we believed the topic, “Big Data, Pharmacogenomics and Real-World Research in Pharmacology”, is a fruitful collection of big data and real-world studies. We wish the readers of Frontiers in Pharmacology would enjoy it. Several big data studies were retrieved from the National Taiwan Insurance Research Database (NHIRD) (Davis and Huang, 2008), a 20-year nationwide, population-based dataset (Hsing and Ioannidis, 2015; Wu and Lee, 2016). Wei et al. reported an increased risk of sulpiride-induced parkinsonism in patients with peptic ulcer and gastroesophageal reﬂux disease. Lin et al. found that ﬂunarizine use might induce parkinsonism in patients with migraine. Tsai S-H et al. described a long-term evidence of incidence of hypothyroidism associated with surgical procedures for thyroid disorders. Big data is also a powerful tool to investigate healthcare costs and utilization (Hsu et al., 2018). For example, Chen et al. published a comparison of healthcare costs and utilization in rheumatoid arthritis (RA) patients receiving biological and conventional synthetic disease- modifying drugs, which shows a solid pharmaco-economic evidence of RA therapies. AUTHOR CONTRIBUTIONS All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. ACKNOWLEDGMENTS The Topic Editors thank all authors for submitting their work, the reviewers, and the handling editors. REFERENCES 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. Davis, K., and Huang, A. T. (2008). Learning from Taiwan: experience with universal health insurance. Ann. Intern Med. 148 (4), 313–314. doi: 10.7326/ 0003-4819-148-4-200802190-00011 Copyright © 2020 Wei, Chang and Mushiroda. 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. Hsing, A. W., and Ioannidis, J. P. (2015). Nationwide Population Science: Lessons From the Taiwan National Health Insurance Research Database. JAMA Intern Med. 175 (9), 1527–1529. doi: 10.1001/jamainternmed.2015.3540 Hsu, J. C., Wu, H. C., Feng, W. C., Chou, C. H., Lai, E. C., and Lu, C. Y. (2018). Disease and economic burden for rare diseases in Taiwan: A longitudinal study using Taiwan’s National Health Insurance Research Database. PLoS One 13 (9), e0204206. doi: 10.1371/journal.pone.0204206 Wu, Y. T., and Lee, H. Y. (2016). National Health Insurance database in Taiwan: A resource or obstacle for health research? Eur. J. Intern Med. 31, e9–e10. doi: 10.1016/j.ejim.2016.03.022 August 2020 \| Volume 11 \| Article 1239 Frontiers in Pharmacology \| www.frontiersin.org
https://openalex.org/W3094566441	https://www.e3s-conferences.org/10.1051/e3sconf/202019603004/pdf	English	null	Principles of organizing earthquake forecasting based on multiparameter sensor-WEB monitoring data	E3S web of conferences	2,020	cc-by	6,721	Principles of organizing earthquake forecasting based on multiparameter sensor-WEB monitoring data Sergey Pulinets1,2, Dimitar Ouzounov3, Dmitry Davidenko4 and Pavel Budnikov5 1Institute of Applied Physics RAS, 603950, 46 Ulianova str., Nizhni Novgorod, Russia 2Space Research Institute RAS, 117998, 84/32 Profsoyuznaya str., Moscow, Russia 3CEESMO, Chapman University, One University Drive, Orange CA 92866, USA 4Korolev Rocket and Space Public Corporation Energia, Korolev, Moscow region, Russia 5Fedorov Institute of Applied Geophysics, 129128, 9 Rostokinskaya str., Moscow, Russia Abstract. The paper describes an approach that allows, basing on the data of multiparameter monitoring of atmospheric and ionospheric parameters and using ground-based and satellite measurements, to select from the data stream a time interval indicating the beginning of the final stage of earthquake preparation, and finally using intelligent data processing to carry out a short-term forecast for a time interval of 2 weeks to 1 day before the main shock. Based on the physical model of the lithosphere-atmospheric- ionospheric coupling, the precursors are selected, the ensemble of which is observed only during the precursory periods, and their identification is based on morphological features determined by the physical mechanism of their generation, and not on amplitude selection based on statistical data processing. Basing on the developed maquette of the automatic processing service, the possibility of real-time monitoring of the situation in a seismically active region will be demonstrated using the territory of the Kamchatka region and the Kuril Islands. © 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/). Corresponding author: pulse@rssi.ru E3S Web of Conferences 196, 03004 (2020) STRPEP 2020 E3S Web of Conferences 196, 03004 (2020) STRPEP 2020 https://doi.org/10.1051/e3sconf/202019603004 1 Precursory period identification At the same time, efforts were made not only to develop technology for the unambiguous identification of precursors, but also to automate this process. That would allow real-time precursor monitoring [7]. It should be noted that the procedure for identifying precursors is based not on the assessment of the amplitude of the deviation of the ionospheric parameters from the unperturbed value, but on the recognition of their morphological characteristics due to the physical mechanism of their generation. The process actually boils down to the recognition of the precursor image, where, for example, in the case of ionospheric precursors, an image was developed  the “mask” of the precursor, which manifests itself in the form of a strong positive variation of the electron concentration over the earthquake preparation zone [8]. This is the only parameter for the interpretation of which human intervention is required, since it is the image that is recognized in the form of a “stalactite-stalagmite” structure - vertical structures on the “mask” image corresponding to the evening and morning positive variations (Fig. 1). that the combination of both approaches is quite acceptable, moreover, it is natural. Here's a simple analogy. An egg lying on the edge of a table is always in danger of falling to the floor. We do not know when and for what reason this will happen, whether from a sharp stream of air when opening the door of the apartment, or whether a person passing by will touch him with the hollow clothes, or a small child will be interested in a white ball lying on the table. But when this happens, the end is inevitable, the egg will fall to the floor and break. So, the period of time until the egg flies from the edge of the table to the floor, we can call the precursor period, meaning the presence of short-term precursors of earthquakes. In this case, the main task is the timely detection and identification of those precursors that are characteristic to a period of time when a return to a state of equilibrium is impossible, a state of irreversibility. In fact, all the studies of physical precursors in recent years, conducted by our group, were aimed at identifying the most reliable precursors (and their combinations), which would unequivocally indicate the approach of an earthquake within the next few days from the moment the precursor was discovered. 1 Precursory period identification In [1], short-term precursors of earthquakes are listed, the generation of which is due to the development of a cascade process of the Earth's outer geo-shells coupling and which we use to create a short-term forecast (if data on these parameters are available). As shown in [2]. the processes that cause the generation of these precursors are genetically related to processes in the earth's crust and used in seismology as precursors, in particular, a decrease in the value of the parameter b in the Gutenberg-Richter relation [3], foreshocks [4], etc. At the same time, continuously there are discussions about the problem of correlation between the probabilistic approach to forecasting and determinism conditioned by the presence of precursors. In comparison with the uncompromising position of the seismologists of the 90s of the last century [5], the positions of the convergence of seismologists and scientists engaged in the study of physical precursors of earthquakes have emerged [6]. It seems to us https://doi.org/10.1051/e3sconf/202019603004 E3S Web of Conferences 196, 03004 (2020) STRPEP 2020 that the combination of both approaches is quite acceptable, moreover, it is natural. Here's a simple analogy. An egg lying on the edge of a table is always in danger of falling to the floor. We do not know when and for what reason this will happen, whether from a sharp stream of air when opening the door of the apartment, or whether a person passing by will touch him with the hollow clothes, or a small child will be interested in a white ball lying on the table. But when this happens, the end is inevitable, the egg will fall to the floor and break. So, the period of time until the egg flies from the edge of the table to the floor, we can call the precursor period, meaning the presence of short-term precursors of earthquakes. In this case, the main task is the timely detection and identification of those precursors that are characteristic to a period of time when a return to a state of equilibrium is impossible, a state of irreversibility. In fact, all the studies of physical precursors in recent years, conducted by our group, were aimed at identifying the most reliable precursors (and their combinations), which would unequivocally indicate the approach of an earthquake within the next few days from the moment the precursor was discovered. 1 Precursory period identification At the same time, efforts were made not only to develop technology for the unambiguous identification of precursors, but also to automate this process. That would allow real-time precursor monitoring [7]. It should be noted that the procedure for identifying precursors is based not on the assessment of the amplitude of the deviation of the ionospheric parameters from the unperturbed value, but on the recognition of their morphological characteristics due to the physical mechanism of their generation. The process actually boils down to the recognition of the precursor image, where, for example, in the case of ionospheric precursors, an image was developed  the “mask” of the precursor, which manifests itself in the form of a strong positive variation of the electron concentration over the earthquake preparation zone [8]. This is the only parameter for the interpretation of which human intervention is required, since it is the image that is recognized in the form of a “stalactite-stalagmite” structure - vertical structures on the “mask” image corresponding to the evening and morning positive variations (Fig. 1). Fig. 1. Averaged for the period 2006-2011 ionospheric precursor mask of GPS TEC variations during the preparation of earthquakes with М≥6 in Greece according to the data of the noa1 receiver Fig. 1. Averaged for the period 2006-2011 ionospheric precursor mask of GPS TEC variations during the preparation of earthquakes with М≥6 in Greece according to the data of the noa1 receiver Fig. 1. Averaged for the period 2006-2011 ionospheric precursor mask of GPS TEC variations during the preparation of earthquakes with М≥6 in Greece according to the data of the noa1 receiver 2 E3S Web of Conferences 196, 03004 (2020) STRPEP 2020 https://doi.org/10.1051/e3sconf/202019603004 In fact, this is an ideal picture, and characteristic, only for this region, because nighttime positive variations can appear not only a day before the earthquake, but also several days (up to 10), and not only during one night, but several nights in a row. In Fig. 2 shows the mask of ionospheric variations at Tsukuba station during the preparation of the M9.1 mega- earthquake in Japan on March 11, 2011. Fig. 2. Ionospheric precursor mask for the Tohoku earthquake, Japan, March 11, 2011 according to the Tsukuba GPS receiver Fig. 2. 1 Precursory period identification Ionospheric precursor mask for the Tohoku earthquake, Japan, March 11, 2011 according to the Tsukuba GPS receiver As one can see, positive nighttime variations begin to appear a week before the earthquake and continue until the event itself and the next night after it. The question arises as to what caused the positive variations during the day (we see them between the morning and evening variations). The thing is that the whole week before the Tohoku earthquake and on the day of the earthquake itself was characterized by very high solar and geomagnetic activity. From February 27 (50 DOY) to March 8 (67 DOY), the solar radio flux index F10.7 increased from 90 to 155, and, as is known, there is a very high correlation between the total electron content and F10.7, with the electron content lagging by about 2 days in relation to F10.7 [9]. In addition, two moderate magnetic storms took place on March 3 and 11. These two factors contributed to the daytime TEC disturbances. Variations of F10.7 and the global equatorial geomagnetic index Dst are shown in Fig. 3. Fig. 3. Left panel - variations of the F10.7 index, right panel - variations of the Dst index Fig. 3. Left panel - variations of the F10.7 index, right panel - variations of the Dst index It was mentioned above that the precursor period is more reliably distinguished using a combination of precursors. As such a combination, we use variations in the total electron content (or the critical frequency foF2 in the presence of an ionosonde inside the earthquake preparation zone), anomalous flux of outgoing infrared radiation in the long-wavelength frequency range OLR [10] and corrections for the chemical potential of water vapor in the 3 3 E3S Web of Conferences 196, 03004 (2020) STRPEP 2020 https://doi.org/10.1051/e3sconf/202019603004 surface layer of the atmosphere [11]. Both of these parameters are associated with the process of ionization of the surface layer of the atmosphere by alpha particles emitted by radon during the intensification of its release from the earth's crust before an earthquake. Condensation of water vapor occurs on the new formed ions, and latent heat is released, which is the source of OLR. Hydration of ions leads to a decrease in relative humidity and an increase in air temperature, causing a failure of the equilibrium state of the functions of temperature and pressure in the atmosphere [12]. 1 Precursory period identification surface layer of the atmosphere [11]. Both of these parameters are associated with the process of ionization of the surface layer of the atmosphere by alpha particles emitted by radon during the intensification of its release from the earth's crust before an earthquake. Condensation of water vapor occurs on the new formed ions, and latent heat is released, which is the source of OLR. Hydration of ions leads to a decrease in relative humidity and an increase in air temperature, causing a failure of the equilibrium state of the functions of temperature and pressure in the atmosphere [12]. p p [ ] Fig. 4 shows the dynamics of the spatial distribution of anomalies of the outgoing flow of long-wave infrared radiation OLR during the preparation of the Tohoku earthquake [13]. Fig. 4 E_index of the rate of rise of the flow OLR from 1 to 12 March 2011 over the region of the Honshu island Fig. 4 E_index of the rate of rise of the flow OLR from 1 to 12 March 2011 over the region of the Honshu island One can see that the anomalies appear on March 7, 4 days before the earthquake and continue until March 12, and the maximum values of the index are recorded on March 10 and 12. In units of the ordinal day of the year, these are 66-71 days. Now let's turn to the variations of the chemical potential correction. Unfortunately, satellite data with a sufficiently high time resolution (3 hours) appeared only in September 2011, so we will use the data of the Ishinomaki ground based meteorological station (Fig. 5). Quite a distinct maximum is observed on March 8 (67 DOY). One of the well-proven precursors revealed by multi-year analysis of precursors is the cross-correlation coefficient between ionospheric monitoring data (ground-based ionosondes or GPS receivers) for stations located at different distances from the epicenter [14]. For the Tohoku case, we used data from two Japanese ionosondes: Kokubunji near the epicenter and Yamagawa in the very south of Japan, Kyushu island. As an earthquake approaches, the 4 https://doi.org/10.1051/e3sconf/202019603004 E3S Web of Conferences 196, 03004 (2020) STRPEP 2020 cross-correlation coefficient according to [14] should decrease, which we observe in Fig. 6. We see that the minimum is also reached on March 8 (67 DOY). Fig. 5. Variations in the chemical potential correction from the Ishinomaki Meteorological Observatory, Japan Fig. 5. 2 Determination of the magnitude and position of the epicenter of an earthquake As the data of long-term researchers of various groups working in the field of monitoring of the physical precursors of earthquake show, the assessment of the earthquake preparation zone obtained back in 1979 [15] has been brilliantly confirmed in practice. Surprisingly, it was obtained in those times when remote sensing data did not exist, and an empirical estimate was made from ground-based point measurements and an estimate of the zone of elastic deformation at the level10-8. Using the Dobrovolsky formula, we can determine the magnitude of the future earthquake as M = [log (R)]/0.43, where R is the radius of the preparation zone in kilometers. It turned out that using remote sensing data, this area can literally be seen. There is a technology for detecting surface thermal anomalies, which allows building a map of their distribution in real time [16]. In Fig. 7 are shown two similar distributions for two earthquakes with different magnitudes: the left panel is the earthquake in Aquila M6.3 on April 6, 2009, the right panel is the earthquake in Gujarat, India M7.7 on January 26, 2001 [1]. In the figure, the preparation zone is marked in blue according to the Dobrovolsky formula, R = 100.43M, and in red, the so-called activation zone according to [17] R = 100.44M. Fig. 7. Left panel - surface heat anomalies recorded before the earthquake in L'Aquila, Italy М6.3, 04/06/2009, right panel - before the earthquake in Gujarat, India М7.7 01/26/2001. Fig. 7. Left panel - surface heat anomalies recorded before the earthquake in L'Aquila, Italy М6.3, 04/06/2009, right panel - before the earthquake in Gujarat, India М7.7 01/26/2001. As one can see, the anomalies practically do not go beyond the boundaries of the designated zones, although they do not completely fill the circle (which corresponds to the definition of a preparation zone). However, the disadvantage of this technology is that anomalies of this type are visible only in cloudless weather, which imposes serious restrictions on its application. At the same time, this is a great opportunity to visualize and prove the reality of the concept of an earthquake preparation zone. As it turned out, the scale of ionospheric variations before earthquakes also has an order of magnitude determined by the Dobrovolsky formula. 1 Precursory period identification Variations in the chemical potential correction from the Ishinomaki Meteorological Observatory, Japan Fig. 6. Variations in the cross-correlation coefficient between the daily changes in the critical frequency foF2 at Kokubunji and Yamagawa stations in the period from February 22 to March 24, 2011. Fig. 6. Variations in the cross-correlation coefficient between the daily changes in the critical frequency foF2 at Kokubunji and Yamagawa stations in the period from February 22 to March 2 2011. Fig. 6. Variations in the cross-correlation coefficient between the daily changes in the critical frequency foF2 at Kokubunji and Yamagawa stations in the period from February 22 to March 24, 2011. Let us summarize. To determine the precursor period, we have data from four sources: variations in the GPS TEC (63-71 DOY), variations in the OLR E_index (66-71 DOY), variations in the chemical potential (67 DOY), and variations in the cross-correlation coefficient (67 DOY). We can say with confidence that the main day giving grounds for a short-term forecast is March 8 (3 days before the earthquake), and we can determine the precursor period (for reliability, we take the coincidence of OLR and GPS TEC) days from March 7 to 11, 2011. The precursor period is a reference point for determining the date of an earthquake. To improve the accuracy, a multi-year analysis of short-term precursors in the selected region is required, from which we choose the interval between the day of the 5 E3S Web of Conferences 196, 03004 (2020) STRPEP 2020 https://doi.org/10.1051/e3sconf/202019603004 earthquake and the first day of the precursor period. As we can see, for Greece this period is very short - 1 day, and for Italy it is 5-6 days. For different regions, this interval can be from 10 to 1 day. Therefore, when making a forecast, a period of time typical for a given region is indicated, and is determined from multi-year observation data. earthquake and the first day of the precursor period. As we can see, for Greece this period is very short - 1 day, and for Italy it is 5-6 days. For different regions, this interval can be from 10 to 1 day. Therefore, when making a forecast, a period of time typical for a given region is indicated, and is determined from multi-year observation data. 2 Determination of the magnitude and position of the epicenter of an earthquake The third parameter that makes it possible to estimate the size of the earthquake preparation zone is the spatial distribution of the chemical potential [2]. In the Fig. 9 an example is shown from the publication [2] where the estimation of the earthquake preparation zone for the M6.4 earthquake on March 20, 2016 off the eastern coast of Kamchatka is given. However, it is possible to assess the magnitude of an earthquake not by the geometric factors of the registered precursors, but based on the physical analysis of the dynamics of the precursor before the earthquake. This is the OLR heat flow. The rapid increase in radiation and a transient change in OLR were recorded at the top of the atmosphere over seismically active regions. In the first approximation, we can define the atmospheric anomaly in the Euler frame of reference by subtracting the mean value. The mean can be defined as the average for the same day of the year, local time, and location over more than 12 years (larger than 11 years, one solar cycle). The advantage of this approach is its simplicity and effectiveness with However, it is possible to assess the magnitude of an earthquake not by the geometric factors of the registered precursors, but based on the physical analysis of the dynamics of the precursor before the earthquake. This is the OLR heat flow. The rapid increase in radiation and a transient change in OLR were recorded at the top of the atmosphere over seismically active regions. In the first approximation, we can define the atmospheric anomaly in the Euler frame of reference by subtracting the mean value. The mean can be defined as the average for the same day of the year, local time, and location over more than 12 years (larger than 11 years, one solar cycle). The advantage of this approach is its simplicity and effectiveness with 2 Determination of the magnitude and position of the epicenter of an earthquake Maps of the distribution of ionospheric anomalies can be built both according to the data of local networks of stationary GPS/GLONASS receivers (in the case of a moderate earthquake magnitude), and according to the IONEX index, which present ready-made maps of the global distribution of the total electron content, but with low spatial resolution and restrictions, superimposed by heterogeneous distribution of IGS network receivers. 6 6 https://doi.org/10.1051/e3sconf/202019603004 E3S Web of Conferences 196, 03004 (2020) STRPEP 2020 Despite the low resolution of IONEX maps (also called GPS GIM), today they are the most suitable option in terms of data availability and efficiency. IGS data in IONEX format is a matrix whose elements are TEC values multiplied by 10. The matrix resolution is 2.5 degrees in latitude and 5 degrees in longitude. TEC values are calculated by IGS every 2 hours (at present, the transition to a time resolution of 1 hour is underway). The calculation and construction of differential maps of the global TEC TECGIM, which represent the deviation of the current TECGIM of the TEC values from the background TECGIMA, is performed according to the formula: TECGIM = TECGIM – TECGIMA. The deviation from the background value is expressed in TECU units. As an example, Fig. 8 shows a difference map obtained on March 8, 2011 (the maximum of ionospheric deviations before the Tohoku earthquake). For a magnitude of 9.1, the preparation zone radius will be ~ 8200 km. In fact, we are talking about global variation in the ionosphere, which occupies half of the earth's global longitude range. Fig. 8. Differential map of total electron content, obtained from the data of the IONEX index, 08.03.2011, 06:00 UT. The Tohoku earthquake epicenter is marked with a white cross Fig. 8. Differential map of total electron content, obtained from the data of the IONEX index, 08.03.2011, 06:00 UT. The Tohoku earthquake epicenter is marked with a white cross The third parameter that makes it possible to estimate the size of the earthquake preparation zone is the spatial distribution of the chemical potential [2]. In the Fig. 9 an example is shown from the publication [2] where the estimation of the earthquake preparation zone for the M6.4 earthquake on March 20, 2016 off the eastern coast of Kamchatka is given. 7 7 https://doi.org/10.1051/e3sconf/202019603004 E3S Web of Conferences 196, 03004 (2020) STRPEP 2020 the availability of long-time satellite observations. Following this, the OLR anomalous variations were defined as an E_index [18]. Fig. 9. Distribution of the chemical potential correction 10 days before the M6.4 earthquake on March 20, 2016 off the eastern coast of Kamchatka. The star marks the position of the future epicenter. Fig. 9. Distribution of the chemical potential correction 10 days before the M6.4 earthquake on March Fig. 9. Distribution of the chemical potential correction 10 days before the M6.4 earthquake on March 20, 2016 off the eastern coast of Kamchatka. The star marks the position of the future epicenter. This index is similar to the definition of an anomalous thermal field proposed by Tramutoli et al. 2003 with the two significant differences: (1) the analyses are in different physical domain - only in the spectral long wave range (8-12 microns) and (2) the OLR field anomaly was computed at the Top of the Atmosphere (TOA) around 300Mb, not on Earth's surface. The E_index was constructed as statically estimated variability in OLR values for specific locations and periods: (1) E_Indexi, j(t) (S(xi, j, yi, j,t)S(xi, j, yi, j,t))/i. j (1) Where: t=1, K days, S (xij, yij ,t) is the current OLR and is the computed mean of the OLR field, defined for multiple years of observations over the same location and same local time, ti,j is the standard deviation. The Magnitude estimation is based on the calculation of the speed of change of E_ind The Magnitude estimation is based on the calculation of the speed of change of E_index. We use the Magnitude Assessment a modified version of E_Index (1), representing the regional calibration of E_Index estimates we apply for the Kamchatka region and defines as: We use the Magnitude Assessment a modified version of E_Index (1), representing the regional calibration of E_Index estimates we apply for the Kamchatka region and defines as: Magnitude Assessment = (A E_Index)/B Magnitude Assessment = (A* E_Index)/B (2) Where A and B are regional calibration coefficients; A – is a mask, mainly defined by the regional seismo-tectonic patterns and frequency of appearance of OLR anomalies for the historical events and B – regional normalization of thermal flux energy, normalizes each of NOAA satellites 15 and 18 – time series of OLR data to the same time coverage (twelve years or more). 7 The Kamchatka region A values vary around 0.5-0.7; B – range 1-3. The 8 https://doi.org/10.1051/e3sconf/202019603004 E3S Web of Conferences 196, 03004 (2020) STRPEP 2020 Magnitude assessment data have been computed with a resolution of 2.5° × 2.5°. The final step includes additional preprocessing to avoid aliasing short wavelengths and spatial filtering based on a “minimum curvature” algorithm [10]. At present, the accuracy of determining the magnitude is 0.5 on the Richter scale. Experience shows that determining the position of the epicenter of a future earthquake is the most difficult task. Actually, we use two methods for determining the position of the epicenter: finding the coordinates of the center of the identified earthquake preparation zone and the position of the OLR anomaly in its maximum development. The distribution of the chemical potential correction shown in Fig. 9 is the exception rather than the rule, and this is natural. Not necessarily the maximum radon flux before an earthquake is observed at a point coinciding with the epicenter of a future earthquake. Then, not always observed anomalies both in the ionosphere and near the earth's surface are symmetrical with respect to the position of the epicenter. The low resolution of the IONEX index (2.5 in latitude and 5 in longitude) should also be noted, which greatly coarsens the accuracy of determining the position of the epicenter from global TEC maps. As one can see from Fig. 4, the OLR anomaly drifts along active tectonic faults and tectonic plate boundaries, but its position of the OLR anomaly at its maximum value is always close to the epicenter position, and the deviation is no more than 2.5-3. In principle, the accuracy of determining the epicenter position from the data on the distribution of the chemical potential also lies within these limits. p Summing up, we can say that at the moment the accuracy of our short-term forecasting technology is: • Time of the earthquake (10-1 days) • Magnitude of an earthquake ± 0.5 on the Richter scale • Position of epicenter 2.5-3 in latitude and longitude It should be noted that OLR has two periods of leading time: middle term 20-5 days, and short term 5-1 days. 3 Creation of a model of automated monitoring and short-term forecasting of strong earthquakes in the Kamchatka region To monitor the state of the atmosphere and ionosphere and identify the precursors of earthquakes in the Kamchatka region, it is required to continuously process data from selected receivers of signals from global navigation satellite systems (GNSS) and a ground- based vertical sounding ionosonde in Petropavlovsk-Kamchatsky simultaneously with data on solar and geomagnetic activity, data from monitoring atmospheric parameters , and only their joint analysis allows us to make an informed conclusion about the possibility of a seismic event in a certain place, at a certain time and with a certain magnitude [1]. The logistics of data processing automation can be conditionally divided into three stages: primary processing, data validation and intelligent processing, as a result of which, in fact, precursors are recognized. This division does not necessarily mean the same sequence in time (some operations can be performed simultaneously), but it helps to understand the process of processing itself. p g Data from receivers of global navigation satellite systems (GPS/GLONASS) in the RINEX format (ftp://garner.ucsd.edu/archive/garner/rinex/; ftp://cddis) are used as input data for monitoring the ionospheric situation over seismic regions. gsfc.nasa.gov/pub/gps/data/daily/; ftp://data-out.unavco.org/pub/rinex/), global ionospheric maps (GIM) of TEC in IONEX format (ftp://cddis. nasa.gov/gps/products/ionex/). In addition to ionospheric data for the analysis of the heliogeomagnetic conditions, it is planned to use data on the geomagnetic conditions - the Ap, Kp and Dst indices 9 E3S Web of Conferences 196, 03004 (2020) STRPEP 2020 https://doi.org/10.1051/e3sconf/202019603004 (https://www.gfz-potsdam.de; http://wdc.kugi.kyoto-u.ac.jp /dst_realtime/index.html) and solar radio flux data F10.7 (ftp://ftp.swpc.noaa.gov/pub/indices/old_indices/), freely available on the Internet and updated in real time. (https://www.gfz-potsdam.de; http://wdc.kugi.kyoto-u.ac.jp /dst_realtime/index.html) and solar radio flux data F10.7 (ftp://ftp.swpc.noaa.gov/pub/indices/old_indices/), freely available on the Internet and updated in real time. Simultaneously with ionospheric data for the implementation of a synergistic approach in accordance with a complex model of lithosphere-atmospheric-ionosphere-magnetospheric coupling [11, 19], it is proposed to use meteorological data (temperature and humidity of atmospheric air), for example, from the site https://meteoinfo.ru/ archive-pogoda, by which a generalized parameter is calculated, called the correction of the chemical potential of water vapor in the atmosphere [11]. If there are other sources of data on the impending earthquake in this region, for example, remote sensing data on thermal anomalies [18], they are also included in the process of intelligent processing. The primary processing phase is the most expensive in terms of machine time, since it involves downloading large amounts of data from various sources simultaneously. 3 Creation of a model of automated monitoring and short-term forecasting of strong earthquakes in the Kamchatka region Typically, data on source servers is stored in a packed form, so it needs to be unpacked, read in the source format, and converted to a format convenient for analysis. In this case, it is necessary to bring data from different sources to a common time scale: data from various geophysical indices can be provided with a time resolution of one hour, three hours, etc. Vertical sounding data can also be both with a resolution of 15 minutes and 1 hour, and etc. At the same time, TEC can be calculated both with a resolution of 15 s, 30 s, and with any given resolution at large time intervals. Equally important is the validation phase. Unfortunately, not all receivers work flawlessly. There are whole periods when corrupted data comes in. The receiver may not work at all for some time, there may be outliers of values far beyond the permissible limits. Sometimes TEC processing algorithms give negative values, which is physically impossible. If all this is put into the recognition system, then we will face severe disappointment. Therefore, already at this stage, it is necessary to use sufficiently intelligent algorithms for recognizing failures in the data stream. Only after bringing the data into proper form we can start machine processing. And, if automatic processing processes have been successfully used for a long time, then machine analysis systems for data containing complex patterns have become popular in geophysics only recently with the development of machine learning methods. At present, the accuracy of data analysis using artificial intelligence methods is not inferior to classical methods [20, 21], while significantly exceeding them in terms of speed and usability. Among the numerous machine learning methods for recognizing anomalies, convolutional neural networks (CNNs) have found the greatest application. The application of these methods is widespread in geological exploration problems [22]. A number of similar machine learning methods have also been used to recognize ionospheric earthquake precursors based on two-dimensional TEC distribution maps [23]. For automated processing of ionospheric monitoring data and analysis of ionospheric precursors, a machine data processing system is proposed (see Fig. 10), which is described below. Processing includes proven algorithms, including: 1. Analysis of the TEC (or foF2) data sets using the pattern recognition method  for the correspondence of the ionospheric precursor mask to the current changes in the ionosphere over the seismically active region [8]. 1. 3 Creation of a model of automated monitoring and short-term forecasting of strong earthquakes in the Kamchatka region Construction of maps of distribution of the correction of the chemical potential according to the data of local temperature and relative humidity to determine the position of the epicenter of the future earthquake and estimate its magnitude [1]. 8. In the case of low-latitude earthquakes, analysis of the dynamics of the equator anomaly (EA) in order to detect the absolute anomaly and the longitudinal effect in EA [1 y y g 9. Multiparameter analysis using operational data on other physical precursors, if a (radon activity, crustal conductivity, OLR, anomalous cloud structures) [1]. ( y y ) [ ] As a result of the analysis, the predicted values of the earthquake magnitude for a given region and their probability are estimated. Based on the forecasts, the final assessment of the probability of an earthquake is made based on machine learning data. This does not exclude the operator's expert judgment. Thus, the system carries out a multivariate analysis of the state of the ionosphere, capable of recognizing a unique image of an earthquake precursor. More detailed description of the process of multiparameter data processing and interpretation is provided in [7]. Fig. 10. Computer processing diagram of geophysical monitoring data for the purpose of automatic identification of earthquake precursors Fig. 10. Computer processing diagram of geophysical monitoring data for the purpose of automatic identification of earthquake precursors The authors thank NASA’s Goddard Space Flight Center and UNAVCO for the GPS (Rinex) and GPS (IONEX) data, NOAA’s Climate Prediction Center (USA), NASA Goddard Earth Science Center (GES 3 Creation of a model of automated monitoring and short-term forecasting of strong earthquakes in the Kamchatka region Analysis of the TEC (or foF2) data sets using the pattern recognition method  for the correspondence of the ionospheric precursor mask to the current changes in the ionosphere over the seismically active region [8]. 2. Correlation analysis of arrays of daily TEC values (or critical frequency foF2) between a pair of adjacent GPS/GLONASS receivers (or ground stations for vertical radio sounding of the ionosphere) [14]. 2. Correlation analysis of arrays of daily TEC values (or critical frequency foF2) between a pair of adjacent GPS/GLONASS receivers (or ground stations for vertical radio sounding of the ionosphere) [14]. 3. In the presence of a dense local network of stationary GPS/GLONASS receivers, the culation of the coefficient of regional variability of the ionosphere can be provided [24]. 10 E3S Web of Conferences 196, 03004 (2020) STRPEP 2020 https://doi.org/10.1051/e3sconf/202019603004 4. Calculation and construction of difference maps of the global TEC TECGIM in order to determine the position of the epicenter of the future earthquake and its magnitude) [1]. If there is a dense local network of fixed GPS/GLONASS receivers, differential maps can be calculated using local data rather than GPSGIM. 4. Calculation and construction of difference maps of the global TEC TECGIM in order to determine the position of the epicenter of the future earthquake and its magnitude) [1]. If there is a dense local network of fixed GPS/GLONASS receivers, differential maps can be calculated using local data rather than GPSGIM. 5. Comparison of variations of the global TEC [25] with the local TEC with reference to the solar activity index F10.7 [1]. 5. Comparison of variations of the global TEC [25] with the local TEC with reference to the solar activity index F10.7 [1]. 6. Calculation of the correction of the chemical potential of water vapor according to the local temperature and relative humidity data to determine the time of a seismic event [11]. 6. Calculation of the correction of the chemical potential of water vapor according to the local temperature and relative humidity data to determine the time of a seismic event [11]. 7. Construction of maps of distribution of the correction of the chemical potential according to the data of local temperature and relative humidity to determine the position of the epicenter of the future earthquake and estimate its magnitude [1]. 7. 4 Conclusion The proposed approach is based on multi-years experience in analysing both ionospheric precursors of earthquakes and other physical precursors, which together create a generalized image of the final stage of preparation of strong earthquakes. The creation of the proposed system for processing ionospheric monitoring data and analysing ionospheric precursors will make it possible to create an earthquake prediction service capable of recognizing and identifying geophysical variations that are precursors of earthquakes in an automatic mode. The authors thank NASA’s Goddard Space Flight Center and UNAVCO for the GPS (Rinex) and GPS (IONEX) data, NOAA’s Climate Prediction Center (USA), NASA Goddard Earth Science Center (GES 11 https://doi.org/10.1051/e3sconf/202019603004 E3S Web of Conferences 196, 03004 (2020) STRPEP 2020 DAAC) for OLR data and the International Research Institute for Climate and Society for SLHF data, WDC Kyoto, GFZ-Potsdam and SWPC NOAA for geophysical indices data. Special thanks go to the US Geological Survey and European‐Mediterranean Seismological Centre for providing earthquake information services and data. The work of S. Pulinets and D. Davidenko was supported by the Russian Science Foundation, project no. 18-12-00441. References 1. S. Pulinets, D. Ouzounov, The possibility of earthquake forecasting. Learning from Nature (IOP Publishing, Bristol, 2018) 2. S. Pulinets, D. Ouzounov, D. Davidenko, A. Petrukhin, E3S Web of Conf. 11, 00019 (2016) 3. D. Schorlemmer, S. Wiemer, M. Wyss, JGR, 109, B12307, (2004) 4. G.A. Papadopoulos, M. Charalampakis, A. Fokaefs, G. Minadakis, Nat. Haz. Earth Syst. Sci., 10, 19 (2010) 5. R.J. Geller, Geophys. J. Int., 131, 425 (1997) 6. P. Shebalin, Combining probabilistic seismicity models with precursory information, in Pre‐Earthquake Signals Generation in: Pre‐Earthquake Processes: A Multidisciplinary Approach to Earthquake Prediction Studies, 173 (AGU/Wiley, 2018) 7. S.A. Pulinets, D.V. Davidenko, P.A. Budnikov, Geomagn. Aeronom., 61, 45 (2021) 8. S.A. Pulinets, D.V. Davidenko, Geomagn. Aeronom., 58, 559 (2018) 7. S.A. Pulinets, D.V. Davidenko, P.A. Budnikov, Geomagn. Aeronom., 61, 45 (2021) 7. S.A. Pulinets, D.V. Davidenko, P.A. Budnikov, Geomagn. Aeronom., 61, 45 (2021) 8. S.A. Pulinets, D.V. Davidenko, Geomagn. Aeronom., 58, 559 (2018) 8. S.A. Pulinets, D.V. Davidenko, Geomagn. Aeronom., 58, 559 (2018) 9. N. Bergeot, I. Tsagouri, C. Bruyninx, J. Legrand, J.-M. Chevalier, P. Defraigne, Q. Baire, E. Pottiaux, J. Space Weather Space Clim. 3, A25 (2013) 10. D. Ouzounov, S. Pulinets, M.C. Kafatos, P. Taylor, Thermal Radiation Anomalies Associated with Major Earthquakes in Pre‐Earthquake Signals Generation in: Pre‐ Earthquake Processes: A Multidisciplinary Approach to Earthquake Prediction Studies, 259 (AGU/Wiley, 2018) 11. S.A. Pulinets, D.P.Ouzounov, A.V. Karelin, D.V. Davidenko, Geomagn. Aeronom., 55, 521 (2015) 12. M.A. Sadovsky, Yu.A. Bannov, K.M. Merzoev, S.Kh. Negmatullaev, Earthquake prediction 6, 242 (1985) (in Russian) 13. D. Ouzounov, S. Pulinets, A. Romanov, A. Romanov (Yr), K. Tsybulya, D. Davidenko, M. Kafatos, P.Taylor, Earthq. Sci., 24, 557 (2011) 14. S.A. Pulinets, T.B. Gaivoronska, A. Leyva Contreras, L. Ciraolo, Nat. Haz. Earth Syst..Sci., 4, 697 (2004) 15. I.P. Dobrovolsky, S.I. Zubkov, V.I. Myachkin, Pageoph., 117, 1025 (1979) 16. V. Tramutoli, R. Corrado, C. Filizzola, N. Genzano, M. Lisi, N. Pergola, Bollet. di Geof. Teor. Applic. 56, 167 (2015) 17. D.D. Bowman, G. Ouillon, C.G. Sammis, A. Sornette, D. Sornette, J. Geophys. Res. 103,24359 (1998) 18. D. Ouzounov, D. Liu, C. Kang, G. Cervone, M. Kafatos, P.Taylor, Tectonophysics, 431, 211 (2007) 19. S. Pulinets, D. Ouzounov, A. Karelin, D. 20. Y. Kim, N. The Leading Edge, 37, 866 (2018) References Davidenko, Lithosphere–Atmosphere– Ionosphere–Magnetosphere Coupling  A Concept for Pre‐Earthquake Signals 12 E3S Web of Conferences 196, 03004 (2020) STRPEP 2020 https://doi.org/10.1051/e3sconf/202019603004 Generation in Pre‐Earthquake Signals Generation in: Pre‐Earthquake Processes: A Multidisciplinary Approach to Earthquake Prediction Studies, 77 (AGU/Wiley, 2018) Generation in Pre‐Earthquake Signals Generation in: Pre‐Earthquake Processes: A Multidisciplinary Approach to Earthquake Prediction Studies, 77 (AGU/Wiley, 2018) 20. Y. Kim, N. The Leading Edge, 37, 866 (2018) 21. A. Alipour, J. Yarahmadi, M. Mahdavi, J. Climatology. 2014. Article ID 839205 (2014) 22. J. Sun, S. Slang, T. Elboth, T. Larsen Greiner, S. McDonald, L.-J. Gelius, Geophysics 85 WA13 (2020) 23. O. Arikan, F. Arikan, Machine learning based detection of earthquake precursors using ionospheric data, in Proceedings of 42nd COSPAR Scientific Assembly. Held 14-22 July 2018, in Pasadena, California, USA, Abstract id. C1.4-16-18 18 (2018) 24. S.A. Pulinets, A.N. Kotsarenko, L. Ciraolo, I.A. Adv. Space Res., 39, 970 (2007) 25. E.L Afraimovich., E.I. Astafyeva, A.V. Oinats, Y.V. Yasukevich, I.V. Zhivetiev, Ann. Geophys. 26, 335 (2008) 13
https://openalex.org/W2907668711	https://scindeks-clanci.ceon.rs/data/pdf/2217-8139/2018/2217-81391804037M.pdf	English	null	Application of numerical methods in analysis of timber concrete composite system	Građevinski materijali i konstrukcije	2,018	cc-by-sa	11,975	PRIMENA NUMERIČKIH METODA U ANALIZI SPREGNUTIH KONSTRUKCIJA DRVO- BETON APPLICATION OF NUMERICAL METHODS IN ANALYSIS OF TIMBER CONCRETE COMPOSITE SYSTEM ORIGINALNI NAUČNI RAD ORIGINAL SCIENTIFIC PAPER UDK:624.016.072.2 doi:10.5937/GRMK1804037M Dragan MANOJLOVIĆ Tatjana KOČETOV MIŠULIĆ Aleksandra RADUJKOVIĆ 1 UVOD Kompozitni konstrukcijski sistemi podrazumevaju racionalno spajanje elemenata od odgovarajućih mate- rijala, tako da se optimalno iskoriste njihova svojstva. Spregnute konstrukcije imaju najširu primenu u inže- njerskim konstrukcijama velikog raspona [12], ali mogu se uspešno primeniti i u stambenim i poslovnim objek- tima. Adekvatnim spajanjem konstrukcijskih elemenata istih ili različitih fizičko-mehaničkih karakteristika u integralni poprečni presek, postiže se osnovni cilj postupka, tj. povećava se nosivost sistema u odnosu na pojedinačne elemente. U zavisnosti od primenjenih materijala, spregnute konstrukcije koje se često prime- njuju u građevinarstvu uglavnom su tipa drvo–drvo, beton–beton, čelik–beton i drvo-beton. Composite construction systems consider the rational structural composition of the right materials at the right places in order to optimally exploit their properties. Composite structures have the widest application in large-span engineering constructions [12], but they can be applied successfully in residential and commercial buildings. By adequate coupling of the constructive elements of the same or different physical- mechanical characteristics into an integral cross-section, the basic goal of the procedure is achieved, i.e. the capacity of the system is increased in relation to the individual elements. Depending on the applied materials, composite structures that are often in use in the construction industry generally are timber-timber, concrete-concrete, steel-concrete and timber-concrete. Budući da su spregnute konstrukcije – napravljene od različitih materijala i različitim načinima spajanja – dostigle veoma visok stepen primene u građevinarstvu u poslednjih nekoliko decenija, neophodna je njihova preciznija analiza, kao i preciznije projektovanje. Poznato je da upotrebljeni tip sredstava za sprezanje najviše utiče na globalno ponašanje spregnutih konstrukcija. Stoga, od ključnog značaja je to kako da se uvede problem ponašanja veze između spregnutih materijala u analizi i proračunu. Since the composite structures, made by different materials and methods of joining, have reached very high level of application in the construction industry in last several decades, there is a demand for their more precise analysis and design. It is known that the type of used fasteners mostly influence the overall behaviour of the coupled structures. Therefore, it is of crucial impor- tance how to introduce the problem of the connection behaviour between coupled materials into the analysis and design. Dragan Manojlovic, MSc, civ.eng., Department of Civil Engineering and Geodesy, Faculty of Technical Sciences, University of Novi Sad, e-mail: manojlovic.dragan@uns.ac.rs Tatjana Kocetov-Misulic, PhD, civ.eng., Department of Civil Engineering and Geodesy, Faculty of Technical Sciences, University of Novi Sad, e-mail: tanya@uns.ac.rs Aleksandra Radujkovic, PhD, civ.eng., Department of Civil Engineering and Geodesy, Faculty of Technical Sciences, University of Novi Sad, e-mail: leksa@uns.ac.rs Dragan Manojlović, MSc, dig, Departman za građevinarstvo i geodeziju, Fakultet tehničkih nauka, Univerzitet u Novom Sadu, e-mail: manojlovic.dragan@uns.ac.rs Tatjana Kočetov-Mišulić, Dr, dig, Departman za građevinarstvo i geodeziju, Fakultet tehničkih nauka, Univerzitet u Novom Sadu, e-mail: tanya@uns.ac.rs Aleksandra Radujković, Dr, dig, Departman za građevinarstvo i geodeziju, Fakultet tehničkih nauka, Univerzitet u Novom Sadu, e-mail: leksa@uns.ac.rs 1 UVOD Dragan Manojlović, MSc, dig, Departman za građevinarstvo i geodeziju, Fakultet tehničkih nauka, Univerzitet u Novom Sadu, e-mail: manojlovic.dragan@uns.ac.rs Tatjana Kočetov-Mišulić, Dr, dig, Departman za građevinarstvo i geodeziju, Fakultet tehničkih nauka, Univerzitet u Novom Sadu, e-mail: tanya@uns.ac.rs Aleksandra Radujković, Dr, dig, Departman za građevinarstvo i geodeziju, Fakultet tehničkih nauka, Univerzitet u Novom Sadu, e-mail: leksa@uns.ac.rs Dragan Manojlovic, MSc, civ.eng., Department of Civil Engineering and Geodesy, Faculty of Technical Sciences, University of Novi Sad, e-mail: manojlovic.dragan@uns.ac.rs Tatjana Kocetov-Misulic, PhD, civ.eng., Department of Civil Engineering and Geodesy, Faculty of Technical Sciences, University of Novi Sad, e-mail: tanya@uns.ac.rs Aleksandra Radujkovic, PhD, civ.eng., Department of Civil Engineering and Geodesy, Faculty of Technical Sciences, University of Novi Sad, e-mail: leksa@uns.ac.rs Dragan Manojlovic, MSc, civ.eng., Department of Civil Engineering and Geodesy, Faculty of Technical Sciences, University of Novi Sad, e-mail: manojlovic.dragan@uns.ac.rs Tatjana Kocetov-Misulic, PhD, civ.eng., Department of Civil Engineering and Geodesy, Faculty of Technical Sciences, University of Novi Sad, e-mail: tanya@uns.ac.rs Aleksandra Radujkovic, PhD, civ.eng., Department of Civil Engineering and Geodesy, Faculty of Technical Sciences, University of Novi Sad, e-mail: leksa@uns.ac.rs GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 61 (2018) 4 (37-53) BUILDING MATERIALS AND STRUCTURES 61 (2018) 4 (37-53) GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 61 (2018) 4 (37-53) BUILDING MATERIALS AND STRUCTURES 61 (2018) 4 (37-53) 37 Coupling of the constitutive elements can be achieved in different ways, where one of the most common procedure is the use of number of individual shear connectors (mechanical fasteners, anchors,...). Shear connectors should ensure bond of two different materials, transferring the shear forces between two elements, enabling the composite action of the structure. The interest of researchers and constructors as well as numerous studies and research works refer to these types of fasteners since the application of dowel type connectors is the most common in timber-concrete composite structures (TCC), and additionally the behaviour of the overall construction depends on their behaviour. The use of mechanical fasteners for coupling two different materials such as timber and concrete shows that the behaviour of the TCC system is very complex, since the fasteners allow certain interlayer slip that leads to partially interaction (elastic composite action). Therefore, the analysis and design of TCC structures requires consideration of the interlayer slip between the sub-elements. Spajanje konstitutivnih elemenata može se postići na različite načine, pri čemu jedan od najčešćih postupaka jeste upotreba diskretno postavljenih sredstava za sprezanje (npr. mehanička spojna sredstva, sidra). 1 UVOD Spojna sredstva treba da obezbede vezu dva različita materijala, prenoseći smičuće sile između dva elementa, s ciljem obezbeđenja kompozitnog sadejstva konstruk- cije. S obzirom na to što je primena štapastih spojnih sredstava najčešća u spregnutim konstrukcijama drvo– beton (SDB), a pošto ponašanje celokupne konstrukcije zavisi od njihovog ponašanja, interes istraživača i projektanata, kao i brojnih studija i istraživačkih radova, odnosi se na ove tipove sredstava za sprezanje. Upotreba mehaničkih spojnih sredstava za sprezanje dva različita materijala, kao što su drvo i beton, ukazuje na to da je ponašanje SDB konstrukcija veoma složeno, budući da spojna sredstva dozvoljavaju izvesno klizanje u spoju koje dovodi do delimične interakcije (elastično sprezanje). Prema tome, analiza i proračun SDB konstrukcija zahteva da se imaju u vidu klizanja u spoju između elemenata. The interest of researchers and constructors as well as numerous studies and research works refer to these types of fasteners since the application of dowel type connectors is the most common in timber-concrete composite structures (TCC), and additionally the behaviour of the overall construction depends on their behaviour. The use of mechanical fasteners for coupling two different materials such as timber and concrete shows that the behaviour of the TCC system is very complex, since the fasteners allow certain interlayer slip that leads to partially interaction (elastic composite action). Therefore, the analysis and design of TCC structures requires consideration of the interlayer slip between the sub-elements. Considering one-dimensional problem, the first theories for partial composite action for beams subjected to static loads were developed by Newmark (1943,1951), Granholm (1949), Pleshov (1952) and Goodman (1967). The application of partial composite action theory was performed by Girhammar and Gopu (1991) in analysis of columns with interlayer slip subjected to one particular axial loading case which was extended and generalized in their further work. Based on previous research and analysis, they presented an exact static analysis of partial composite structures with interlayer slip [7] and afterwards in papers [8] , [9] , [10] they proposed an exact and simplified methods for analysis of the partial composite structures applied to the beams and columns. In Serbia, in the field of timber- concrete composites, the theoretical basis for analysis of partially composite system was given by B.Stevanović (1994) [17] and later on by Lj.Kozaric [11] and R.Cvetkovic [3], which was followed by experimental data. 1 UVOD Razmatrajući jednodimenzionalni problem, prve teorije elastičnog sprezanja kod greda izloženih statičkom dejstvu razvili su Newmark (1943,1951), Granholm (1949), Pleshov (1952) i Goodman (1967). Teoriju elastičnog sprezanja primenili su Girhammar i Gopu (1991) u analizi stubova s klizanjem u spoju, izloženih odgovarajućem slučaju aksijalnog opterećenja, koja je kasnije proširena i generalizovana u njihovom daljem radu. Na osnovu prethodnih istraživanja i analiza, oni su prikazali tačan analitički postupak statičke analize elastično spregnutih nosača s klizanjem u spoju [7], a u narednim radovima [8], [9], [10] predložili su tačne analitičke i pojednostavljene metode za analizu elastično spregnutih sistema s primenom na grede i stubove. U Srbiji, u oblasti sprezanja drvo–beton, teorijske osnove za analizu delimično spregnutih sistema uz eksperimentalne rezultate dao je B. Stevanović (1994) [17], kao i Lj. Kozarić [11] i R. Cvetković [3]. Teorija parcijalnog (elastičnog) sprezanja zasniva se na odgovarajućim pretpostavkama teorije elastičnosti i uzima u obzir klizanje spoja/veze pri njihovom proračunu. Analitički proračun elastičnog sprezanja podrazumeva rešavanje diferencijalnih jednačina, gde se rešenja u zatvorenom obliku mogu formulisati samo za pojedine (jednostavnije) slučajeve konturnih uslova i opterećenja. The theory of partial (elastic) composite action is based on the corresponding assumptions of the theory of elasticity and takes into account the interlayer slip in the connection at their calculation. The exact calculation of the partial composite action implies solving differential equations where closed form solutions can be formulated only for some particular (simple) cases of boundary and loads conditions. p j U EN1995 [5] usvojen je pojednostavljeni manuelni postupak proračuna („γ-metod”), koji se u praksi široko primenjuje. Ovaj metod prvobitno je primenio Mohler (1956), razmatrajući problem klizanja u spoju između spregnutih elemenata (drvo–drvo), s mehaničkim spojnim sredstvima, ali uz odgovarajuće modifikacije, ovaj postupak se može primeniti i na druge tipove spregnutih konstrukcija, kao što su konstrukcije tipa drvo–beton. „Gama” metod razvijen je za statički sistem proste grede izložene sinusoidnom opterećenju q=q0·sin(π·x/L). U ovom slučaju postoji jednostavno rešenje u zatvorenom obliku, koje se može primeniti i na druge vrste opterećenja, a zbog malog odstupanja od tačnog analitičkog rešenja diferencijalne jednačine. Ova metoda zasniva se na efektivnoj krutosti spregnutog sistema i teoriji elastičnog sprezanja, imajući u vidu konzervativni efekat raspodele sila unutar nosača, i skoro u potpunosti pokriva sve parametre koji utiču na y In EN1995 [5] the simplified manual design procedure ("γ-method") widespread in practice is adopted. 1 UVOD Residue methods are in such cases a convenient way to formulate a numerical solution. g j U varijacionoj formulaciji problema, potrebno je naći nepoznatu funkciju ili više funkcija koje zadovoljavaju uslov stacionarnosti funkcionala, gde u ovom slučaju nepoznata funkcija mora da zadovolji odgovarajuće dodatne uslove koji nisu implicitno sadržani u funkcionalu. Da bi se primenila varijaciona formulacija, neophodno je da za razmatrani problem postoji funkcional. In the variational formulation of the problem, it is necessary to find unknown function or several functions that satisfy the requirement of functional stationarity, where the unknown function must also satisfy the corresponding additional conditions that are not implicitly contained in the functional. In order to apply the variational formulation, it is necessary that functional exists for considered problem. Na osnovu diferencijalne i varijacione formulacije problema, razvijene su brojne metode i postupci za određivanje približnih rešenja, pri čemu je od metoda reziduuma najzastupljenija Galerkinova metoda, dok je od varijacionih to Ritz-ova metoda. Numerous methods and procedures for determi- nation of approximate solutions have been developed based on the differential and variational formulation of the problem. The Galerkin method is the most frequently applied one from the residue methods, while the Ritz method is most often used for variational formulation. Metod konačnih elemenata (MKE) jeste jedan od najčešće korišćenih numeričkih metoda u strukturalnoj analizi, pri čemu se formulacija konačnog elementa zasniva na rešenju diferencijalnih jednačina metodama reziduuma ili korišćenjem varijacione formulacije. MKE zasnovana na Galerkinovoj metodi (ili drugim metodama reziduuma) može se primeniti na mnogo širi skup diferencijalnih jednačina, jer nije potrebno imati odgovarajuću varijacionu formu, kao što je slučaj kada se koristi MKE bazirana na Raileigh-Ritz-ovoj metodi [1]. Na osnovu prethodnog izlaganja, može se zaključiti da je primena pojednostavljenih i/ili aproksimativnih numeričkih metoda za analizu i proračun SDB konstrukcija dobrodošla i preporučena. Upravo iz tog razloga, približne metode zasnovane na diferencijalnoj ili varijacionalnoj formulaciji [16] imaju široku primenu, jer mogu biti implementirane u programe za strukturalnu analizu, kako bi se obezbedio poseban alat inženjerima za proračun elastično spregnutih konstrukcija. 1 UVOD This method was originally applied by Mohler (1956), considering the problem of interlayer slip between composite members (timber-timber) coupled with mechanical fasteners, but, with appropriate modifications, this procedure can be applied to the other types of composite constructions such as timber- concrete system. "Gamma" method was developed in the case of simply supported beam subjected to sinusoidal load q=q0·sin(π·x/L). In this case, there is a simple closed-form solution, that could be applied to the other types of loads as well, due to a slight deviation from the exact analytical solution of the differential equation. This method is based on the effective stiffness of the composite system and on the theory of elastic GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 61 (2018) 4 (37-53) BUILDING MATERIALS AND STRUCTURES 61 (2018) 4 (37-53) 38 ponašanje SDB konstrukcija. ponašanje SDB konstrukcija. coupling, taking into account the conservative effect of the distribution of forces within the girders, and so far most fully covers all the parameters that affect the behaviour of TCC. Takođe, za proračun spregnutih sistema, moguće je primeniti aproksimativne metode zasnovane na diferencijalnoj [14] ili varijacionoj formulaciji [13]. Also, for the calculation of composite systems, it is possible to apply approximate methods based on the differential [14] or the variation formulation [13]. Diferencijalna formulacija zasniva se na izvođenju diferencijalnih jednačina koje opisuju problem u određenom domenu, gde rešenje zavisi od graničnih uslova. U rešavanju problema, potrebno je naći nepoznatu funkciju koja će zadovoljiti diferencijalnu jednačinu, kao i granične uslove. Rešavanjem izvedenih diferencijalnih jednačina, dobija se analitičko rešenje problema, gde se rešenja u zatvorenom obliku mogu dobiti samo za ograničen broj jednostavnih proračunskih modela. Ako je proračunski model kompleksan, tada se najčešće primenjuju aproksimativne metode, pogodne za dobijanje prihvatljivog rešenja. Metode reziduuma u takvim slučajevima jesu pogodan način za formulisanje numeričkog rešenja. The differential formulation is based on the derivation of differential equations that describe the problem in a particular domain, where the solution depends on the boundary conditions. In solving the problem, it is necessary to find unknown function that satisfies differential equation as well as the boundary conditions. By solving the derived differential equations, an analytical solution of the problem arises, where the closed-form solution can be obtained only for a limited number of simpler design models. If the design model is complex, then the approximate methods are most commonly used and suitable for obtaining an acceptable solution. 2 GOVERNING EQUATION OF TCC SYSTEM The theory of elastic coupling [17], [11] is used for the calculation of TCC structures, where mechanical fasteners are used. Za proračun spregnutih nosača od drveta i betona, gde se koriste mehanička spojna sredstva, primenjuje se teorija elastičnog sprezanja [17], [11]. The basic assumptions of the theory of elasticity that are introduced are as follows: Osnovne pretpostavke teorije elastičnosti koje se uvode jesu sledeće: Osnovne pretpostavke teorije elastičnosti koje se uvode jesu sledeće: • drvo i beton su izotropni, elastični materijali – važi Hukov zakon; • drvo i beton su izotropni, elastični materijali – važi Hukov zakon; • timber and concrete are isotropic, elastic materials and Hook's law applies, • važi Bernulijeva hipoteza, odnosno ravni preseci i posle deformacije ostaju ravni i upravni na deformisanu osu preseka; • Bernoulli's hypothesis is valid, i.e. plane sections initially perpendicular to the midsurface will remain plane and perpendicular on deformed axis, p • spojna sredstva postavljena su na određenom razmaku i mogu se smatrati ekvivalentnom kontinualnom vezom s konstantnom elastičnosti spoja duž celog nosača; • coupling means are set at certain distances and can be considered as equivalent continuous connection with the constant elasticity along the beam, • cross sections of concrete and timber are constant along the span, • poprečni preseci betona i drveta konstantni su duž raspona; • concrete and timber have equal deflections at each point of the connection, • drvo i beton imaju jednake ugibe u svakoj tački spoja; • axial force acts at the centre of gravity (centroid) of the concrete section. • axial force acts at the centre of gravity (centroid) of the concrete section. • aksijalna sila deluje u težištu betonskog preseka. j j g p Pri savijanju SDB nosača, nastaje pomeranje (klizanje v) u spojnoj ravni dva materijala. Klizanje elemenata sprečeno je spojnim sredstvima, što uzrokuje pojavu sile klizanja (smičuće sile u kontaktnoj ravni) Ts koja izaziva silu pritiska N1 i momenat savijanja M1 – u gornjem, a silu zatezanja N2 i momenat savijanja M2 – u donjem elementu nosača, slika 1 (gde su sa A i I obeležene geometrijske karakteristike poprečnih preseka gornjeg i donjeg elementa, a sa E moduli elastičnosti primenjenih materijala). Intenziteti sila zavise od krutosti i deformabilnosti spojnog sredstva, odnosno njegovog modula pomerljivosti K [2]. In TCC structure, one element slips (v)over the other along TC interface in the case of bending. 1 UVOD Finite element method (FEM) is one of the most used numerical methods in structural analysis where the final element formulations is based on the solution of differential equations by residual methods or using the variation formulation.FEM based on the Galerkin method (or other weighted residual methods) can be applied to a much broader set of differential equations because it is not necessary to have a proper variational form as it is the case when using Rayleigh-Ritz based FEM [1]. Based on the previous exposition, it can be concluded that the application of simplified and/or approximate numerical methods for the analysis and design of TCC structures is welcome and recommended. Therefore, the approximate methods based on differential or variational formulation [16] are widely used, because they can be implemented in structural analysis software in order to provide a specific tool for engineers for designing partial composite structures. U radu je prikazana Galerkinova metoda u analizi SDB konstrukcija [14]. Analiziran je izbor probnih funkcija koje opisuju problem elastičnog sprezanja, kao i njihov uticaj na konačne rezultate. Za poređenje dobijenih rezultata, sprovedene su i analize prema analitičkim rešenju [17] i „gama” postupku [5]. Na osnovu predloženih numeričkih modela, model koji najbolje opisuje problem elastičnog sprezanja izabran je za dodatnu komparativnu analizu sa eksperimentalnim podacima [18]. Pored toga, predstavljena je i upotreba Ritz-ove metode u analizi SDB konstrukcija. Dobijeni rezultati prema Ritz-ovoj metodi, s različitim probnim funkcijama, analizirani su i upoređeni sa analitičkim rešenjem i „gama” postupkom. Sve analize su sprovedene upotrebom programa MATLAB [15]. This paper presents the Galerkin method in the analysis of the TCC system [14]. The selection of trial functions that describe the problem of elastic composite action as well as their influence on the final results was analyzed. For comparison of the obtained results, analysis were performed according to analytical solution [17] and the "gamma" method [5]. On the basis of the proposed numerical models, a model that best describes the problem of elastic coupling was chosen for further comparative analysis with the experimental data [18]. In addition, the use of the Ritz method was also presented in the analysis of the TCC system. The obtained results GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 61 (2018) 4 (37-53) BUILDING MATERIALS AND STRUCTURES 61 (2018) 4 (37-53) 39 according to the Ritz method with different trial functions were analyzed and compared to the analytical and the "gamma" method solutions. 1 UVOD All analysis were performed using MATLAB software [15]. 2 GOVERNING EQUATION OF TCC SYSTEM Sliding of elements is prevented by the coupling means with appearance of interlayer slip (shear in contact interface) force Ts with compression force N1 and the bending moment M1 in the upper and the tensile force N2 and the bending moment M2 in the lower element of the structure, Figure 1 (where notation A and I represent geometrical properties of cross-sections of upper and lower element, while E represent the modulus of elasticity of applied materials). The intensities of forces depend on the stiffness and deformability of the coupling means and its slip modulus K [2]. Slika 1. Klizanje u kontaktnoj ravni SDB nosača [2] Figure 1. Interlayer slip of TCC beam [2] Slika 1. Klizanje u kontaktnoj ravni SDB nosača [2] Figure 1. Interlayer slip of TCC beam [2] Slika 1. Klizanje u kontaktnoj ravni SDB nosača [2] Figure 1. Interlayer slip of TCC beam [2] Slika 1. Klizanje u kontaktnoj ravni SDB nosača [2] Figure 1. Interlayer slip of TCC beam [2] GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 61 (2018) 4 (37-53) BUILDING MATERIALS AND STRUCTURES 61 (2018) 4 (37-53) 40 The problem of partial composite action could be represented with differential equation of the second order in the function of the axial force in concrete while observing the composite timber-concrete simply supported beam system with uniformly distributed load q(x) without an external axial force: Kada se razmatra spregnuta greda drvo–beton, statičkog sistema proste grede, opterećena ravnomerno raspodeljenim opterećenjem q(x), bez spoljašnje aksijalne sile, problem elastičnog sprezanja može se predstaviti diferencijalnom jednačinom drugog reda u aksijalne sile u betonu: (1) where are: (2) (3) (1) (1) where are: (2) (3) (1) where are: where are: gde je: (2) (3) M(x)- the moment of the fully composite section (k→∞), M(x) – moment kruto spregnutog preseka (k→∞); k – krutost spoja („raspodeljen” modul pomerljivosti) [N/m2], k=K/s; M(x) – moment kruto spregnutog preseka (k→∞); k – krutost spoja („raspodeljen” modul pomerljivosti) [N/m2], k=K/s; K – modul pomerljivosti spojnog sredstva [N/m], određen ispitivanjima; ( ) k - the slip modulus per-unit length (“smeared” slip modulus)[N/m2], k=K/s, [ ], ; K – modul pomerljivosti spojnog sredstva [N/m], određen ispitivanjima; )[ ] K-the slip modulus [N/m], determined )[ ] K-the slip modulus [N/m], determined by testing, p j ; s – rastojanje spojnih sredstava za sprezanje; s-the spacing between connections, r - the distance between centroid of flange and web elements. 2 GOVERNING EQUATION OF TCC SYSTEM r – rastojanje između težišta betonskog i drvenog dela preseka. p Takođe, problem SDB nosača može se izraziti putem diferencijalne jednačine četvrtog reda u funkciji vertikalnog pomeranja: In addition, the problem of the TCC beam could be expressed through the differential equation of the fourth order in function of vertical displacement: (4) (4) gde je: where is: (5) (5) (5) (EI)0 i (EI)∞ predstavljaju savojnu krutost za nespregnutu (k→0) i kruto spregnutu (k→∞) gredu, respektivno. (EI)0 and (EI)∞ are the bending stiffness of non- composite (k→0) and fully composite (k→∞) beam, respectively. p Rešavanje diferencijalnih jednačina (1 ili 4) predstavlja složen zadatak, i to naročito za različite slučajeve opterećenja i/ili granične uslove. U literaturi, za različite slučajeve opterećenja i uslove oslanjanja, mogu se pronaći analitička rešenja. U radu [17], analitička rešenja za aksijalnu silu N, silu klizanja u spoju Ts i vertikalno pomeranje w, za statički sistem proste grede i kontinualno opterećenje, data su jednačinama (6–8). Solving the differential equations (1 or 4) is a complex task, especially for different load cases and/or boundary conditions. In the literature, analytical solutions for different load cases and support conditions could be found. According to [17], analytical solutions for axial force N, interlayer slip force Ts and vertical displacement w, for a simply supported beam system and continuous load, are given by the equations (6-8). Aksijalna sila Axial force (6) (6) Slip force (7) Sila klizanja Sila klizanja Sila klizanja (7) GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 61 (2018) 4 (37-53) BUILDING MATERIALS AND STRUCTURES 61 (2018) 4 (37-53) 41 GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 61 (2018) 4 (37-53) BUILDING MATERIALS AND STRUCTURES 61 (2018) 4 (37-53) 41 GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 61 (2018) 4 (37-53) BUILDING MATERIALS AND STRUCTURES 61 (2018) 4 (37-53) 41 Vertical displacement Vertical displacement Vertikalno pomeranje (8) (8) Prikaz kvalitativne promene aksijalne sile N to jest sile klizanja u spoju Ts SDB nosača, dat je na slici 2. A demonstration of the qualitative change of the axial force N i.e. slip force Ts in TCC system is shown in Figure 2 Prikaz kvalitativne promene aksijalne sile N to jest sile klizanja u spoju Ts SDB nosača, dat je na slici 2. A demonstration of the qualitative change of the axial force N i.e. slip force Ts in TCC system is shown in Figure 2. elastično sprezanje elastic composite action kruto sprezanje fully composite action Slika 2. Prikaz kvalitativne promene normalne tj. sile klizanja u SDB sistemu [17] Figure 2. Demonstration of qualitative change of the axial i.e. slip forces in TCC [17] elastično sprezanje elastic composite action kruto sprezanje fully composite action Slika 2. Prikaz kvalitativne promene normalne tj. sile klizanja u SDB sistemu [17] Figure 2. Demonstration of qualitative change of the axial i.e. where is: slip forces in TCC [17] 3 APROKSIMATIVNE METODE Problemi teorije elastičnosti opisani su pomoću diferencijalne formulacije (diferencijalne jednačine i odgovarajućih graničnih uslova) ili u varijacionoj formulaciji u obliku funkcionala. Iako rešenja ovih problema u matematičkom smislu egzistiraju kao jednoznačna, nalaženje analitičkih rešenja predstavlja zahtevan i često nerešiv zadatak. Stoga, približne metode često se koriste prilikom određivanja rešenja za ove probleme. Posebno značajne jesu one metode gde se kao polazna osnova koristi pretpostavka o rešenju u obliku aproksimativnih ili probnih funkcija, pri čemu je jedna od najčešće primenjivanih metoda reziduuma Galerkinova metoda, dok je od varijacionih metoda to najčešće Ritz-ova metoda. The problems of the theory of elasticity are described by means of the differential formulation (differential equations and corresponding boundary conditions) or in the variational formulation in the form of the functional. Although the solutions to these problems in the mathe- matical sense exist as unambiguous, finding analytical solutions is a delicate and often unsolvable task. There- fore, the approximate methods are often used to find solutions to these problems. Of particular interest are those methods in which the assumption of a solution in the form of approximate or trial function is used as the baseline, wherein one of the most commonly applied weight residual methods is Galerkin method, and commonly applied variational method is Ritz method. Nepoznata funkcija u(x) diferencijalne jednačine problema aproksimira se približnim rešenjem ū(x), izraz (9), koje se može predstaviti kao suppozicija proizvoda poznatih baznih funkcija Φm i nepoznatih koeficijenta cm. The unknown function u(x) of problem's differential equation has to be approximated by the approximate solution ū(x), expression (9), that could be represented as a superposition of products of known basis functions Φm and unknown coefficients cm. gde je: Φ where are: Φ (9) (9) (9) where are: where are: gde je: g j Φm – skup izabranih linearno nezavisnih funkcija Φm(xm); g j Φm – skup izabranih linearno nezavisnih funkcija Φm(xm); Φm - set of chosen linearly independent functions Φm(xm), cm – nepoznati parametri, konstante ili funkcije, koje treba odrediti. cm- unknown parameters, constants or functions to be determined. Najčešći oblici probnih funkcija jesu polinomi ili trigonometrijske funkcije. Funkcije Φm unapred se usvajaju, imajući u vidu granične uslove po The most common trial functions are polynomials or trigonometric functions. Functions Φm are adopted in advance by taking into account of essential boundary GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 61 (2018) 4 (37-53) BUILDING MATERIALS AND STRUCTURES 61 (2018) 4 (37-53) 42 pomeranjima. 3 APROKSIMATIVNE METODE Kada je reč o drugim uslovima, izbor funkcija Φm uglavnom je proizvoljan, ali kvalitet rešenja umnogome zavisi baš od izbora funkcija Φm. Poželjno je da funkcije Φm zadovoljavaju i granične uslove po silama, te da njihov oblik kvalitativno odgovara tačnom analitičkom rešenju. Dakle, kvalitativno poznavanje prirode rešenja veoma je korisno da bi se izbegao pogrešan izbor funkcija koje po svom obliku predstavljaju grubo odstupanje od analitičkog rešenja. conditions. As regards other conditions, the choice of functions Φm is generally arbitrary, but the quality of the solution largely depends on the choice of functions Φm. It is desirable that the functions Φm also satisfies natural boundary conditions, and thattheir shape qualitatively corresponds to the exact analytical solution. Therefore, qualitative knowledge of the nature of the solution is very useful in order to avoid the wrong choice of functions that in their form represent a rough deviation from the analytical solution. Galerkinova metoda ima širu primenu od Ritz-ove metode, jer se može primeniti pri rešavanju onih problema za koje funkcional ne postoji. U mehanici deformabilnih tela, ove dve metode su ekvivalentne, jer daju rezultate iste tačnosti. Izborom istih probnih funkcija u Ritz-ovoj i Galerkin-ovoj metodi, dobijaju se isti koeficijenti cm (ista rešenja). Galerkin method has wider application than Ritz's because it can solve even those problems in which the functional does not exist. In the mechanics of defor- mable bodies, these two methods are equivalent, as they give results of the same accuracy. By choosing the same trial functions in Ritz and Galerkin method, the same coefficients of cm (i.e. same solutions) will be obtained. gde je: where are: L - corresponding linear differential operator, L - corresponding linear differential operator, L – odgovarajući linearni diferencijalni operator; u(x) – nepoznata funkcija problema, koja zavisi od koordinate x unutar prostora Ω, pri čemu funkcija u(x) zadovoljava date granične uslove na granicama domena Ω; u(x) - unknown function of the problem, that depends on the coordinate x within the domainΩ, where the functionu(x) satisfy the given boundary conditions at the boundaries of the domain Ω, ; fΩ – vektor slobodnih članova u jednačini u domenu Ω. fΩ – given force term in domainΩ. The unknown function of the problem u(x) is approximated with the approximate function ū(x), equation (9), which satisfies the boundary conditions upon the displacements (essential conditions), but does not have to satisfy the conditions by forces (natural conditions). As ū(x) is approximate solution of the equation (10), the residue or residuum is obtained: Nepoznata funkcija problema u(x) aproksimira se s približnom funkcijom ū(x), izraz (9), koja zadovoljava granične uslove po pomeranjima (esencijalne granične uslove), ali ne mora da zadovoljava i uslove po silama (prirodne granične uslove). Kako je ū(x) približno rešenje jednačine (10), dobija se ostatak ili reziduum: (11) 3.1 Metoda reziduuma Neka je posmatrani fizički problem, u domenu Ω, koji može da bude 1D do 3D, definisan diferencijalnom jednačinom: A physical problem is observed in the domain Ω, which can be 1D to 3D, defined with a differential equation: (10) where are: (10) Kako je jednačina (10) sistem jednačina, odnosno matrična jednačina, ostatak jeste R(ū) vektor. Naravno, kada bi ū(x) bilo tačno analitičko rešenje, onda bi vektor ostatka R(ū) bio jednak nultom vektoru. Ideja metode jeste da se vektor ostatka svede na nulti vektor „u prosečnom smislu”. Stoga, uvode se linearno nezavisne težinske funkcije W(ū), uz uslov da integral skalarnog proizvoda vektora težinskih funkcija i vektora ostatka unutar domena Ω bude jednak nuli: Since equation (10) is a system of equations i.e. a matrix equation, than the residue R(ū) is a vector. Of course, if ū(x) would be the exact solution, then the residue vector R(ū) would be equal to the zero vector. The idea behind the method is to reduce the residue vector to the zero vector "in the average sense". Because of that, linearly independent weight functions W(ū) are introduced with the condition that the integral of the scalar product of the weight function vector and the residual vector within the domain Ω is equal to zero: (12) (12) (12) (12) (12) The scalar product of the two vectors is equal to zero if these vectors are mutually orthogonal. Accordingly, the integral equation (12) is a condition of the orthogonality of the residual vector to the selected vector of weight functions. Skalarni proizvod dva vektora jednak je nuli ukoliko su ti vektori međusobno ortogonalni. Prema tome, integralna jednačina (12) predstavlja uslov ortogonal- nosti vektora ostatka na izabrani vektor težinskih funkcija. The scalar product of the two vectors is equal to zero if these vectors are mutually orthogonal. Accordingly, the integral equation (12) is a condition of the orthogonality of the residual vector to the selected vector of weight functions. j Metode reziduuma sastoje se u nalaženju funkcija ū(x) za koje će integralna jednačina (12) biti zadovo- Residue methods consist of finding functions ū(x) for which the integral equation (12) will be satisfied. If the GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 61 (2018) 4 (37-53) BUILDING MATERIALS AND STRUCTURES 61 (2018) 4 (37-53) 43 equation (12) is satisfied for any weight functions vector, then the residue vector will approach the zero vector. ljena. Ako je jednačina (12) zadovoljena za bilo koji vektor težinskih funkcija, onda će se vektor ostatka približavati nultom vektoru. In this way, the approximate solution ū(x) approximates the exact solution u(x). All solutions ū(x) that satisfy (10) must satisfy (12) regardless of weight functions' choice. The dimension of the weight functions vector corresponds to the number of unknown coefficients cm of the considered problem. Na taj način, približno rešenje ū(x) aproksimira tačno rešenje u(x). Sva rešenja ū(x) koja zadovoljavaju (10) moraju da zadovoljavaju i (12), bez obzira na izbor težinskih funkcija. Dimenzija vektora težinskih funkcija odgovara broju nepoznatih koeficijenata cm razmatranog problema. As one of the basic variants of the residual method, which adopts weight functions as basis functions Φm for which the required solution is approximated, is Galerkin's method [16]. p Kao jedna od osnovnih varijanti metode reziduuma, koja usvaja težinske funkcije kao bazne funkcije Φm kojima je aproksimirano traženo rešenje, jeste Galerkinova metoda [16]. (12) Based on the differential equations of the elastic coupling (1 or 4) and the condition (12), it is possible to define the following relations for determining the problem of the TCC girder trough the axial force in the concrete or trough displacements for the case of a TCC beam loaded with continuous load q, according to fallowing expressions: Na osnovu diferencijalnih jednačina elastičnog spre- zanja (1 ili 4) i uslova (12), moguće je definisati sledeće relacije za određivanje problema SDB nosača u funkciji aksijalne sile u betonu ili u funkciji pomeranja za slučaj SDB grede opterećene kontinualnim opterećenjem q, prema sledećim izrazima: (13) (14) (13) (13) (14) An integral formulation that implicitly contains a differential equation of the problem is called a weak formulation (13 or 14) that expresses the conditions and relations that must be satisfied in the average, or in an integral sense. Integralna formulacija koja u sebi implicitno sadrži diferencijalnu jednačinu problema, naziva se slaba formulacija (13 ili 14) koja izražava uslove i relacije koje moraju biti zadovoljene u prosečnom ili integralnom smislu. Kako je diferencijalna jednačina problema parnog reda (2r = 4), parcijalnom integracijom izraza (12) red izvoda r u probnim funkcijama moguće je smanjiti sa r = 4 na r = 2. Parcijalnom integracijom izraza (13 ili 14) postiže se da odabrane probne funkcije moraju zadovoljavati samo granične uslove po pomeranjima, koji moraju biti zadovoljeni izborom samih probnih funkcija, dok su uslovi po silama već uključeni u formulaciju problema parcijalnom integracijom. g Since the differential equation of the problem is of even order (2r = 4), it is possible to reduce the required order of derivation in the trial functions by partial integration of the expression (12) from r = 4 to r = 2. By partial integration of expressions (13 or 14) is achieving that selected trial functions must satisfy only the essential conditions, that have to be satisfied by the selection of trial functions themselves, while the force conditions are already included into the formulation of the partial integration problem. Rešavanjem integrala, dobija se sistem od n jednačina po nepoznatim koeficijentima cm i približno rešenje za traženu funkciju u(x) može se dobiti određivanjem koeficijenata cm. By solving the integrals, a system of n equations by unknown coefficients cmis obtained andan approximate solution for the required function u(x) could be derived by determination of coefficients cm. 3.2 Varijaciona metoda As the Ritz method is based on a variational formulation, it is necessary to satisfy the requirement of extremum of a functional that describes the problem under consideration. To solve problems in the mechanics of deformable bodies, the functional is equal to the total potential energy, and the stationary value corresponds to its minimum value. In the theory of structures this method is the most famous variation procedure. The reason for that is that there is a functional in the form of potential energy [13]. In case of one-dimensional beam problem with the defined domain x∈[x1,x2], functional (potential energy) is expressed as an integral I(u)over the entire domain: Kako se Ritz-ova metoda zasniva na varijacionoj formulaciji, potrebno je zadovoljiti uslov stacionarnosti funkcionala koji opisuje razmatrani problem. Za rešavanje problema u mehanici deformabilnih tela, funkcional je jednak ukupnoj potencijalnoj energiji, a stacionarna vrednost odgovara njenoj minimalnoj vrednosti. U Teoriji konstrukcija, ovaj metod je najpoznatiji varijacioni postupak. Razlog jeste to što postoji funkcional u obliku potencijalne energije [13]. Kada da se posmatra jednodimenzionalni linijski problem s domenom definisanosti x∈[x1,x2], funkcional (potencijalna energija) izražava se putem integrala I(u) u celom domenu: GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 61 (2018) 4 (37-53) BUILDING MATERIALS AND STRUCTURES 61 (2018) 4 (37-53) 44 (15) (15) where is: gde je: Π (...) - represents the functional of functionsu(x), du(x)/dx, d2u(x)/dx,... g j Π (...) – funkcional funkcija u(x), du(x)/dx, d2u(x)/dx,... Uslov stacionarnosti funkcionala prikazuje se uslovom da je prva varijacija funkcionala jednaka nuli: ( ) ( ) Extremum of a functional is represented by requirements that the first variation of the functional be zero: (16) (16) ili zapisano u razvijenom obliku: or shown in the developed form: (17) (17) Kako su koeficijenti c1,c2,...,cn međusobno nezavisni parametri, onda se δΠ=0 svodi na sledeći uslov: Since c1,c2,...,cn are mutually independent para- meters, then δΠ=0 is represented by following condition: Since c1,c2,...,cn are mutually independent para- meters, then δΠ=0 is represented by following condition: (18) (18) što predstavlja sistem algebarskih jednačina po nepoznatim koeficijentima cm. which represents a system of algebraic equations with unknown coefficients cm. Based on the differential formulation of the partially composite problem, it is possible to define a functional according to variation principles [8]. As the mechanical fasteners are commonly used for coupling in TCC, a certain displacements (i.e. an interlayer slip) occur on the TC interface due to the external load. 3.2 Varijaciona metoda Besides the strain energy due to internal forces (M1, N1, M2 i N2 where N=N1=-N2), it is also necessary to take into account the strain energy due to interlayer slip. Functional, or total potential energy of the composite system, in the case of simply supported beam with uniform distributed load q(x), can be shown in the following form [19]: Na osnovu diferencijalne formulacije problema elastičnog sprezanja, moguće je definisati funkcional na osnovu opštih varijacionih principa [8]. Pošto se najčešće koriste mehanička spojna sredstva za SDB nosače, usled spoljašnjeg opterećenja javljaju se izvesna pomeranja (tj. klizanja u spoju) na kontaktu između drveta i betona. Pored rada unutrašnjih sila (M1, N1, M2 i N2 where N=N1=-N2), potrebno je uzeti u obzir i deformacioni rad usled klizanja u spoju. Funkcional, ili ukupna potencijalna energija spregnutog sistema, u slučaju proste grede na koju deluje raspodeljeno opte- rećenje q(x), može se prikazati u sledećem obliku [19]: (19) (19) (20) (21) (19) (20) (20) (21) (21) (21) where is: where is: gde je: gde je: (22) (22) Wi- strain energy due to internal forces, Wi – potencijalna energija deformacije; We – potencijal sila. We -potential energy due to external forces. As the bending moment M(x) can be expressed by deflection w(x), using the condition of equal rotations of the composite members (timber and concrete), introducing the relation g(x), the expression (20) is represented in the following form: Kako moment savijanja M(x) možemo izraziti preko pomeranja w(x), koristeći uslov jednakih rotacija spregnutih elemenata (drveta i betona), uvodeći odnos g(x), izraz (20) prikazujemo u sledećem obliku: GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 61 (2018) 4 (37-53) BUILDING MATERIALS AND STRUCTURES 61 (2018) 4 (37-53) 45 (23) (23) where: (24) (25) gde je: gde je: (24) (25) Uvedeni odnos g(x) izveden je iz uslova kompatibilnosti pomeranja na spoju dva elementa, koji može da se zapiše u sledećem obliku: The introduced relation g(x) was derived from the compatibility of displacements at the interface of the two subelements, that could be written in the following form: Uvedeni odnos g(x) izveden je iz uslova kompatibilnosti pomeranja na spoju dva elementa, koji može da se zapiše u sledećem obliku: The introduced relation g(x) was derived from the compatibility of displacements at the interface of the two subelements, that could be written in the following form: (26) (26) Poznajući rad unutrašnjih sila Wi, određen je funkcional za elastično spregnuti SDB nosač. 3.2 Varijaciona metoda Kako se u izrazu g(x) javlja normalna sila N(x), za rešavanje problema, pored pretpostavljanja probne funkcije za pomeranje w(x), potrebno je pretpostaviti i probnu funkciju za normalnu silu N(x). Primenom varijacionih principa na funkcional, Ritz-ovom metodom, možemo rešiti problem elastičnog sprezanja, odnosno odrediti pomeranje nosača i unutrašnje sile u spregnutom nosaču. Knowing the strain energy due to internal forces Wi, functional for a partial TCC system is determined. As in the expression g(x) the normal force N(x) appears, for solving the problem, beside assumed trial function of displacement w(x), it is also necessary to assume the trial function for N(x). By applying variation principles to a functional, with Ritz method, the problem of partially composite system can be solved, which means to determine displacement and internal forces in the composite members. 4 APPROXIMATION OF THE SOLUTION – TRIAL FUNCTIONS Probna funkcija i njen prvi izvod Figure 3. Trial function and its first derivative 1. Hiperbolična funkcija / Hyperbolic function: 1. Hiperbolična funkcija / Hyperbolic function: 1. Hiperbolična funkcija / Hyperbolic function: (27) (27) 2. Sinusna funkcija / Sinusoidal function: (28) 2. Sinusna funkcija / Sinusoidal function: (28) (28) 3. Polinom / Polynomial function: 3. Polinom / Polynomial function: (29) (29) Slika 3. Probna funkcija i njen prvi izvod Figure 3. Trial function and its first derivative In Galerkin method, one of three suggested trial functions has to be adopted (for N or w) according to chosen integral formulation (Eq 13 or 14). In Ritz method it is necessary to adopt two trial functions(for N and w). U Galerkinovoj metodi, jednu od tri predložene funkcije treba usvojiti za probnu funkciju (za N ili w) prema odabranoj integralnoj formulaciji (jednačine 13 ili 14). U Ritz-ovoj metodi, potrebno je usvojiti dve probne funkcije (za N i w). 4 APPROXIMATION OF THE SOLUTION – TRIAL FUNCTIONS Pogodne, a samim tim i najčešće, probne funkcije su polinomi ili trigonometrijske funkcije. Probne funkcije treba da zadovolje sledeće uslove: Suitable, and therefore, most often, trial functions are polynomials or trigonometric functions. Trial functions should satisfy the following conditions: • da su neprekidne i do potrebnog reda diferencijabilne; • da su neprekidne i do potrebnog reda diferencijabilne; • to be continuous and differentiable till the necessary order, • to be continuous and differentiable till the necessary order, • in addition to essential, they also have to satisfy natural boundary conditions, • in addition to essential, they also have to satisfy natural boundary conditions, • pored esencijalnih graničnih uslova, treba da zadovolje prirodne granične uslove; • to correspond qualitatively by the form to the analytical solution, • treba da oblikom kvalitativno odgovaraju tačnom rešenju; • da budu potpune, npr. u slučaju polinoma određenog stepena, takođe treba da budu uključeni i svi članovi nižeg stepena. • to be complete, e.g. in the case of polynomials of a certain degree, all members of the lower degrees should also be included. Na osnovu dobro poznatih rešenja iz literature, jednačine (6) i (8), kao i kvalitativnog poznavanja oblika rešenja (slika 2), u ovom radu za probne funkcije izabrane su tri funkcije (hiperbolična, sinusna i funkcija oblika polinoma). Based on the well-known solutions from the literature, equations (6) and (8), as well as on the qualitative flow of the solution (Fig. 2), three functions (hyperbolic, sinusoidal and polynomial functions) were selected for trial functions in this paper. The adopted trial functions describe the law of the change of the axial force N, slip forces Ts (N') and displacement w along the composite girder and qualitatively correspond to the solutions (Fig. 2). By means of expressions (27, 28 and 29), selected trials for Nand/orw are given, while on Fig. 3 the shape of the function and its first derivative along the beam are shown. Usvojene probne funkcije opisuju zakon promene aksijalne sile N, sile klizanja u spoju Ts (N') i pomeranje w duž spregnutog nosača i kvalitativno odgovaraju analitičkim rešenjima (slika 2). Pomoću izraza (27, 28 i 29), date su odabrane probne funkcije za N i/ili w, dok je na slici 3 prikazan oblik funkcije i njen prvi izvod duž nosača. GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 61 (2018) 4 (37-53) BUILDING MATERIALS AND STRUCTURES 61 (2018) 4 (37-53) 46 Slika 3. 5.1 Opis analiziranog modela The TCC floor structure is considered for the numerical analysis by Galerkin and Ritz method. The disposition of the elements and fasteners, as well as their dimensions and properties of the applied materials according to European standards, are shown in Fig. 4. Razmatrana je SDB konstrukcija tavanice za numeričku analizu metodama Galerkina i Ritz-a. Raspored elemenata i spojnih sredstava, kao i njihove dimenzije i svojstva primenjenih materijala prema evropskim standardima, prikazani su na slici 4. Slika 4. Analizirani model međuspratne SDB konstrukcije [13] Figure 4. Analyzed model of the TCC floor structure [13] Slika 4. Analizirani model međuspratne SDB konstrukcije [13] Figure 4. Analyzed model of the TCC floor structure [13] GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 61 (2018) 4 (37-53) BUILDING MATERIALS AND STRUCTURES 61 (2018) 4 (37-53) 47 The floor structure consists of a glulam beams that are coupled with concrete slab by vertically arranged dowel type fasteners. In this paper, the slip modulus K is determined by the Gelfi model[6]. The floor structure is loaded by the self-weight of the structural elements g, by additional permanent load dg, as well as by the imposed load p. It is considered that the timber glulam beams will be supported in the stage of pouring and hardening of the concrete slab, and the composite section will receive imposed and total permanent load. It is possible to analyze the part of the composite floor structure separately (glulam beam with the effective width of the concrete slab), because in analyzed TCC floor system all concrete slabs are one-way and glulam beams are simply supported with uniformly distributed load. Numerical analysis according to Galerkin and Ritz method of TCC structure was performed and several subprograms/codes are written in MATLAB 2014 [15]. The simplified “γ-procedure” was also performed in order to obtain the referent values suggested by Eurocode. Konstrukcija tavanice sastoji se od lameliranih lepljenih drvenih (LLD) greda koje su – zajedno s betonskom pločom – spregnute vertikalno postavljenim štapastim spojnim sredstvima. U ovom radu, modul pomerljivosti K određen je Gelfijevim modelom [6]. Tavanica je opterećena sopstvenom težinom elemenata konstrukcije g, dodatnim stalnim opterećenjem dg, kao i korisnim opeterećenjem p. Smatra se da će LLD grede biti poduprte u fazi izlivanja i očvršćavanja betonske ploče, te će spregnuti presek primati korisno i ukupno stalno opterećenje. 5.2 Numerička analiza primenom Galerkinove metode Za potrebe numeričke analize, definisane su dve grupe modela na osnovu izbora probnih funkcija i integralnih formulacija (jednačine 13 ili 14) za aksijalnu silu N(x) ili ugib w(x): Modeli grupe A (N-HIP, N-SIN, N- POL) i Modeli grupe B (w-HIP, w-SIN, w-POL). ) Vertical displacements (w), moments (M1, M2), axial forces (N1, N2) and stresses (σ1 i σ2) for the cross- section of concrete / timber element (top and bottom) in the middle of the beam span were calculated, as well as shear forces (Fs) in connectors and slip force (Ts) values at the concrete-timber contact in support zones. Results of performed numerical analysis for Models of group A and B were compared with analytical solution and their percentage deviations are shown at Figures 5,6. Results of simplified “γ-procedure” were also compared with analytical solution. ) g p ( ) Sračunata su vertikalna pomeranja (w), momenti savijanja (M1, M2), aksijalne sile (N1, N2) i naponi (σ1 i σ2) u betonu / drvetu (gornje i donje vlakno) za presek u sredini raspona grednog nosača, kao i smičuće sile (Fs) u spojnim sredstvima i sile klizanja (Ts) na kontaktu betona i drveta nad osloncem. Rezultati numeričke analize modela iz grupe A i B upoređeni su s rezultatima analitičkog rešenja, a njihova procentualna odstupanja prikazana su na slikama 5, 6. Rezultati pojedno- stavljenog „γ-postupka” takođe su poređeni sa analitičkim rešenjem. y From Figs. 5 and 6, it can be noticed that numerical results of group A models have smaller differences in relation to the analytical solution than the models of group B. j Sa slika 5 i 6, može se uočiti da rezultati numeričkih modela grupe A imaju manja odstupanja u odnosu na analitičko rešenje nego modeli grupe B. g p Analysis of results obtained by different trial functions for the approximate solution of the normal force N shows that a minimal deviation occurs when a hyperbolic function is adopted, and the maximum one if it is the sinusoidal function. The models N-POL and N-SIN have significant deviations in stresses (σ1,b and σ2,t) even up to 21%, and minor deviations in the slip and shear forces (Ts and Fs) up to 6.5%. All values are smaller than those obtained by analytical solutions. 5.2 Numerical analysis by Galerkin method For the purpose of numerical analysis, two groups of models are defined on the basis of selection of trial functions and integral formulation (eqs. 13 or 14) for axial force N(x) ordeflection w(x):Models of Group A (N- HIP, N-SIN, N-POL) and Models of Group B (w-HIP, w- SIN, w-POL). 5.1 Opis analiziranog modela Moguće je analizirati izdvojen deo spregnute tavanice (LLD greda s betonskom pločom efektivne širine), jer se smatra da u analiziranoj SDB tavanici betonska ploča nosi u jednom pravcu, a da su LLD grede statičkog sistema proste grede opterećene ravnomerno raspodeljenim opterećenjem. Sprovedena je numerička analiza SDB nosača prema Galerkinovoj i Ritz-ovoj metodi primenom programa MATLAB 2014 [15] u kome su napisani potprogrami/kodovi za njihov proračun. Takođe, pojednostavljenim „γ postupkom” izvršena je analiza kako bi se odredile referentne vrednosti predložene Evrokodom. 5.2 Numerička analiza primenom Galerkinove metode Comparing the Group A models with results of the "γ" method, none of the models has greater deviations in absolute sense, but it can be noticed that the N-POL and N-SIN models give smaller values. j g g p Analiza rezultata dobijenih primenom različitih probnih funkcija za aproksimativno rešenje normalne sile N, pokazuje da se minimalno odstupanje javlja kada se usvoji hiperbolična funkcija, a maksimalno ako se usvoji sinusna funkcija. Modeli N-POL i N-SIN imaju znatna odstupanja kod napona (σ1,b and σ2,t) i to čak do 21%, a manja odstupanja za sile klizanja i za smičuće sile (Ts and Fs) do 6,5%. Sve vrednosti su manje od onih dobijenih analitičkim rešenjem. Poredeći modele grupe A sa rezultatima „γ-metode”, nijedan od ovih modela nema veća odstupanja u apsolutnom smislu, ali može se primetiti da modeli N-POL i N-SIN daju manje vrednosti. Analiza rezultata dobijenih primenom različitih probnih funkcija za aproksimativno rešenje pomeranja w, pokazuje da se minimalno odstupanje javlja ukoliko se usvoji funkcija oblika polinoma, a maksimalno ako se usvoji hiperbolična funkcija. Modeli w-HIP i w-SIN imaju značajna odstupanja kod napona (σ1,b and σ2,t) i to čak do 41%, a za sile klizanja i za smičuće sile (Ts and Fs) čak i do 28%. Model w-HIP pokazuje znatno manje Analiza rezultata dobijenih primenom različitih probnih funkcija za aproksimativno rešenje normalne sile N, pokazuje da se minimalno odstupanje javlja kada se usvoji hiperbolična funkcija, a maksimalno ako se usvoji sinusna funkcija. Modeli N-POL i N-SIN imaju znatna odstupanja kod napona (σ1,b and σ2,t) i to čak do 21%, a manja odstupanja za sile klizanja i za smičuće sile (Ts and Fs) do 6,5%. Sve vrednosti su manje od onih dobijenih analitičkim rešenjem. Poredeći modele grupe A sa rezultatima „γ-metode”, nijedan od ovih modela nema veća odstupanja u apsolutnom smislu, ali može se primetiti da modeli N-POL i N-SIN daju manje vrednosti. Analysis of results obtained by different trial functions for the approximate displacement w solution shows that a minimal deviation occurs if the polynomial function is adopted, and the maximum one if it is a hyperbolic function. The w-HIP and w-SIN models have significant deviations in stresses (σ1,b and σ2,t) even up to 41%, and for slip and shear forces (Ts and Fs) even up to 28%. 5.2 Numerička analiza primenom Galerkinove metode Model w-HIP shows significantly smaller values Analiza rezultata dobijenih primenom različitih probnih funkcija za aproksimativno rešenje pomeranja w, pokazuje da se minimalno odstupanje javlja ukoliko se usvoji funkcija oblika polinoma, a maksimalno ako se usvoji hiperbolična funkcija. Modeli w-HIP i w-SIN imaju značajna odstupanja kod napona (σ1,b and σ2,t) i to čak do 41%, a za sile klizanja i za smičuće sile (Ts and Fs) čak i do 28%. Model w-HIP pokazuje znatno manje GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 61 (2018) 4 (37-53) BUILDING MATERIALS AND STRUCTURES 61 (2018) 4 (37-53) 48 vrednosti u poređenju sa analitičkim rešenjem. Poredeći modele grupe B s rezultatima „γ-metode”, može se uočiti je da najbolje podudaranje sa „γ-metodom” ima model w-POL. comparing to analytical. By comparing the models of group B with results of the "γ-method", it can be seen that the best match with the "γ-method" has the w-POL model. vrednosti u poređenju sa analitičkim rešenjem. Poredeći modele grupe B s rezultatima „γ-metode”, može se uočiti je da najbolje podudaranje sa „γ-metodom” ima model w-POL. 0.2% -0.3% -0.3% -0.1% -2.2% -1.1% -0.1% -0.7% -0.7% 0.0% 1.3% -2.4% -2.4% -0.8% -15.7% -8.0% -1.0% -4.9% -4.9% -0.3% 1.6% -3.1% -3.1% -1.0% -20.5% -10.4% -1.3% -6.3% -6.3% -0.3% -1.5% 2.9% 2.9% 1.0% 19.1% 9.7% 1.2% 15.6% 15.6% 0.4% N M1 M2 σ1,t σ1,b σ2,t σ2,b Ts,max Fs,max wmax Model N-HIP Model N-POL Model N-SIN γ-method Slika 5. Procentualna odstupanja modela grupe A u odnosu na analitičko rešenje Figure 5. Percentage deviations of models of group A in relation to the analytical solution Slika 5. Procentualna odstupanja modela grupe A u odnosu na analitičko rešenje Figure 5. Percentage deviations of models of group A in relation to the analytical solution 3.2% -6.0% -6.0% -2.0% -39.6% -20.1% -2.5% -28.2% -28.2% -0.9% -1.5% 2.9% 2.9% 1.0% 19.3% 9.8% 1.2% 15.6% 15.6% 0.4% -3.3% 6.2% 6.2% 2.0% 40.9% 20.8% 2.6% 27.7% 27.7% 0.7% -1.5% 2.9% 2.9% 1.0% 19.1% 9.7% 1.2% 15.6% 15.6% 0.4% N M1 M2 σ1,t σ1,b σ2,t σ2,b Ts,max Fs,max wmax Model w-HIP Model w-POL Model w-SIN γ-method Slika 6. Procentualna odstupanja modela grupe B u odnosu na analitičko rešenje Figure 6. Percentage deviations of models of group B in relation to the analytical solution Slika 6. Procentualna odstupanja modela grupe B u odnosu na analitičko rešenje Figure 6. Percentage deviations of models of group B in relation to the analytical solution Slika 6. 5.3 Numerical analysis by Ritz method Takođe, može se primetiti da model (w-POL,N-SIN) ima najbolje poklapanje s pojednostavljenim „γ-metodom”, predloženim u EN 1995. Iako varijantni model (w-POL,N- POL) daje manja odstupanja od varijantnog modela (w- SIN,N-SIN), poredeći ih sa analitičkim rešenjem, može se uočiti da dobijene vrednosti precenjuju ili potcenjuju analitičke, te ovi modeli nisu na strani sigurnosti. From Fig. 7, it can be noticed that the minimum deviation from the analytical solution shows the variant model (w-POL,N-SIN), while the max deviation occurs in variant model (w-SIN,N-SIN). The results obtained by variant model (w-POL,N-SIN) are on the safe side because they give a slightly higher values (up to 3%) for internal forces and displacements, while deviations for normal stresses and slip/ shear forces, arise up to 19% and 15% respectively, comparing to analytical solution. The reason for such increase lays in the fact that normal stresses and slip/ shear forces are derived values from baseline unknowns w(x) and N(x), so the cumulative errors are higher. It is obvious that the selection of trial functions for w(x) and N(x) has the significant impact on final result, as well as on derived statical values. It can be also noted that model (w-POL,N-SIN) has the best match with the approximate "γ-method" proposed in EN 1995. Although variant model (w-POL,N-POL) shows smaller deviations from variant model (w-SIN,N-SIN), comparing these two models with analytical solutions it can be seen that obtained values overestimate or underestimate analytical ones, but both models are not on the safe side. From Fig. 7, it can be noticed that the minimum deviation from the analytical solution shows the variant model (w-POL,N-SIN), while the max deviation occurs in variant model (w-SIN,N-SIN). The results obtained by variant model (w-POL,N-SIN) are on the safe side because they give a slightly higher values (up to 3%) for internal forces and displacements, while deviations for normal stresses and slip/ shear forces, arise up to 19% and 15% respectively, comparing to analytical solution. The reason for such increase lays in the fact that normal stresses and slip/ shear forces are derived values from baseline unknowns w(x) and N(x), so the cumulative errors are higher. It is obvious that the selection of trial functions for w(x) and N(x) has the significant impact on final result, as well as on derived statical values. It can be also noted that model (w-POL,N-SIN) has the best match with the approximate "γ-method" proposed in EN 1995. 5.2 Numerička analiza primenom Galerkinove metode Procentualna odstupanja modela grupe B u odnosu na analitičko rešenje Figure 6. Percentage deviations of models of group B in relation to the analytical solution GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 61 (2018) 4 (37-53) BUILDING MATERIALS AND STRUCTURES 61 (2018) 4 (37-53) 49 5.3 Numerical analysis by Ritz method Za potrebe numeričke analize, definisana je grupa modela na osnovu istovremenog izbora dve probne funkcije za aksijalnu silu N(x) i ugib w(x): Modeli grupe C (w-POL,N-POL; w-POL,N-SIN, w-SIN,N-SIN). Svi uticaji analizirani Galerkinovom metodom sračunati su i prema Ritz-u, a njihova procentualna odstupanja – u odnosu na analitičko rešenje – prikazana su na slici 7. Rezultati pojednostavljenog „γ-postupka” takođe su prikazani i upoređeni sa analitičkim rešenjem. For the purpose of numerical analysis, the group of models is defined on the basis of simultaneous selection of two trial functions for axial force N(x) anddeflection w(x): Models of Group C (w-POL,N-POL; w-POL,N-SIN; w-SIN,N-SIN). All effects analyzed by Galerkin method are calculated according Ritz as well and their percentage deviations compared to analytical solution are presented on Fig 7. Results of simplified “γ- procedure” were also compared with analytical solution. 1.3% 4.7% 4.7% 3.2% 17.4% 10.0% 3.4% -4.9% -4.9% 0.6% -1.8% 2.6% 2.6% 0.6% 19.0% 9.4% 0.9% 15.2% 15.2% 0.03% 1.6% 6.2% 6.2% 4.2% 23.1% 13.3% 4.5% -6.3% -6.3% 0.7% -1.5% 2.9% 2.9% 0.9% 19.2% 9.7% 1.2% 15.6% 15.6% 0.4% N M1 M2 σ1,t σ1,b σ2,t σ2,b Ts,max Fs,max wmax Model w-POL,N-POL Model w-POL,N-SIN Model w-SIN,N-SIN γ-method Slika 7. Procentualna odstupanja modela grupe C u odnosu na analitičko rešenje Figure 7. Percentage deviations of models of group C in relation to the analytical solution Slika 7. Procentualna odstupanja modela grupe C u odnosu na analitičko rešenje Figure 7. Percentage deviations of models of group C in relation to the analytical solution GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 61 (2018) 4 (37-53) BUILDING MATERIALS AND STRUCTURES 61 (2018) 4 (37 53) 50 Na slici 7, može se primetiti da minimalno odstupanje od analitičkog rešenja pokazuje varijantni model (w- POL,N-SIN), dok se maksimalna odstupanja javljaju kod varijantnog modela (w-SIN,N-SIN). Rezultati određeni varijantnim modelom (w-POL,N-SIN) jesu na strani sigurnosti, jer daju neznatno veće vrednosti (do 3%) za unutrašnje sile i pomeranja, dok odstupanja za normalne napone i sile klizanja / smičuće sile, dostižu vrednosti do 19% i 15% respektivno, u odnosu na analitičko rešenje. Razlog za takvo povećanje leži u činjenici da su normalni naponi i sile klizanja / smičuće sile izvedene veličine osnovnih nepoznatih w(x) i N(x), pa su kumulativne greške veće. Očigledno je da izbor probnih funkcija za w(x) i N(x) ima značajan uticaj na konačni rezultat, kao i na izvedene statičke veličine. 5.3 Numerical analysis by Ritz method Although variant model (w-POL,N-POL) shows smaller deviations from variant model (w-SIN,N-SIN), comparing these two models with analytical solutions it can be seen that obtained values overestimate or underestimate analytical ones, but both models are not on the safe side. Na slici 7, može se primetiti da minimalno odstupanje od analitičkog rešenja pokazuje varijantni model (w- POL,N-SIN), dok se maksimalna odstupanja javljaju kod varijantnog modela (w-SIN,N-SIN). Rezultati određeni varijantnim modelom (w-POL,N-SIN) jesu na strani sigurnosti, jer daju neznatno veće vrednosti (do 3%) za unutrašnje sile i pomeranja, dok odstupanja za normalne napone i sile klizanja / smičuće sile, dostižu vrednosti do 19% i 15% respektivno, u odnosu na analitičko rešenje. Razlog za takvo povećanje leži u činjenici da su normalni naponi i sile klizanja / smičuće sile izvedene veličine osnovnih nepoznatih w(x) i N(x), pa su kumulativne greške veće. Očigledno je da izbor probnih funkcija za w(x) i N(x) ima značajan uticaj na konačni rezultat, kao i na izvedene statičke veličine. Takođe, može se primetiti da model (w-POL,N-SIN) ima najbolje poklapanje s pojednostavljenim „γ-metodom”, predloženim u EN 1995. Iako varijantni model (w-POL,N- POL) daje manja odstupanja od varijantnog modela (w- SIN,N-SIN), poredeći ih sa analitičkim rešenjem, može se uočiti da dobijene vrednosti precenjuju ili potcenjuju analitičke, te ovi modeli nisu na strani sigurnosti. GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 61 (2018) 4 (37-53) BUILDING MATERIALS AND STRUCTURES 61 (2018) 4 (37-53) 50 5.4 Potvrda Galerkinove metode Radi provere Galerkinove metode u analizi SDB konstrukcija, upoređene su dobijene numeričke vred- nosti sa eksperimentalnim podacima. Kako se pokazalo da N-HIP model na najbolji način opisuje problem SDB, ovaj model izabran je za komparativnu analizu sa eksperimentalnim rezultatima SDB greda (EP1 and EP2) [18], gde su upotrebljena mehanička spojna sredstva. Dijagrami na slici 8 predstavljaju napone u poprečnom preseku konstitutivnih elemenata SDB greda za presek u sredini raspona, u odnosu na intenzitet sile tokom faza nanošenja opterećenja (F = 6, 12, 18, 24, 30 i 36 kN). Osenčene površine predstavljaju anvelopu eksperimen- talno dobijenih rezultata za grede EP1 i EP2, dok pune i isprekidane linije predstavljaju rezultate numeričke analize pomoću N-HIP modela i „γ-metode”, respektivno. Razmatrajući napone (σ1,b and σ2,t) na kontaktu dva materijala, znatno odstupanje od eksperimentalnih rezul- tata može se primetiti za napon σ2,t, pri opterećenjima F = 18 i 24 kN. Primetno je odlično poklapanje nume- ričkih vrednosti sa eksperimentalnim rezultatima kod gornjeg i donjeg vlakna spregnutog poprečnog preseka, kao i donjeg vlakna u betonskoj ploči. In order to verify the application of Galerkin's method in TCC system, the comparison of numerical obtained values and experimental data was done. As it was shown that the N-HIP modeldescribes the problem of TCC partial composite action on the best way, this model was applied for comparative analysis with experimental test results of TCC (EP1 and EP2) beams [18], where mechanical fasteners were used. The diagrams in Figure 8 show the stresses in cross-section of constitutive elements of TCC beams in the middle of the span, in relation to force intensity throughout experimental loading phases (F = 6, 12, 18, 24, 30 and 36 kN). The shaded surfaces represent the envelopes of the results obtained from experiments for beams EP1 and EP2, while the full and dashed lines represent the results of the numerical analysis by N-HIP model and by "γ-method" respectively. Considering the stresses (σ1,b and σ2,t) on the contact of two materials, deviations from the experimental results can be noticed, significantly for stressσ2,t at loads F = 18 and 24 kN. A good match with experimental results on the top and bottom sides of the cross-section as well as in bottom of the concrete slab is obvious. Slika 8. Uporedna analiza rezultata dobijenih N-HIP modelom i eksperimentalnih podataka Figure 8. Comparative analysis of numerical model N-HIP and experimental data Slika 8. 6 ZAKLJUČAK 6 Na osnovu predstavljenih analiza primenom Galerkinove i Ritz-ove metode, može se zaključiti da izbor probnih funkcija u formulaciji problema ima najveći uticaj na konačne vrednosti dobijenih i prikazanih rezultata. U Galerkinovoj metodi, značajan uticaj ima i sam izbor osnovnih nepoznatih (w i N). Takođe, kvalitativno poznavanje prirode rešenja može značajno doprineti smanjenju odstupanja rezultata (greške) od analitičkog rešenja. U prikazanoj analizi primenom Galerkinove metode, predloženi N-HIP model smatra se najpogodnijim za rešavanje problema elastičnog sprezanja. Kada se primenjuje varijaciona formulacija, funkcional može biti definisan putem jedne promenljive ili više njih (sila/pomeranje), dok će nepoznate koje su izabrane za osnovne biti određene s većom tačnošću od ostalih izvedenih veličina. Based on the presented analysis using Galerkin's method and Ritz method, it can be concluded that the selection of trial functions in problem formulation has the major influence on the final effect values of obtained and presented results. An important influence is also the choice of baseline unknowns (w and N) for Galerkin's method. Also, the qualitative knowledge of solution nature can significantly contribute tothe reduction of errors in obtained results related to analytical solution. In presented analysis by Galerkin's method the proposed N-HIP model qualifies as the most appropriate in order to solve the problem of partial coupling. When using a variational formulation, functional could be defined trough one or more unknowns (forces/displacements), while the unknowns that are chosen as basic will be determined with more accuracy than the derived ones. Kako su metode reziduuma ili varijacione metode prilično uobičajeni oblik formulacije u MKE, predstavljene metode Galerkina i Ritz-a mogu se uspešno primeniti prilikom definisanja spregnutog KE za elastično spregnute konstrukcije drvo–beton, a time se dobija efikasan inženjerski alat u praksi. Uvođenjem Evrokoda za spregnute sisteme drvo–beton [4], očekuje se preciznije definisanje osnovnog ulaznog parametra za različita sredstva spajanja – modula pomerljivosti spojnog sredstva K, a time i realniji odgovor spregnutih nosača u numeričkim analizama. As the weighted residual method or the variation formulation in the FEM is quite usual form, presented the Galerkin and Ritz methods could be successfully applied when defining a composite FE for partially timber- concrete composite systems, thereby enabling an efficient engineering tool in practice. ZAHVALNOST The work has been done within the scientific research project TR 36043 "Development and application of a comprehensive approach to the design of new and safety assessment of existing structures for seismic risk reduction in Serbia", which is funded by the Ministry of Science of Serbia. Ovaj rad proistekao je iz istraživačkog projekta TR 36043 „Razvoj i primena sveobuhvatnog pristupa u projektovanju novih i proceni sigurnosti postojećih konstrukcija u cilju smanjenja seizmičkog rizika u Srbiji”, koji je finansiralo Ministarstvo nauke Republike Srbije. 6 ZAKLJUČAK With introduction of Eurocode for timber-concrete composite structures [4], it is expected that more precise definition of basic input parameter - slip modulus for different types of fasteners K, will contribute to the more realistic response of composite beams in numerical analysis. 5.4 Potvrda Galerkinove metode Uporedna analiza rezultata dobijenih N-HIP modelom i eksperimentalnih podataka Figure 8. Comparative analysis of numerical model N-HIP and experimental data GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 61 (2018) 4 (37-53) BUILDING MATERIALS AND STRUCTURES 61 (2018) 4 (37-53) 51 51 7 LITERATURA REFERENCES [7] Girhammar U.A., Gopu V.K.A. (1993): Composite beam-columns with interlayer slip – Exact analysis, ASCE Journal of Structural Engineering, 119(4), pp. 1265–1282. [1] Cassel K. W. (2013): Variational Methods with Applications in Science and Engineering University Press, Cambridge. g [2] Ceccotti A. (2002): Composite concrete–timber structures, Progress Structural Engineering and Material (4), pp. 264–275. [8] Girhammar U.A., Pan D. (2007): Exact static analysis of partially composite beams and beam- columns, International Journal of Mechanical Sciences, 49, pp. 239–255. [3] Cvetković R. (2016): Mehaničko ponašanje spregnutih konstrukcija tipa drvo-beton, Doktorska disertacija, Građevinsko-arhitektonski fakultet, Univerzitet u Nišu, Niš, 204 str. [9] Girhammar U.A. (2008): Composite beam-columns with interlayer slip – Approximate analysis, International Journal of Mechanical Sciences, 50(12), pp. 1636–1649. [4] Dias A., Schänzlin J., Dietsch P. (eds.) (2018): Design of timber-concrete composite structures: A state-of-the-art report by COST FP1402/WG 4, Shaker Verlag Aachen. [10] Girhammar U.A. (2009): A simplified analysis method for composite beams with interlayer slip, International Journal of Mechanical Sciences, 51(7), pp. 515–530. g [5] EN 1995-1. (2008): Eurocode 5: Design of timber structures – Part 1–1: General – Common rules and rules for buildings, CEN, Brussels. ( ) pp [11] Kozarić Lj. (2016): Vibracije izazvane ljudskim delovanjem kod spregnutih međuspratnih konstrukcija tipa drvo-laki beton, Doktorska disertacija, Građevinski fakultet Subotica, Univerzitet u Novom Sadu, Subotica, 112 str. [6] Gelfi P., Giuriani E., Marini A. (2002): Stud Shear Connection Design for Composite Concrete Slab and Wood Beams, Journal of Structural Engineering, pp. 1544–1550. GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 61 (2018) 4 (37-53) BUILDING MATERIALS AND STRUCTURES 61 (2018) 4 (37-53) 52 [16] Reddy J. N. (2002): Energy Principles and Variational Methods in Applied Mechanics, 2nd Edition, John Wiley & Sons, Inc., New York, pp. 608. [12] Manojlović, D., Kočetov Mišulić, T. (2016): Analiza i modeliranje spregnutih sistema drvo–beton – primena na proračun mostovske konstrukcije prema EN, Građevinski materijali i konstrukcije, 59(4), pp. 47–74. [17] Stevanović B. (1994): Analiza spregnutih nosača od drveta i betona, Magistarska teza, Građevinski fakultet Univerziteta u Beogradu, Beograd, 119 str. [13] Manojlović D., Radujković A., Kočetov Mišulić T. (2017): Application of Ritz method in analysis of timber-concrete composite system, 17. International Symposium MASE, Ohrid, Faculty of Civil Engineering, pp. 721–728. [18] Stevanović B. (2004): Eksperimentalna i teorijska analiza spregnutih nosača drvo-beton izvedenih mehaničkim spojnim sredstvima, Materijali i konstrukcije 47, str. 29–46. [14] Manojlović D., Kočetov Mišulić T., Radujković A. APPLICATION OF NUMERICAL METHODS IN ANALYSIS OF TIMBER CONCRETE COMPOSITE SYSTEM Dragan MANOJLOVIĆ Tatjana KOČETOV MIŠULIĆ Aleksandra RADUJKOVIĆ Dragan MANOJLOVIC Tatjana KOCETOV MISULIC Aleksandra RADUJKOVIC Analiza i proračun spregnutih drvo–beton (SDB) konstrukcija, gde je veza konstitutivnih elemenata ostvarena mehaničkim spojnim sredstvima, predstavlja kompleksan zadatak zbog uzimanja u obzir pomerljivosti sredstava za sprezanje tj. klizanja na kontaktu dva materijala. Primena pojednostavljenih postupaka i metoda u analizi SDB nosača predstavlja pogodan i poželjan način proračuna, koji inženjerima u praksi omogućava efikasan alat. Široko rasprostranjen, pojednostavljen proračun tzv. γ-metod dat je u EN 1995. Metode zasnovane na diferencijalnoj ili varijacionoj formulaciji često su u upotrebi kada su u pitanju programi za strukturalnu analizu konstrukcija. U radu je prikazana Galerkin-ova i Ritz-ova metoda za analizu i proračun SDB nosača za slučaj proste grede izložene raspodeljenom opterećenju. Analiziran je izbor probnih funkcija koje opisuju problem elastičnog sprezanja, kao i njihov uticaj na konačne rezultate. Za potrebe numeričke analize, na osnovu predloženih numeričkih modela, napisani su kodovi u MATLAB-u. Model primenjen u analizi Galerkinovom metodom, koji najbolje opisuje problem elastičnog sprezanja, izabran je za komparativnu analizu sa eksperimentalnim podacima. The analysis and design of composite timber- concrete (TCC) structures, where the connection of the constituent elements is achieved by dowel type fasteners, is a complex task due to taking into account the slip of the coupling means, i.e. interlayer slip on the contact surface of two materials. The application of simplified procedures and methods in the analysis of the TCC system is a convenient and desirable way of design that enables efficient tool for engineering practice. Widespread simplified calculation procedure, so called "γ-method", is adopted in EN 1995. Methods based on differential or variational formulation are commonly applied when software for structural analysis are used. Galerkin's and Ritz's methods for analysis and design of TCC systems in the case of simply supported beam loaded with uniformly distributed load are shown in this paper. The selection of trial functions that describe the problem of elastic composite action as well as their influence on the final results were analyzed. For the purposes of numerical analysis, based on the proposed numerical models, several codes are written in MATLAB. The model applied in analysis by Galerkin's method, that best describes the problem of elastic coupling, was chosen for further comparative analysis with experimental data. Ključne reči: Ritz-ova metoda, Galerkinova metoda, γ-metod, slaba formulacija, numerička analiza, MKE, sprezanje drvo–beton, elastično sprezanje, klizanje u spoju. REZIME SUMMАRY 7 LITERATURA REFERENCES (2018): Weighted residual method in analysis of timber concrete composite system, Zbornik radova sa nacionalnog kongresa, 15. Kongres Društva građevinskih konstruktera Srbije, Zlatibor, pp. 573- 582. [19] Szabó B. (2006): Influence of shear connectors on the elastic behaviour of composite girders, Doctoral dissertation, Helsinki University of Technology, Espoo, Finland, pp. 124. [15] MATLAB R2014a (2014): MathWorks Inc. The Language of Technical Computing. APPLICATION OF NUMERICAL METHODS IN ANALYSIS OF TIMBER CONCRETE COMPOSITE SYSTEM Key words: Ritz's method, Galerkin's method, γ- method, Weak form, Numerical analysis, FEM, Timber- concrete composite, Partial interaction, Interlayer slip. GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 61 (2018) 4 (37-53) BUILDING MATERIALS AND STRUCTURES 61 (2018) 4 (37-53) 53
https://openalex.org/W4283759783	https://hal.science/hal-03874607/file/Benjamin%20et%20al.%20-%202022%20-%20Tracking%20transitional%20probabilities%20and%20segmenting.pdf	English	null	Tracking transitional probabilities and segmenting auditory sequences are dissociable processes in adults and neonates	Developmental science	2,022	cc-by	14,109	Tracking transitional probabilities and segmenting auditory sequences are dissociable processes in adults and neonates Lucas Benjamin, Ana Fló, Marie Palu, Shruti Naik, Lucia Melloni, Ghislaine Dehaene-Lambertz Lucas Benjamin, Ana Fló, Marie Palu, Shruti Naik, Lucia Melloni, Ghislaine Dehaene-Lambertz To cite this version: Lucas Benjamin, Ana Fló, Marie Palu, Shruti Naik, Lucia Melloni, et al.. Tracking transitional probabilities and segmenting auditory sequences are dissociable processes in adults and neonates. Developmental Science, 2022, 2022, pp.e13300. 10.1111/desc.13300. hal-03874607 Tracking transitional probabilities and segmenting auditory sequences are dissociable processes in adults and neonates KEY WO RDS KEY WO RDS EEG, language learning, neonates, prosody, sequence learning, statistical learning Tracking transitional probabilities and segmenting auditory sequences are dissociable processes in adults and neonates Lucas Benjamin1 Ana Fló1 Marie Palu1 Shruti Naik1 Lucia Melloni2,3 Ghislaine Dehaene-Lambertz1 1Cognitive Neuroimaging Unit, CNRS ERL 9003, INSERM U992, CEA, Université Paris-Saclay, NeuroSpin Center, Gif-sur-Yvette, Île-de-France, France R E S E A R C H A R T I C L E R E S E A R C H A R T I C L E HAL Id: hal-03874607 https://hal.science/hal-03874607v1 Submitted on 28 Nov 2022 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 - NonCommercial - NoDerivatives 4.0 International License Distributed under a Creative Commons Attribution - NonCommercial - NoDerivatives 4.0 International License Received: 9 November 2021 Revised: 31 May 2022 Accepted: 11 June 2022 Received: 9 November 2021 Revised: 31 May 2022 Accepted: 11 June 2022 DOI: 10.1111/desc.13300 DOI: 10.1111/desc.13300 Correspondence KEY WO RDS EEG, language learning, neonates, prosody, sequence learning, statistical learning Developmental Science. 2022;e13300. https://doi.org/10.1111/desc.13300 Abstract Since speech is a continuous stream with no systematic boundaries between words, how do pre-verbal infants manage to discover words? A proposed solution is that they might use the transitional probability between adjacent syllables, which drops at word boundaries. Here, we tested the limits of this mechanism by increasing the size of the word-unit to four syllables, and its automaticity by testing asleep neonates. Using markers of statistical learning in neonates’ EEG, compared to adult behavioral performances in the same task, we confirmed that statistical learning is automatic enough to be efficient even in sleeping neonates. We also revealed that: (1) Success- fully tracking transition probabilities (TP) in a sequence is not sufficient to segment it. (2) Prosodic cues, as subtle as subliminal pauses, enable to recover words segmenting capacities. (3) Adults’ and neonates’ capacities to segment streams seem remarkably similar despite the difference of maturation and expertise. Finally, we observed that learning increased the overall similarity of neural responses across infants during expo- sure to the stream, providing a novel neural marker to monitor learning. Thus, from birth, infants are equipped with adult-like tools, allowing them to extract small coher- ent word-like units from auditory streams, based on the combination of statistical analyses and auditory parsing cues. 2Department of Neuroscience, Max Planck Institute for Empirical Aesthetics, Frankfurt am Main, Hessen, Germany Correspondence Lucas Benjamin, Cognitive Neuroimaging Unit, CNRS ERL 9003, INSERM U992, CEA, Université Paris-Saclay, NeuroSpin center, 91191 Gif/Yvette, France. Email: lucas.benjamin@cea.fr 2 of 16 1 INTRODUCTION 2 of 16 BENJAMIN ET AL. One of the limitations of statistical learning, already reported in the literature, is its interaction with alternative segmentation cues (Black & Bergmann, 2017), especially its embedding in prosodic units. A word cannot straddle a prosodic boundary. Therefore, even if two syllables are always presented in succession, they are not attributed to the same word if a prosodic boundary separates them. This property is observed in adults (Shukla et al., 2007) and in 5–8-month-old babies (Johnson & Tyler, 2010; Shukla et al., 2011). This result is not surprising given the importance of prosody to structure the speech signal. A hierarchy of prosodic units (Nespor & Vogel, 2006) roughly parallel to the syntactic tree is used to improve speech comprehension in adults and to favor language acquisition in infants. For example, even at 2 months of age, infantsmemorize better the phonetic contentofa sentence witha well- formed prosodic contour relative to a word list (Mandel et al., 1994). This advantage can be explained because statistical computations are limited to a few elements within the prosodic unit, relieving memory. Prosodic units also provide perceptual anchors, which help infants note the reproducible location of certain words at their edges, such as arti- cles or proper name. Finally, the higher frequency of function words relative to content words has also been proposed as anchors favor- ing word discovery (Hochmann et al., 2010). To succeed in the complex task of constructing a lexicon from natural speech, infants have a tool- box of procedures at their disposal, whose relative contributions are currently underspecified. One of the main challenges encountered by infants to learn their native language and construct their lexicon is that words are rarely produced in isolation. Instead, words are embedded in sentences with no system- atic silence or clear acoustic boundaries between them. Subtle acous- tical markers such as the lengthening of the last syllable, pitch change, slowing-down of the syllabic rate and less coarticulation between syl- lables can signal words ending. But adults rely mainly on lexical knowl- edge and sentential context to retrieve words in their native language (Mattys et al., 2005) and in an unknown language, they have great dif- ficulty in correctly segmenting sentences into words (Wakefield et al., 1974). 2 of 16 1 INTRODUCTION However, when the experimental task is simplified by using an artificial stream of concatenated words, these acoustical cues can be used to discover the possible words as shown by their above-chance accuracy in forced-choice tasks (Bagou & Frauenfelder, 2018). Simi- larly, neonates can detect these subtle variations in a binary situation in which they have to discriminate pseudo-words constituted of syllables either coming from inside a word (e.g., /mati/ from mathematicien) or from two successive words (e.g., /mati/ from pyjama tissé) (Christophe et al., 1994). However, the relative weights of these markers vary across languages (Ordin et al., 2017) and within a language (i.e., they depend on the position of the word in the sentence and interact with other prosodic features such as lexical stress). Therefore, the robust- ness of these word-boundary cues is commonly estimated as insuffi- cient for infants to segment natural speech in successive word units. Here we investigated another putative limitation of the statistical learning mechanism: the size of the words that can be learned. In fact, most, if not all, studies in infants have used tri-syllabic words. Is it due to particular experimental choices? Or is there a hard limit to segmenta- tion based on statistical computation, especially in immature infants? If the latter, can subtle prosodic cues rescue segmentation and word learning, allowing memory processes to deploy (Fló et al., 2022)? To investigate these questions, we created a first artificial stream con- sisting of four quadri-syllabic words, pseudo-randomly concatenated without any prosodic cue, and a second one strictly identical to the first one but with a 25 ms pause between each word, every four syllables. In previous studies of artificial streams with this short pause, adults reported not noticing it and were at chance when they had to choose which of the two streams had pauses (Peña et al., 2002). Neverthe- less, pauses significantly improved their performances (Buiatti et al., 2009; Peña et al., 2002). The pause was probably perceived as a vowel lengthening, a universal ending cue for words and musical segments (Tyler & Cutler, 2009). In adults, final syllable lengthening improved tri- syllabic word segmentation (Saffran et al., 1996). The authors proposed a putative hierarchy in using these cues, i.e., infants first rely on transi- tionalprobabilities,thennoticethatsyllablelengtheningcoincideswith a word ending to finally learn this new cue. Research Highlights ∙Successfully tracking transitional probabilities in a sequence is not always sufficient to segment it. ∙Word segmentation solely based on transitional probability is limited to bi- or tri- syllabic elements. ∙Prosodic cues, as subtle as subliminal pauses, enable to recover chunking capacities in sleeping neonates and awake adults for quadriplets. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2022 The Authors. Developmental Science published by John Wiley & Sons Ltd. Developmental Science. 2022;e13300. wileyonlinelibrary.com/journal/desc 1 of 16 https://doi.org/10.1111/desc.13300 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2022 The Authors. Developmental Science published by John Wiley & Sons Ltd. D l t l S i 2022 13300 il li lib /j l/d 1 f 16 2 of 16 1 INTRODUCTION 3 of 16 FIGURE 1 (a) Experimental design: Participants were first exposed to a structured stream comprising four quadri-syllabic words (called ABCD) then presented with 3 types of isolated test words. (b) Experimental procedure: Neonates were tested asleep using high-density EEG (128 channels) while they were presented with random stream, structured stream, and isolated words. Short Str: short structured streams were presented to the neonates to maintain learning. Adults were tested on a web platform. After familiarization with the structured stream, adults were asked to rank the familiarity on a scale (1–6). To avoid a bias to quadri-syllabic words, they were also presented with foils corresponding to three other types of bi-syllabic test words (see Section 5 for more details) BENJAMIN ET AL. 3 of 16 FIGURE 1 (a) Experimental design: Participants were first exposed to a structured stream comprising four quadri-syllabic words (called FIGURE 1 (a) Experimental design: Participants were first exposed to a structured stream comprising four quadri-syllabic words (called ABCD) then presented with 3 types of isolated test words. (b) Experimental procedure: Neonates were tested asleep using high-density EEG (128 channels) while they were presented with random stream, structured stream, and isolated words. Short Str: short structured streams were presented to the neonates to maintain learning. Adults were tested on a web platform. After familiarization with the structured stream, adults were asked to rank the familiarity on a scale (1–6). To avoid a bias to quadri-syllabic words, they were also presented with foils corresponding to three other types of bi-syllabic test words (see Section 5 for more details) response, which is observed as a power increase at the frequency of the syllable presentation. If the syllables are perceived grouped in a quadri-syllabic word, the power should increase at ¼ of the syllable frequency (⅓if tri-syllabic words are used). Such a power increase at the word frequency has indeed been reported for tri-syllabic words in adults (Batterink & Choi, 2021; Benjamin et al., 2021; Buiatti et al., 2009), in 6–8-month-old infants and in neonates (Choi et al., 2020; Fló et al., 2022; Kabdebon et al., 2015). We also presented neonates with “random” streams constituted of pseudo-randomly concatenated sylla- bles, with and without a pause every four syllables, to control whether the pause itself was sufficient to induce a 4-syllable-rhythm. 2 of 16 1 INTRODUCTION Yet, this hypothesis remains untested because the relative contribution of transitional probabilities and this subtle prosodic cue was not assessed in this study. A second mechanism, based on the analysis of the transitions between syllables, has thus been proposed. Within a word, syllables have a fixed order, whereas any syllable can follow the last syllable of a word. Thus, a word boundary corresponds to a drop in the tran- sition probabilities (TP) between consecutive syllables. To prove that the concept could apply for word learning in infancy, Saffran, Aslin,and Newport (1996) used a mini-language of four tri-syllabic words and tested 8-month-old infants who listened for 3 mins to a continuous stream in which these words were concatenated with a flat intonation. The authors reported that infants were subsequently able to distin- guish two different lists of isolated tri-syllables pseudo-words: one corresponding to the Words (i.e., ABC:TP were equal to 1 between each syllable) and the other to PartWords formed by the two last syl- lables of a word and the first syllable of the next word for example (i.e., BCA’:TP were equal to 1 and 0.33). This result has been replicated mul- tiple times (Black & Bergmann, 2017) and extended to non-linguistic stimuli (Saffran et al., 1999; Schön et al., 2008) and to the visual domain (Fiser & Aslin, 2002). Sensitivity to statistics in sequences is also observed in animals (Hauser et al., 2001; James et al., 2020; Toro & Trobalón, 2005) indicating that the capacity of extracting transitional probabilities between subsequent elements is a robust general mech- anism. Additionally, it has been reported in asleep neonates (Fló et al., 2019; Fló, Benjamin, et al., 2022; Teinonen et al., 2009); and to some extent, in inattentive adults (Batterink & Choi, 2021; Benjamin et al., 2021; Fernandes et al., 2010; Toro et al., 2005). Yet the limits of this mechanism and the influence of development and expertise on the performances are still poorly known. We used high-density EEG (128 channels) to evaluate segmenta- tion processes in neonates. EEG allows not only to observe different responses to test-words after learning, but also to track learning while neonates are listening to the artificial stream. As the syllables have exactly the same length, their perception creates a regular evoked BENJAMIN ET AL. 2 of 16 1 INTRODUCTION In adults, inserting such a pause in a random stream did not produce any increase of power at the pause frequency (Buiatti et al., 2009). Therefore, in the case of successful segmentation, we expected a significant power increase at the word frequency in the structured streams relative to the random streams. No change, or perhaps a decrease, was expected at the syllabic rate, in line with previous reports in adults in which per- ceiving the word induced a decrease of the entrainment at the syllabic rate (Batterink & Choi, 2021; Benjamin et al., 2021). indicates an ill-formed word. In ShuffleWords, the two middle sylla- bles of a Word were inverted, violating local position. Thus, while all the transitional probabilities were zero, all syllables were always heard in close proximity during the learning stream. This temporal proximity might induce memory errors and a wrong recognition of ShuffleWords as possible words. Indeed, in longer words of six-syllables, neonates are not able to detect a shuffle of the middle syllables, whereas they detect a shuffle of the edges syllables (Ferry et al., 2016). Thus, our experimental design provides several markers of transi- tional probability computation and word segmentation that might be differently associated, opening the possibility to disentangle several steps or hypotheses of this classical learning task. (H1: TP computa- tion) If infants computed TP and memorized the TP matrix, they should reject Words from Shuffle- Words (1+1+1 vs. 0+0+0) but marginally Words from PartWords (1+1+1 vs. 1+0.33+1). (H2: segmentation) Stream segmentation should create an increase of neural entrainment at the word frequency. (H3: complete memorization of the word) should create a difference between words on one side and PartWords and ShuffleWords on the other side. (H4: memory errors) If Words are seg- mented and swap errors occur, ShuffleWords should not differ from Words due to the temporal proximity of the syllables belonging to the same Word. (H5: first syllable memorization only). This hypothesis could explain why words are preferred over PartWords in many statistical learning studies. As the typical trisyllabic paradigm compares Words (ABC) to PartWords (BCA’), the difference observed could result from After the learning phase, three types of test-words were presented in isolation: Words, PartWords, and ShuffleWords (Figure 1). Success- ful word segmentation is commonly revealed by a significant difference between the measured response to Words and PartWords. 2.1 Adults As described in other studies on neural entrainment (Fló, et al., 2022; Hochmann et al., 2010; Kabdebon et al., 2015), there was a signifi- cant increase in power and Phase Locking Value (PLV) at the frequency of the syllables presentation compared to neighboring frequencies in both groups (continuous and with pauses) and stream types (random and structured) (all ps < 0.05 FDR corrected). No interaction was observed between groups and streams indicating a similar signal-to- noise ratio and comparable experimental conditions in the two groups (all ps > 0.05 FDR corrected). The power analysis for each condition and group is presented in the supplementary material. Two groups of adults were tested online on a web platform (n = 43). After having listened to the continuous stream, or to the stream with 25 ms subliminal pauses depending on the group, the participants had to judge the familiarity of three types of words (Words, Part-Words and Shuffle-Words). Exposure (3.3 mn) and test were performed two times and results of the two tests were aggregated (see Section 5 and Figure S5 for results in each test). We analyzed the responses by items in a linear mixed-effects model in each group with FDR correction. For the stream without subliminal pause at the end of the word, Words and Part-Words were similarly rated (p = 0.26) and estimated more famil- iar than the ShuffleWords (W vs. SW p < 0.001, PW vs. SW p < 0.01). When subliminal pauses were added at the end of the words in the stream, all types of words were ranked differently (all p < 0.001) with the following order: Words were judged more familiar than PartWords themselves more familiar than ShuffleWords (Figure 1a,b). To better visualize the difference in segmentation performances between the two groups, we calculated the difference in mean familiarity ranking given by each participant to Words and PartWords, and performed an unpaired unidirectional, t-test t(41) = 2.3, p = 0.013. The segmentation effect, seen as a positive value in Figure 2c, was larger when subliminal pauses were present (Figure 2c). Stream segmentation should be revealed by a significant increase of power and/or PLV at the frequency of the words (i.e., ¼ of the syllabic frequency) relative to the random stream. by a subliminal acoustic cue. Yet even when there was no pause, they rejected ShuffleWords because of null transitional probabilities. the encoding of the first syllable only (A vs. B). In a recent study with tri-syllabic words, we indeed observed an ERP difference between words and PartWords for the first syllable, whereas no difference was recorded when the last syllable was incorrect (Fló, et al., 2022). 2.2 Finally, comparing the two groups of neonates, one listening to the stream without pauses (continuous group) and the other to the stream with pauses (with pauses group), should clarify the relative contribution of auditory parsing cues and transitional probabilities to word segmentation at that age. This comparison should disentangle whether pauses rescue segmentation, subsequently allowing the com- putation of transitional probabilities on smaller segments, or whether the computation of TP is done independently of the segmentation process. EEG was recorded in two groups of healthy full-term neonates (n = 52 after rejection procedure, see Section 5) while they were listening to streams without pauses for the first group and with 25 ms pauses every four syllables for the second group. For each group, neonates were exposed first to ∼7 min of a “random” stream in which syllables were randomly concatenated with a flat TP of 0.33 with and without a subliminal pause every four syllables depending on the group, fol- lowed by ∼13.5 min of the word-structured stream. After the exposure learning phase, a test phase followed in which they were exposed to isolated quadri-syllables sequences (Words, PartWords, and Shuffle- Words). To avoid interference with learning in the testing phase and to reinforce the learning of the structured materials, 40s-short segments of the structured stream were presented every 12 words during this phase. Finally, another ∼7 min of the random stream was presented again after this testing phase. The division of the random stream into two periods was done to avoid a time confound in the comparison between random and structured streams. We used a longer exposure time than usual statistical learning experiments in order to perform pattern similarity analyses as done by Henin et al (2021). Neonates are two-decades far from a mature state in terms of linguistic abilities but also in terms of memory capacities. To our knowl- edge, no adult equivalent of the paradigm proposed here was available. Thus, we collected adult behavioral data as a mature model of the mechanisms we explored in neonates. We adapted the paradigm to collect behavioral responses via a web-based procedure and short- ened the habituation to avoid over-learning already reported in similar experiments in adults (Peña et al., 2002) (see Figure 1). Despite different procedures and learning indicators, the results were sur- prisingly congruent with those obtained in infants, especially showing comparable limitations. 4 of 16 BENJAMIN ET AL. 2 of 16 1 INTRODUCTION In Words, all transitional probabilities between syllables equal 1, while in Part- Words (straddling two words), a drop in transitional probabilities 2.2.3 Syllable pattern similarity analysis In a recent paper, Henin et al. (2021) proposed that pattern similar- ity between syllables can vary with learning in a similar task. More specifically, using electrocorticography in epileptic adult patients who listened to a structured stream composed of the concatenation of four trisyllabic words, they computed different patterns of similar- ity between the 12 syllables. They took advantage of the high spatial resolution of electrocorticography and observed different clusters of electrodes sensitive either to TP transitions (low vs. high TP), the ordi- nal position (1st vs. 2nd vs. 3rd syllable), or the word identity (word 1 vs. word 2 vs. word 3 vs. word 4) in different brain areas. We computed a similar analysis on the responses to the syllables during the structured stream and showed that the similarity pattern for syllables belonging to the same words was significantly increased in the pause group com- pared to the continuous group (p = 0.012). However, we failed to find an increase in pattern similarity for low TP (DA’) and ordinal position (AA’,BB’,CC’,andDD’)betweenthetwogroups.Toinvestigateifthesig- nificant increase in similarity for syllables belonging to the same word was only due to an increase in high TP pairs or all pairs belonging to the same word, we separated the word condition in two sub-conditions: Consecutive (AB, BC, CD) and non-Adjacent (AC, BD & AD). Interest- ingly,Consecutiveandnon-adjacentpairsshowedasignificantincrease BENJAMIN ET AL. 5 of 16 BENJAMIN ET AL. 5 of 16 FIGURE 2 Adult behavioral results. (a,b) Results of the familiarity ranking tests in adults subjects for each item. Both test sessions were aggregated. Results for each session are presented in Figure S5. (c) Interaction at the subject level between both groups on the main effect of segmenting (Word - PartWord). Dots represent familiarity ranking difference between Words and PartWords in each subject. p-Value is estimated using one-way unpaired t-test FIGURE 2 Adult behavioral results. (a,b) Results of the familiarity ranking tests in adults subjects for each item. Both test sessions were aggregated. Results for each session are presented in Figure S5. (c) Interaction at the subject level between both groups on the main effect of segmenting (Word - PartWord). Dots represent familiarity ranking difference between Words and PartWords in each subject. p-Value is estimated using one-way unpaired t-test continuous group (Figure 4a Right). No difference was observed during the random streams (Figure 4b Right). frequencies of interest over sliding time windows of 2 min with a 1.5 s step, similarly to Fló, et al. (2022). We observed no change at the word frequencyalong time for either group (Figure S3). The poor signal/noise ratio at these low frequencies might explain the poor sensitivity of this analysis. FIGURE 2 Adult behavioral results. (a,b) Results of the familiarity ranking tests in adults subjects for each item. Both test sessions were aggregated. Results for each session are presented in Figure S5. (c) Interaction at the subject level between both groups on the main effect of segmenting (Word - PartWord). Dots represent familiarity ranking difference between Words and PartWords in each subject. p-Value is estimated using one-way unpaired t-test 2.1 Adults In the first group (continu- ous stream), we failed to find this result, contrary to the second group (stream with pause), in whom a significant increase of both power and PLV was observed in several electrodes. Finally, the interaction between groups and streams was significant for both power and PLV on several electrodes (Figure 3, all ps < 0.05 cluster corrected, Figure S2 for the results for each stream in each group). It has been described that the power at the syllabic rate decreased when adults segment the stream (Buiatti et al., 2009). However, we did not find any modulation of the power or PLV at the syllabic fre- quency in the structured stream compared to the random one. We also performed a time-course analysis of the neural entrainment at the Thus, adults were able to distinguish Words from PartWords, indi- cating that they had correctly segmented the stream only if helped 5 of 16 s FIGURE 3 Neural entrainment analyses at the word frequency in the structured stream minus the random stream (Power and phase locking value [PLV]) in the two groups of neonates (continuous and with pauses). Top rows: the presentation of stimuli with a fixed duration evoked a reproducible time-locked neural response that can be recovered as a neural oscillation at the frequency of stimulation. If infants segment the structure streams based on the quadri-syllabic words, an increase at the word frequency should be observed relative to the random stream. It is what is seen in the second group of neonates (with pauses) who listened to the streams with sub-liminal pauses. The acoustical effect of pauses was controlled by also adding a pause every four syllables in the random stream in this group. The last column shows the interaction between groups and frequencies. Dots locate the electrodes showing a significant result at p < 0.05 uncorrected, and larger dots after cluster correction (cluster p < 0.05). Power during structured and random streams are presented separately in each group in Figure S2 ing in the occipital ROI (0.05 < p < 0.1). Informed by the adult results, we compared Words and PartWords against ShuffleWords—the only condition rejected by the adults. This contrast revealed a significant difference in the frontal ROI (time: [1253–1644]ms, p = 0.025), and a trend was observed in the occipital ROI (0.05 < p < 0.1) (Figure 5 first panel). In the group with pauses, as shown in the second row of Figure 5, the response to Words seems to stand out from the other two conditions. ERPs to Words indeed significantly differed from those to PartWords in the two ROIs (all p < 0.05 Frontal: [1224–1780]ms, Occipital: [1196–1768]ms), and from those to Shuffle words in the occipital ROI ([1196–1768]ms) whereas two clusters were showing a trend (0.05 < p < 0.1) in the frontal ROI. in pattern similarity (both p < 0.05 FDR corrected) with pauses com- pared to the continuous group. The differences in pattern similarity between the two groups for each condition are reported in Figure 4c. 6 of 16 BENJAMIN ET AL. FIGURE 3 Neural entrainment analyses at the word frequency in the structured stream minus the random stream (Power and phase locking value [PLV]) in the two groups of neonates (continuous and with pauses). Top rows: the presentation of stimuli with a fixed duration evoked a reproducible time-locked neural response that can be recovered as a neural oscillation at the frequency of stimulation. If infants segment the structure streams based on the quadri-syllabic words, an increase at the word frequency should be observed relative to the random stream. It is what is seen in the second group of neonates (with pauses) who listened to the streams with sub-liminal pauses. The acoustical effect of pauses was controlled by also adding a pause every four syllables in the random stream in this group. The last column shows the interaction between groups and frequencies. Dots locate the electrodes showing a significant result at p < 0.05 uncorrected, and larger dots after cluster correction (cluster p < 0.05). Power during structured and random streams are presented separately in each group in Figure S2 BENJAMIN ET AL. 6 of 16 6 of 16 BENJAMIN ET A FIGURE 3 Neural entrainment analyses at the word frequency in the structured stream minus the random stream (Power and phase locking value [PLV]) in the two groups of neonates (continuous and with pauses). Top rows: the presentation of stimuli with a fixed duration evoked a reproducible time-locked neural response that can be recovered as a neural oscillation at the frequency of stimulation. If infants segment the structure streams based on the quadri-syllabic words, an increase at the word frequency should be observed relative to the random stream. It is what is seen in the second group of neonates (with pauses) who listened to the streams with sub-liminal pauses. The acoustical effect of pauses wa controlled by also adding a pause every four syllables in the random stream in this group. The last column shows the interaction between groups and frequencies. Dots locate the electrodes showing a significant result at p < 0.05 uncorrected, and larger dots after cluster correction (cluster p < 0.05). Power during structured and random streams are presented separately in each group in Figure S2 6 of 16 BENJ FIGURE 3 Neural entrainment analyses at the word frequency in the structured stream minus the random stream (Power and phase value [PLV]) in the two groups of neonates (continuous and with pauses). Top rows: the presentation of stimuli with a fixed duration evok reproducible time-locked neural response that can be recovered as a neural oscillation at the frequency of stimulation. If infants segmen structure streams based on the quadri-syllabic words, an increase at the word frequency should be observed relative to the random stre what is seen in the second group of neonates (with pauses) who listened to the streams with sub-liminal pauses. The acoustical effect of p controlled by also adding a pause every four syllables in the random stream in this group. The last column shows the interaction between and frequencies. Dots locate the electrodes showing a significant result at p < 0.05 uncorrected, and larger dots after cluster correction p < 0.05). Power during structured and random streams are presented separately in each group in Figure S2 2.2.2 Between-subjects correlation analysis Because the exact same stream was used in each participant, we were able to analyze whether learning increased the global neural synchro- nization between neonates beyond neural entrainment. To do so, we tested whether the correlation between participants increased over time more when they listened to the structured stream. We then com- pared at each time the topography of each subject with the average of the other subjects’ topographies at that time. We observed a pro- gressive increase of the mean correlation between subjects in neural activity only in the second group with pauses (Figure 4a Left). Indeed, the increase was higher for the second group (with pauses) than the first one (continuous) (cluster corrected p < 0.01, time [88-820]s; Figure 4a). During the random streams, the correlations were flat rel- ative to baseline in both groups (Figure 4b Left). To confirm this effect, we computed a linear regression of the variation of subject correlation with the group with time at the subject level during each stream and compared the slopes in the two groups. Only when neonates listened to the structured stream with pauses (second group), the slope was signif- icantly positive and significantly greater than the same measure in the 2.2.4 ERP analysis For this analysis, we used the auditory localizer to determine regions of interest (ROI) for each group (see Section 5). A click preceding each word allows to isolate the two poles of the auditory response in each group and the time series of the electrodes of these data-driven ROIs were averaged together before we performed permutation cluster- based analyses on time as implemented in Fieldtrip (Oostenveld et al., 2011). In the continuous group, the response was flatter for Shuffle- Words compared to the other two conditions. When the conditions were contrasted two by two, we only observed that Word versus Shuf- fleWord tended to differ during two time-periods in the frontal ROI ([1308–1652]ms, p = 0.03; [1780–2000]ms, p = 0.05) but only trend- We also tested the interaction between groups and the main effect Words – Part Words and found significant interactions (p < 0.05) on both the frontal and occipital ROIs (Figure 5 last panel). Using a permu- tation cluster-based method (field trip cluster analysis) between 250 and 2000 ms without previous ROI extractions, we obtained similar results (Figure S4). BENJAMIN ET AL. 7 of 16 FIGURE 4 Correlation analysis. (a) Comparison of the correlation between neonates in the two groups during the structured stream. Left: Evolution of the correlation across neonates with time. Right: comparison of the slopes of the linear regression with time in each group (orange: ontinuous, blue: with pauses). (b) Similar analysis for the first (plain lines) and second random (dotted lines) streams in both groups. RND = Random. (c) Pattern Similarity analysis: We computed the increase of pattern similarity between the EEG response to each syllable in the wo groups during the structured stream. The similarity significantly increased for syllables belonging to the same word (for adjacent pairs: AB, BC, CD, and non-adjacent pairs AC, BD, and AD) BENJAMIN ET AL. 7 of 16 FIGURE 4 Correlation analysis. (a) Comparison of the correlation between neonates in the two groups during the structured stream. Left: Evolution of the correlation across neonates with time. Right: comparison of the slopes of the linear regression with time in each group (orange: continuous, blue: with pauses). (b) Similar analysis for the first (plain lines) and second random (dotted lines) streams in both groups. RND = Random. 3 DISCUSSION ous group contrary to what we obtained in a similar paradigm with tri-syllabicwords.Becausethe“noise”levelduetothebackgroundneu- ral activity is exponentially growing with low frequencies in EEG data, notably in neonates, the lack of neural entrainment for quadruplets might have been due to a lack of sensitivity of the method at 1 Hz, com- pared to 1.33 Hz in the case of triplets. However, the significant word entrainment in the group listening to the stream with pauses and the interaction between both groups confirm that neural entrainment is a sensitive method even at these low frequencies. In natural speech, many signal-derived cues might assist segmenta- tion (Wakefield et al., 1974), but none is robustly consistent to be systematically used by infants. Therefore, the computation of the transitions probabilities between syllables has been proposed as a possible solution (Saffran, Aslin, et al., 1996). We presented here a stream comprising quadri-syllabic words to investigate the efficiency of this strategy for longer words. Whereas tri-syllabic words are eas- ily extracted from a flat speech stream using TP between syllables in adults (Saffran, Newport, et al., 1996), infants (Saffran, Aslin, et al., 1996), and even sleeping neonates (Fló, et al., 2022), this single cue seemed insufficient here for quadri-syllabic words even in awake vigilant adults, revealing a clear limitation of the statistical learning mechanism in a segmentation task. Whereas the power increase at the syllabic frequency was large during all streams, we did not record a significant neural entrainment at the word frequency in the continu- In natural speech, many signal-derived cues might assist segmenta- tion (Wakefield et al., 1974), but none is robustly consistent to be systematically used by infants. Therefore, the computation of the transitions probabilities between syllables has been proposed as a possible solution (Saffran, Aslin, et al., 1996). We presented here a stream comprising quadri-syllabic words to investigate the efficiency of this strategy for longer words. Whereas tri-syllabic words are eas- ily extracted from a flat speech stream using TP between syllables in adults (Saffran, Newport, et al., 1996), infants (Saffran, Aslin, et al., 1996), and even sleeping neonates (Fló, et al., 2022), this single cue seemed insufficient here for quadri-syllabic words even in awake vigilant adults, revealing a clear limitation of the statistical learning mechanism in a segmentation task. 2.2.4 ERP analysis (c) Pattern Similarity analysis: We computed the increase of pattern similarity between the EEG response to each syllable in the two groups during the structured stream. The similarity significantly increased for syllables belonging to the same word (for adjacent pairs: AB, BC CD, and non-adjacent pairs AC, BD, and AD) FIGURE 5 ERP analysis. Grand average ERPs to the test-words in both groups and to the word minus part word difference. The ROIs correspond to the two poles of the response to the auditory localizer preceding the test word in each group. Dark gray areas identify significant temporal clusters. Light shaded areas surrounding the thick lines represent the standard error across neonates. W = Words (ABCD), PW = Part Words (CDA’B’) and SW = ShuffleWords (ACBD). Gray lines at the bottom of the plots indicate the time windows on which statistics were performed FIGURE 5 ERP analysis. Grand average ERPs to the test-words in both groups and to the word minus part word difference. The ROIs correspond to the two poles of the response to the auditory localizer preceding the test word in each group. Dark gray areas identify significant temporal clusters. Light shaded areas surrounding the thick lines represent the standard error across neonates. W = Words (ABCD), PW = Part Words (CDA’B’) and SW = ShuffleWords (ACBD). Gray lines at the bottom of the plots indicate the time windows on which statistics were performed of a pause but to a genuine learning process, as it was not observed for the random stream that also included pauses every four sylla- bles. Second, neural synchrony increased between participants only for the structured stream with pauses, further suggesting that neonates were following a similar learning process constraining their brain state in this condition. Again, this phenomenon was only observed for the structured stream and notfor the random stream withpauses. Itunder- scores that it was not a general difference between the two groups of neonates but was related to the learning process engaged when they listened tothe structured stream. Third, ERPstoWordsand PartWords were significantly different after the stream with pauses, a classi- cal indicator of word segmentation in this type of paradigm. Finally, adults also ranked Words higher than PartWords when they listened to streams with pauses relative to streams without pauses, confirming an undeniable advantage for the former over the latter. All these indica- tors of successful segmentation were not only lacking when the pause cue was not present, but for all of them, the differences between the two groups were significant in both infants and adults. 3 DISCUSSION Whereas the power increase at the syllabic frequency was large during all streams, we did not record a significant neural entrainment at the word frequency in the continu- The word segmentation failure is also not explained by the higher number of syllables to be memorized (16 syllables here vs. 12 for tri- syllabic words) and the larger word size since the same material, just with the addition of sub-liminal pauses, rescued the word extraction process. Furthermore, we recorded several other indicators of learning in the second group who listened to the stream with pauses: First, the increase in power and phase locking-value observed at the word fre- quency in the structured stream was not related to the mere presence BENJAMIN ET AL. 8 of 16 3.1 Word segmentation based on statistical learning is limited by the word size Part-Words created by the pauses faded away (Figure S5) suggest- ing that the weight attributed to each parameter might not be strictly hierarchized but dependent on the strength of the evidence provided. It was proposed that the computation of the TP might be used to seg- ment a speech stream, either through boundary markers—a TP drop creates a prediction error, and the surprise allows to memorize the syl- lable following the drop (i.e., the first syllable of the following word)—or because adjacent events acquired a similar representation. However, neither the local drop of TP nor the temporal proximity within a chunk were sufficient to structure the stream, not even after 13 mins of expo- sure when the unit size was four syllables (1s long). On the contrary, sleeping neonates perceived a tri-syllabic rhythm in the same circum- stances and only after 2 mins of exposure, and memorized the set of possible first syllables (Fló, et al., 2022). It remains possible that longer exposure to the continuous stream might eventually allow word segmentation. However, compared to the segmentation of tri-syllabic words tested under similar conditions, both neonates and adults had considerable difficulty performing the task with quadri-syllabic words. We also observed in infants that similarity between syllables within the word was increased relative to the continuous stream without pauses (Figure 4c). Not only similarity between adjacent syllables within a word was stronger in the stream with pauses than without pauses, but similarity also increased between more distant syllables belonging to the same word. We cannot disentangle whether this increase in similarity between syllables in a word induced the seg- mentation as in a clustering strategy that is opposed to a bracketing strategy in which splitting points are looked for (Swingley, 2005), or the reverse, that is, because syllables were perceived in the same chunk, their similarity increased. It is also interesting to note that perceiving the stream at a more complex level of representations increased neural synchrony between the neonates. Whereas the syllabic rate itself, which affects many chan- nels (see Figure 2 in appendix), already creates a strong and similar entrainment across participants, it is not this low-level cue that was predominant in the neural synchrony between neonates but the per- ception of a higher level of organization of the stream. 3.2 Rescuing segmentation with sub-liminal pauses Adding a subliminal pause at the end of the word radically affects the performances at both ages. Although not consciously perceived, pauses act as other word boundary markers (e.g., lengthening of the last syllable, pitch drop) that neonates can perceive (Christophe et al., 1994). Our result demonstrates that such a word boundary marker is not only perceived but is effectively used to segment a stream from birth on, that is, before infants have perceived many isolated words. The use of word-ending cues, at least when it is a pause as here, does not need that infants first learn words as it was postulated (Saffran, Newport, et al., 1996) but is part of the auditory/linguistic percep- tive system. This observation is in agreement with the proposal of a hierarchical framework in weighting the multiple segmentation cues (Wakefield et al., 1974) and the subordination of statistical learning to many other cues, such as coarticulation (Fernandes et al., 2007), prosodic contour (Shukla et al., 2007; Shukla et al., 2011), and top- down contextual parsing (Wang et al., 2020). However, in adults as exposure lengthens and the absolute frequency of all possible tran- sitions increased, the familiarity advantage for Words relative to Adding a subliminal pause at the end of the word radically affects the performances at both ages. Although not consciously perceived, pauses act as other word boundary markers (e.g., lengthening of the last syllable, pitch drop) that neonates can perceive (Christophe et al., 1994). Our result demonstrates that such a word boundary marker is not only perceived but is effectively used to segment a stream from birth on, that is, before infants have perceived many isolated words. 3.1 Word segmentation based on statistical learning is limited by the word size This synchrony measure probably captures a wider cross-subject convergence beyond neural entrainment at the two frequencies of interest in specific chan- nels. It reveals that neonates’ brain states are not purely entrained by the physical features of the stimulation, which remain similar along the stream but also constrained by learning mechanisms that led to more synchronous responses across neonates. The opposite hypothesis is that the neonates might have learned as quickly as in the case of trisyllabic words but that as time passed, this learning faded away because even low probability transitions became familiar. This overlearning effect has been reported in adults (Peña et al., 2002). The analysis of the neural entrainment along time of expo- sure was not sensitive to figure out the learning timeline even in the group with pauses, probably because of the very low signal to noise ratio in low frequencies. However, the group difference in the corre- lation between neonates’ recordings increased from around the first minute of exposure showing that the two groups were diverging early on between a learning condition (stream with pauses) and a no-learning condition (continuous stream). Finally, the performances between the test phase during which iso- lated quadri-syllabic sequences were presented were also massively affected by the stream condition, suggesting that once segmentation was done, memory encoding was improved. Words and PartWords were indeed only discriminated after the stream with pauses. However, in a similar experimental paradigm but after a stream of concatenated tri-syllabic words, Words were recognized since the first syllable (Fló et al., 2022), whereas here, the difference was developing from around 500 ms to become significant only after the end of the word. The lack of first syllable effect was confirmed by the absence of difference between PartWords and ShuffleWords, although the latter started with a correct first syllable. It is also consistent with the lack of sim- ilarity increase between both groups within the set of first syllables (Figure 4c right), which contrasts with the result reported in adults by Henin et al. (2021). Thus, contrary to the tri-syllabic stream, the ordinal position of the syllables was not encoded, and the difference between correct and incorrect chunks took longer to develop. Yet, even if participants were not able to segment the words in the continuous structured stream, both adults and neonates rejected Shuf- fleWords, which contained the exact same syllables as the Words, but in the wrong order. This result reveals that the participants computed TP and were not misled by the temporal proximity of the syllables, but this computation was not sufficient to trigger stream segmenta- tion. Interestingly, attentive adults appeared not better than sleeping neonates in the task: They also failed to segment the stream without the help of acoustical cues. Thus, tracking TP does not always result in word segmentation. 9 of 16 BENJAMIN ET AL. 3.4 Similarity between adults and neonate cognitive abilities The similar drop in performance in neonates and awake linguisti- cally productive adults suggests a structural limitation in the number of items that can be stored in the STM. This limit of four in STM has been proposed as explaining several higher order linguistic observations, such as the size of phrasal verbs and idioms predominantly used in spo- ken languages such as English, the mean length of continuous discourse without pauses (Green, 2017), and the drop in mutual information scores after four words in many languages (Pothos & Juola, 2007). It also seems compatible with the observed word length inferior to four syllables in many languages (Zipf, 1935, Sigurd et al, 2004), suggesting that this chunk size limitation we observe here might be fine-tuned to real language word size. This limitation also reveals that TP computa- tion might not be robust enough to be the proposed general-purpose mechanism for word segmentation in all speech streams without being complemented by other indices. Despite very different measuring methods and attentional state in this set of experiments, the results in neonates and adults pointed to similar successes and failures in terms of TP computation and stream segmentation. This is somehow surprising given the fact that many of the structures that support sequence learning (Henin et al., 2021)—hippocampus, dorsal linguistic pathway, the superior temporal region—change rapidly in the first year of life; but the classic assump- tion that immature means poorly functional is increasingly challenged by brain imaging methods that provide markers of learning in young children. FMRI remains difficult in infants, but some results support the hypothesis of early efficiency despite immaturity. In a recent, paper Ellis et al. tested 3–24 month-old infants on a statistical learning task in the visual domain with fMRI and reported activation in the hippocam- pus associated with segmentation. Dehaene-Lambertz et al. (2002) reported activations in temporal and frontal areas in 3-month-olds listening to speech showing that regions usually reported in adults dur- ing statistical learning tasks (Henin et al., 2021) are, to some extent, already functional in infants. If neural entrainment during the stream reflects the chunking and word encoding, the ERPs to the isolated chunks in the test phase tested the participants’ recognition and familiarity with the different condi- tions. to the global familiarity of the word rather than noticing a particular error. In adults, Henin et al. (2021) confirmed using similarity analy- ses on ECOG recordings, that the ordinal position of the syllables was encoded. Adults are nevertheless better than neonates, encoding not only which syllables were first but also which were second and which were last. Here, we tried similar analyses in the neonates’ data despite the sparser resolution of EEG. We observed an increase of similarity of the ERPs to the adjacent and non-adjacent syllables belonging to the same word in the stream with pause compared to the continuous stream. However, we found no evidence of an increase of similarity between the words first syllables. Thus, the particular status of the first syllables observed in neonates in the case of tri-syllablic words (Fló, et al., 2022) had no support in this study when quadri-syllabic words were used. This result might just reflect a lack of power of our analy- sis, or it might be explained by the difference in perception of the drop of TP in a tri-syllabic word stream. The TP drop, which can induce a surprise following a prediction error, might favor encoding these syl- lables at a particular position (i.e., the first position of the next word). These results might thus suggest that depending on the segmenting cue, different memory processes are engaged in neonates and that TP computation might favor a more precise encoding of the chunking ele- ments, starting with the first syllable and progressing from one syllable to the next. Such an intriguing hypothesis should be further tested in experiments specifically designed to contrast these two cues and the word-size at this age and also in adults. 3 to 4 syllables, raised interesting questions regarding both word seg- mentation during the stream and subsequent memory encoding of the word unit. Although this experiment does not directly test this question, we propose that recovering words in a stream is based on short-term memory (STM). Indeed, if TP between syllables can be locally computed within the auditory cortex, the integration of the successive syllables within a word requires a longer temporal window of integration. The sharp difference between tri- and quadri-syllabic words seems remi- niscent of the 4 ± 1 unit limit of the auditory STM (Cowan, 2001) and suggests that the TP drop leading to word segmentation might only be noticeable when all the elements of a word plus the next syllable are present at once in the STM. Several studies suggest that adults use STM, and more specifically working memory, in such statistical learn- ing tasks. For instance, their performance improves when speech is slowed down, an observation at odds with a decay-time in a purely sensory buffer that should be detrimental as the time between sylla- bles increases, but in favor of maintenance of the successive syllabic items (Palmer & Mattys, 2016). Performances also drop when partic- ipants perform a concurrent two-back task (Palmer & Mattys, 2016) suggesting competition for general resources. These observations are coherent with the activations reported by Henin et al. (2021) along the dorsal linguistic pathway, and notably in the inferior frontal region. In the neonates, no explicit rehearsal was possible because they were asleep and, in any case, unable at that age to repeat syllables, but even in adults, STM effects may remain implicit (Hassin et al., 2009). If statis- tical learning is improved when adult participants are actively doing the task, the task remains feasible when they are distracted and unaware of the task (Fernandes et al., 2007; Palmer & Mattys, 2016). 3.3 Why a sharp distinction between tri and quadri-syllabic words? The differences, in terms of neural entrainment during familiarization and ERPs during test in infants as the drop of performances in adults, between our two word-segmentation studies (Fló, etal., 2022), in which we used a similar paradigm except that the word size increased from BENJAMIN ET AL. 10 of 16 4 CONCLUSION Human neonates display sequence learning abilities even during sleep, based on TP computations and segmenting helped by acous- tic/prosodic cues. The similarities with adults’ successes and fail- ures were remarkable, revealing early powerful capacities to process speech. A speech stream is not a uniform landscape for infants, but different cues might help them to chunk it into smaller units, opening the possibility to discover the linguistic regularities and the productive properties of speech. Human neonates display sequence learning abilities even during sleep, based on TP computations and segmenting helped by acous- tic/prosodic cues. The similarities with adults’ successes and fail- ures were remarkable, revealing early powerful capacities to process speech. A speech stream is not a uniform landscape for infants, but different cues might help them to chunk it into smaller units, opening the possibility to discover the linguistic regularities and the productive properties of speech. 5.1.1 Participants A total of 43 adults were recruited via social media and mailing (21 males, age distribution = [18-25]: 9, [25–40]:16, [40–60]: 17, 60+: 1]) with no reported auditory issue or language related troubles. They were randomly assigned to one of the streams with the instruction to carefully listen for ∼3 min to a nonsense language composed of non- sense words that they have to learn because they will have to answer questions on the words afterward. The learning/test procedure was repeated twice. The study was coded in javascript using jspsych toolbox (de Leeuw, 2015) and played audio mp3 pre-loaded and pre-created in MATLAB (see below) to avoid latencies during the presentation. Subjects vol- untarily participated on their computer. They were asked to wear headphones, sit in a quiet environment, and stay focused during the whole task. The Ethical research committee of Paris Saclay University approved the protocol under the reference CER-Paris-Saclay-2019-063. 5 MATERIALS AND METHODS Together, our results show that behavioral subjective ranking and EEG analyses provide powerful tools to investigate statistical learning and segmenting tasks. There was a neat congruency between the behav- ioral results in adults and the neural markers observed in neonates. Moreover, EEG data enables the investigation of such questions in pre- verbal and non-verbal subjects with different levels of attention (e.g., neonates, sleeping subjects, comatose patients). Power and PLV during the stream as well as ERP during isolated test words, were already pro- posed as reliable neural markers in this task (Kabdebon et al., 2015; Fló, Benjamin, et al., 2022). However, to our knowledge, between-subject correlation as a function of time had not been shown to capture learn- ing in infants successfully. Our results confirm that despite the noise in infant EEG data, a significant part of the variance cannot be only explained by low-level bottom-up activation to external stimuli but instead by a more sustained learning effect. Although this first attempt might have been still noisy, we might hope that this method could more accurately quantify the average amount of learning of a group over time or even characterize learning at the subject level. One drawback of this method is that, to compare across subjects, all subjects have to be exposed to the exact same stimuli, which presents a risk of con- found in the experimental design. Here we minimized this risk by taking two precautions. We first carefully designed and balanced the auditory material on acoustic aspects (see SI). Secondly, we ran two groups with a minimal change (a subliminal pause every four syllables) so that, if any bias persists, it would be the same in both groups and thus cannot explain differences between groups. sleeping adults even for tri-syllabic words (Farthouat et al., 2018; Bat- terink&Zhang, 2022),andtheirlearningremainedlimitedtobi-syllabic words, that is, to classical associative learning. Infants might perform better than adults during sleep due to the different organization of sleep-wake cycles. At this age, sleep comprises only two clear stages, quiet (∼40% of a sleep cycle at birth) and active sleep (50%–60% of a sleep-cycle at birth) with many micro-arousal periods within and between sleep stages (Scher, 2008). The short periods of wakefulness are immediately followed by active sleep, which is the equivalent of REM sleep in adults. In adults, learning has been shown to exist dur- ing REM (Andrillon & Kouider, 2016) and also that a task started during wake can continue during REM (Andrillon et al., 2016), opening the possibilities that neonates might learn and consolidate more efficiently than later, thanks to the closer wake-REM sleep alternations. such as the placement of the net on the infant’s head which is more variable at this age due to birth-related head deformation, and can introduce between groups differences; (2) eventually long-tail effects of the previous trials on the topography that can affect the baseline. BENJAMIN ET AL. 11 of 16 3.4 Similarity between adults and neonate cognitive abilities In the tri-syllabic experiment (Fló et al., 2022), neonates during the test-phase were no more sensitive to TP (i.e., no distinctive ERP response for triplets containing a TP = 0) and were reacting to an incor- rect first syllable. Here, they were rejecting ShuffleWords, thus were still sensitive to TP, but did not react particularly fast to the incorrect first syllable (Word vs. Part-Word), suggesting a more general response A major distinction between adults and neonates seems to be the capacity of computing such a task during sleep. Indeed, with both three and quadri-syllabic experiments, we showed that sleeping neonates were able to process and segment the streams, under the correct circumstances. However, recent studies report a learning failure in 5.1.3 Procedure After listening to the structured stream, participants were asked to rank the familiarity of the individual words (from “Completely unfa- miliar” to “Completely familiar” on a six-step scale). Learning and test phases were repeated twice. Data of the two tests sessions were aggre- gated in the main analysis (see separated analysis of each session in Figure S5). Six conditions (three bi-syllabic as foils and three quadri- syllabic conditions) were used to avoid any bias based on the length of the test words, with four trials in each of the six conditions. To avoid phonological similarity effects that could bias toward one or the other condition, participants were assigned to one of two groups where con- ditions were reversed. Four different pairs of structured streams per group were also generated, and participants were randomly assigned to one pair to avoid any given particularity of a stream driving the results. Three conditions were studied: Words, PartWords, and Shuf- fleWords. Words corresponded to the words that were embedded in the structured streams (ABCD), while PartWords corresponded to the two last syllables of a word and the two first of another word (CDA’B’). Thus, although PartWords were heard during the structured stream, they violated chunking based on TP. ShuffleWords corresponded to words in which the second and third syllables were inverted, creating a null TP between all syllables (none of the transitions were heard dur- ing the structured stream). However, the first and last syllables were correct. 5.2.1 Participants Two groups of healthy full-term neonates were tested between days 1 and 3. There was no problem during pregnancy and delivery, birth- weight > 2500 g, term > 38 wGA, APGAR ≥6 and 8 at 1′ and 5′, normal audition tested with otoacoustic emission. Parents provided informed consent, and the Ethical Committee (CPP Tours Region Centre Ouest 1) approved the study (EudraCT/ID RCB: 2017-A00513-50). In the first group (continuous), 34 neonates were tested. Among them, seven were excluded because they did not complete the experimental protocol or technical issues leaving 27 infants (14 males). In the second group, 34 infants were tested (with pauses). Nine were excluded because they did not complete the experimental protocol or technical issues, leaving 25 infants (13 males). 5.2.2 Stimuli We used the same 16 isolated syllables generated with MBROLA as in the adult experiment to construct four different streams (structured and random, with and without pause). The random stream consisted of 1600 pseudo-randomly concatenated syllables (6.7 mn). Each sylla- ble could be followed by three others from the pool leading to a flat TP during the stream. This pseudo-random stream offers a more con- trolled stimulus than the random streams used previously because the TPs were fixed to 1/3 (instead of 1/15), a similar value than the TP between words in the structured stream. The structured stream was comprised of 3200 syllables (13.3 mn). All streams were ramped up and down during the first and last 5 s to prevent the beginning and the end of the streams from being used as anchors. We created only one syllabic order for each stream to obtain learning markers better comparable between infants. For the second group, a pause was added every four syllables in both the structured (duration: 13.7 mn) and the random streams (duration: 6.8 mn). Thus, the sequences were identi- cal for all infants in both groups except for the 25-ms subliminal pauses every four syllables in the second group. Because all subjects had the same auditory materials, we carefully controlled for low-level acoustic- phonetic properties. We equilibrated the characteristics of consonants and vowels in the different words and at the different syllabic positions within words to avoid learning based on low-level acoustic cues (See Figure S1 for more details). As in adults, three types of test words were created: Word (ABCD), PartWord (CBA’B’), and ShuffleWords (ACBD) (Table 1 and Figure S1). 5.2 Infant EEG experiment In a previous experiment with similar streams with and without 25 ms pauses, Peña et al. (2002) showed that participants were at chance when they had to choose which of the two streams had pauses. To confirm that the pauses were not consciously perceived, eight adults listened to 20 streams (40 syllables – 10 s) presented randomly (10 without pauses and 10 with a 25 ms-pause every four syllables) and were unable to indicate which stream had pauses or not (mean = 49% (range [40, 59]%); p = 0.89). 12 of 16 BENJAMIN ET AL. mean familiarity ranking for each condition in each subject and sub- tracted the PartWord ranking from the word ranking within each subject. We then performed a one-way unpaired t-test between the two groups. pause, a 25-ms pause was inserted every 4-syllables (total duration 3.4 mn). All streams were ramped up and down during the first and last 5 s to avoid the start and end of the streams serving as perceptual anchors. We used the same syllables and words for the infant experiment. To avoid phonological similarity effects that could bias toward one or the other condition, Words and PartWords were reversed for half of the subjects. 5.1.2 Stimuli All speech stimuli were generated with the MBROLA text-to-speech software (Dutoit et al., 1996) using French diphones. The duration of all syllables was equalized to 250 ms with flat intonation and no coar- ticulation between syllables. Each experiment was composed of 800 syllables (3.3 mn) of an artificial monotonous stream of concatenated syllables that correspond to the four possible words randomly con- catenated with the only restriction that the same word could not be presented twice in a row. The same vocabulary (sixteen syllables) was used in the two streams, with and without pause. In the stream with We also implemented what we believe to be an improvement for ERP analysis. Before the presentation of isolated words, we presented a short audio click as an auditory localizer. In this way, we were able to extract ROIs for analysis with a data-driven approach instead of liter- ature driven. We performed a cluster based-permutation analysis on all data against zero during the click presentation to extract the audi- tory ERP ROI. Moreover, this localizer cluster was representative of the auditory response in this particular group of subjects taking into account non-relevant variations due to (1) Experimental conditions 5.2.5 Neural entrainment The recordings from the structured and random streams were seg- mented into consecutive non-overlapping epochs of 15 words (corre- sponding to 15 s in the continuous group and 15.375 s in the pause group). All subjects having 10 good epochs or more in each condition were included in this analysis (25 neonates in the continuous group, 21 in the pause group). We averaged the activity over artifact-free epochs for each neonate and electrode and computed the Fourier Transform using the fast Fourier transform algorithm (FFT) as implemented in MATLAB. We then computed the power of the FFT. The PLV between trials was computed on the FFT of single trials. Those values were nor- malized with neighboring frequency bins [−8:1,1:8]. The frequencies of interest were selected as the inverse of the duration of a word (f = 1 Hz for the continuous group f = 0.975 for the second group with pauses) and one-quarter of a word (i.e., roughly a syllabic rate, f = 4 Hz for the first group, f = 3.9 Hz for the second). To assess the significance of the power/PLV at the two frequencies of interest, we computed a con- trast between the power/PLV during the structured stream compared to the random streams for each electrode. As we expect learning dur- ing the structured stream to elicit a word rate oscillation, we computed a one-way (structured > random) paired t-test on each electrode. We corrected for multiple comparisons using a cluster corrected approach (alpha = 0.05). To look for a potential difference between groups, we computed an interaction between the previously described contrasts of both groups (difference of the structure minus random contrast in each group). Specifically, we ran a one-way unpaired t-test on each electrode and the clustering approach for the interaction. Both groups followed the same procedure (Figure 1). A first con- trol stream of a pseudorandom concatenation of 1600 syllables was followed by a structured stream composed of 3200 syllables grouped in words of four syllables. Infants then heard eight repetitions of short structuredstreams(160syllables)followedby12testwordspresented in isolation (four in each condition: Word, PartWords and Shuffle- Words, ISI 2-2.5s) for a total of 96 test-words (32 in each condition). The short streams were added to maintain learning because 2/3 of the test-words violated the learned structure. Each test word was pre- ceded by a short click 200 ms before its onset. TABLE 1 Words used in the experiment s were presented in isolation, four words, four ShuffleWords and four PartWords out of the eight possible. Note: During each small test block, 12 test words were presented in isolation, four words, four ShuffleWords and four PartWords out of the eight possible. Following a reviewer’s requirement, we tested adults on the same material with the same exposure duration (∼13mn). Their behavioral results are presented in Figure S5. lished for each subject and electrode as two interquartile ranges away from the 3rd quartile. This gave a logical matrix of the size of the recording, indicating bad data. Electrodes were definitely rejected if they were marked as bad more than 50% of the recording time, and time-samples were marked as bad if more than 35% of the electrodes were marked bad at this time-sample. For the ERP analysis, we then performed spatial interpolation of missing channels, and the data were mathematically referenced to the average of the 128 channels. 5.2.5 Neural entrainment The click was added as a task unrelated auditory localizer and to reset the baseline with a neutral event to avoid long-range drifts following the words. Finally, a second control stream was presented. Thus, the two random-streams were sandwiching the structured stream to control for habituation to the auditory stimulation, change in sleep stage, and any confounding time effect. 5.2.3 Procedure EEG was recorded with 128 electrodes (EGI geodesic sensor net), care- fully placed on the neonates’ heads by trained researchers to increase the consistency of the net placement. Three nets with different radii were used to fit infants’ heads. For the continuous group, infants were tested while asleep in the experimenter or parent’s arms. Due to COVID restrictions, the second group of babies was tested asleep in the crib. This slightly increased the noise level in the second group and might have marginally decreased the sensitivity of our analysis for this group. 5.1.4 Data processing Each answer was converted to a numerical value from 1 (completely unfamiliar) to 6 (completely familiar). The responses to the bisyllabic trials were not considered. All data, from both test sessions, were aggregated together in each group to compute a linear mixed-effects model on items (y ∼condition + (1\|subject)) to take the subject effect into account. The p-values were then FDR corrected. To compare sub- jects’ segmenting performances for both streams, we computed the BENJAMIN ET AL. 13 of 16 TABLE 1 Words used in the experiment Words (W) Part words (PW) Shuffle words (SW) RaFiBouNeu BouNeuNonLo/BouNeuVouDon RaBouFiNeu GuMaReuZo ReuZoVouDon/ReZoNonLo GuReuMaZo NonLoSanBi SanBiGuMa/SanBiRaFi NonSanLoBi VouDonMuLan MuLanRaFi/MuLanGuMa VouMuDonLan Note: During each small test block, 12 test words were presented in isolation, four words, four ShuffleWords and four PartWords out of the eight possible. 13 of 16 BENJAMIN ET AL. 5.2.6 Correlation analysis In both experiments, all subjects heard the exact same auditory mate- rial avoiding differences in stimulation between participants. We could thus compute the instantaneous correlation between each participant and the others. For each subject at each time during the streams, we computed the correlation at the topographical level between the topography of subject i at time t (a vector of 128 voltage values at time t corresponding to the 128 electrodes) and the topogra- phy of the grand average excluding subject i at time t (a vector of 128 values corresponding to the average across the other subjects at time t for each of the 128 electrodes). It gave, for each sub- ject, a vector of correlation between its own topography and the mean topography of all other subjects throughout time. Bad data were replaced by zeros and not taken into account for the average topographies across subjects. Time points with only bad data gave NaN correlation results. We hypothesized that learning should lead to an increase with time in the correlation between neonates as they learn the same material. To test it, we used two different methods. In the first one, we smoothed the correlation signal using a 400s- sliding-average-window in each neonate and stream, then computed a cluster-based analysis to reveal a significant cluster of time during which one stream showed a greater correlation than the other one. In the second one, we computed the slope of the linear regression with time in each subject and then considered the slope as a vari- able for the structured and random conditions in t-test comparing both groups. To extract ROI corresponding to the functional auditory localizer of each group, we measured the auditory event-related potential associated with the click presentation at the beginning of each trial by running a cluster-based analysis against zero to extract auditory ERP (5000 randomizations, two-tailed t-test, alpha < 0.01, cluster- alpha < 0.01, between −200 and 0 ms). This procedure identified a positive frontal and a negative occipital cluster in each group, on which we restricted the ERP analyses. Therefore, the voltage was averaged across electrodes in each of the two clusters in each neonate and condition. A cluster-based analysis was performed on the obtained time- series (10000 randomizations two-tailed t-test alpha < 0.05, cluster alpha < 0.05) between 250 ms (end of the first syllable) and 2000 ms to compare all pairs of conditions. ACKNOWLEDGMENTS We thank Simon Henin for help and comments about pattern similarity analysis. We also thank all the families who participated in our study as well as the maternity hospitals of Port-Royal and Orsay. This research has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No. 695710). 5.2.8 ERP analysis shorter experiments at the expense of more data manipulations. Both approaches were quite similar, confirming the validity of both that can be better adapted depending on the amount of available data. Data were segmented in 2850 ms long epochs ([−750 +2100]ms relative to word onset), averaged in the three conditions (Words, PartWords, and ShuffleWords), and baseline-corrected with the mean voltage value inthe interval [−750 to0] ineachneonate. Neonateswith less than 20 remaining trials in total were excluded from analysis (none in the continuous group, 1 in the pause group). 5.2.6 Correlation analysis Because of the adults’ behavioral results, we added the contrast "heard" (average of Word and Part- Word) vs. "non heard" (ShuffleWord) in the continuous group. Finally, we computed the interaction between groups and conditions (Word- PartWord) during the time window in which the previous analysis revealed a significant effect. 14 of 16 14 of 16 ETHIC APPROVAL Ethical research committee of Paris Saclay University under the ref- erence CER-Paris-Saclay-2019-063 & Ethical Committee CPP Tours Region Centre Ouest 1 (EudraCT/ID RCB: 2017- A00513-50) COMPETING INTERESTS To compute pattern similarity between syllables, we epoched each syllable from the structured stream from −100 ms to 350 ms. We removed the 100 first syllables to give enough time for participants to learn the task. The remaining epochs were averaged by syllables for each subject and a correlation matrix between each pair of syllables was computed with all the electrodes between 0 and 350 ms. We then separated the pairs of syllables into five conditions: First syllable (AA’), Ordinal position (BB’ or CC’ or DD’), Word and TP (AB or BC or CD), Word only (AC or AD or BD) and Low TP (DA’). We then averaged the similarity per condition and subtracted the correlation between all the other pairs. We then compared if pattern similarity between groups of syllables was increased differently across groups (One-way t-test with pauses > continuous). All other authors declare they have no competing interests. All other authors declare they have no competing interests. DATA AND MATERIALS AVAILABILITY All data and analysis are available upon request. BENJAMIN ET AL. 5.2.4 Data processing EEG recordings were band-pass filtered between 0.2 and 15 Hz for all analyses. Artifact rejection was performed on the non-epoched record- ing session using APICE pipeline (Fló, et al., 2022) based on the EEGLAB toolbox (Delorme & Makeig, 2004). Artifacts were identified on contin- uous data, based on voltage amplitude, variance, first derivative, and running average. The variance algorithm was applied in sliding time windowsof500mswith100mssteps. Adaptivethresholdswereestab- Additionally, we also replicated the neural entrainment effects with a slightly different method as proposed in Fló, et al. (2022). With this approach, the signal is decomposed on 1s long epochs and reconstructed in longer meta-epochs composed of several non- necessarily consecutive segment. It allows to save more data for REFERENCES (2009). Investigating the neu- ral correlates of continuous speech computation with frequency-tagged neuroelectric responses. Neuroimage, 44(2), 509–519. Choi, D., Batterink, L. J., Black, A. K., Paller, K. A., & Werker, J. F. (2020). Preverbal infants discover statistical word patterns at similar rates as adults: Evidence from neural entrainment. Psychological Science, 31(9), 1161–1173. Hochmann, J. R., Endress, A. D., & Mehler, J. (2010). Word frequency as a cue for identifying function words in infancy. Cognition, 115(3), 444–457. James, L. S., Sun, H., Wada, K., & Sakata, J. T. (2020). Statistical learning for vocal sequence acquisition in a songbird. Scientific Reports, 10(1), 1–18. Christophe, A., Dupoux, E., Bertoncini, J., & Mehler, J. (1994). Do infants perceive word boundaries? An empirical study of the bootstrapping of lexical acquisition. Journal of the Acoustical Society of America, 95(3), 1570–1580. Johnson, E. K., & Tyler, M. D. (2010). Testing the limits of statistical learning for word segmentation. Developmental Science, 13(2), 339–345. Kabdebon, C., Pena, M., Buiatti, M., & Dehaene-Lambertz, G. (2015). Electrophysiological evidence of statistical learning of long-distance de- pendencies in 8-month-old preterm and full-term infants. Brain and Language, 148, 25–36. Cowan, N. (2001). The magical number 4 in short-term memory: A recon- sideration of mental storage capacity. Behavioral and Brain Sciences, 24(1), 87–114. Mandel, D. R., Jusczyk, P. W., & Kemler Nelson, D. G. (1994). Does senten- tial prosody help infants organize and remember speech information?, Cognition, 53(2), 155–180. Dehaene-Lambertz, G., Dehaene, S., & Hertz-Pannier, L. (2002). Functional neuroimaging of speech perception in infants. Science, 298(5600), 2013– 2015. Mattys, S. L., White, L., & Melhorn, J. F. (2005). Integration of multiple speech segmentation cues: A hierarchical framework. Journal of Experimental Psychology General, 134(4), 477–500. de Leeuw, J. R. (2015). jsPsych: A JavaScript library for creating behavioral experiments in a Web browser. Behav Res, 47, 1–12. https://doi.org/10. 3758/s13428-014-0458-y Nespor, M., & Vogel, I. (2006). Prosodic phonology. Delorme, A., & Makeig, S. (2004). EEGLAB: An open source toolbox for analysis of single-trial EEG dynamics including independent component analysis. Journal of Neuroscience Methods, 134(1), 9–21. Oostenveld, R., Fries, P., Maris, E., & Schoffelen, J. M. (2011). FieldTrip: Open source software for advanced analysis of MEG, EEG, and Invasive Electrophysiological Data. Computational Intelligence and Neuroscience, 2011(2011), Article ID 156869, https://doi.org/10.1155/2011/156869 Dutoit, T., Pagel, V., Pierret, N., Bataille, F., & van der Vrecken, O. (1996). REFERENCES Fiser, J., & Aslin, R. N. (2002). Statistical learning of new visual feature com- binations by infants. Proceedings of the National Academy of Sciences of the United States of America, 99(24), 15822–15826. Andrillon, T., & Kouider, S. (2016). Implicit memory for words heard during sleep. Neuroscience of Consciousness, 2016(1), niw014. Fló, A., Benjamin, L., Palu, M., & Dehaene-Lambertz, G. (2022). Sleeping neonates track transitional probabilities in speech but only retain the first syllable of words. Scientific Reports, 12(1), 4391. https://doi.org/10. 1038/s41598-022-08411-w Andrillon, T., Poulsen, A. T., Hansen, L. K., Léger, D. L., & Kouider, S. (2016). Neural markers of responsiveness to the environment in human sleep. Journal of Neuroscience, 36(24), 6583–6596. Bagou, O., & Frauenfelder, U. H. (2018). Lexical segmentation in artificial word learning: The effects of converging sublexical cues. Language and Speech, 61(1), 3–30. Fló, A., Brusini, P., Macagno, F., Nespor, M., Mehler, J., & Ferry, A. L. (2019). Newborns are sensitive to multiple cues for word segmentation in continuous speech. Developmental Science, 22(4), e12802. Batterink, L. J., & Choi, D. (2021). Optimizing steady-state responses to index statistical learning: Response to Benjamin and colleagues. Cortex, 142, 379–388. Fló, A., Gennari, G., Benjamin, L., & Dehaene-Lambertz, G. (2022). Auto- mated Pipeline for Infants Continuous EEG (APICE): A flexible pipeline for developmental cognitive studies. Developmental Cognitive Neuro- science, 54, 101077. https://doi.org/10.1016/j.dcn.2022.101077 Batterink, L. J., & Zhang, S. (2022). Simple statistical regularities pre- sented during sleep are detected but not retained. Neuropsychologia, 164, 108106. https://doi.org/10.1016/j.neuropsychologia.2021.108106 Green, C. (2017). Usage-based linguistics and the magic number four. Cognitive Linguistics, 28(2), 209–237. Benjamin, L., Dehaene-Lambertz, G., & Fló, A. (2021). Remarks on the analysis of steady-state responses: Spurious artifacts introduced by overlapping epochs. Cortex, 142(2), 370–378. Hassin, R. R., Bargh, J. A., Engell, A. D., & McCulloch, K. C. (2009). Implicit working memory. Consciousness and Cognition, 18(3), 665–678. Hauser, M. D., Newport, E. L., & Aslin, R. N. (2001). Segmentation of the speech stream in a non-human primate: Statistical learning in cotton-top tamarins. Cognition, 78(3), 53–64. Black, A., & Bergmann, C. (2017). Quantifying infants’ statistical word seg- mentation: A meta-analysis. Proceedings of the 39th Annual Conference of the Cognitive Science Society, (3), 124–129. Henin, S., Turk-Browne, N. B., Friedman, D., Liu, A., Dugan, P., Flinker, A., Doyle, W., Devinsky, O., & Melloni, L. (2021). Learning hierarchi- cal sequence representations across human cortex and hippocampus. Science Advances, 7(8), 1–13. Buiatti, M., Peña, M., & Dehaene-Lambertz, G. BENJAMIN ET AL. 15 of 16 ORCID LucasBenjamin https://orcid.org/0000-0002-9578-6039 AnaFló https://orcid.org/0000-0002-3260-0559 16 of 16 BENJAMIN ET AL. Scher, M. S. (2008). Ontogeny of EEG-sleep from neonatal through infancy periods. Sleep Medicine, 9, 615–636. Wakefield, J. A., Doughtie, E. B., & Lee Yom, B. H. (1974). The identifi- cation of structural components of an unknown language. Journal of Psycholinguistic Research, 3(3), 261–269. Schön, D., Boyer, M., Moreno, S., Besson, M., Peretz, I., & Kolinsky, R. (2008). Songs as an aid for language acquisition. Cognition, 106(2), 975–983. Wang, F. H., Zevin, J. D., Trueswell, J. C., & Mintz, T. H. (2020). Top-down grouping affects adjacent dependency learning. Psychonomic Bulletin & Review, 27(5), 1052–1058. Shukla, M., Nespor, M., & Mehler, J. (2007). An interaction between prosody and statistics in the segmentation of fluent speech. Cognitive Psychology, 54(1), 1–32. Zipf, G. K. (reprinted 1965). (1935). The psycho-biology of language. MIT Press. Shukla, M., White, K. S., & Aslin, R. N. (2011). Prosody guides the rapid mapping of auditory word forms onto visual objects in 6-mo-old infants. Proceedings of the National Academy of Sciences of the United States of America, 108(15), 6038–6043. REFERENCES The MBROLA project: Towards a set of high quality speech synthesizers free of use for non commercial purposes. Proceeding of Fourth Interna- tional Conference on Spoken Language Processing. ICSLP ’96, 3, 1393–1396. https://doi.org/10.1109/ICSLP.1996.607874 Ordin, M., Polyanskaya, L., Laka, I., & Nespor, M. (2017). Cross-linguistic dif- ferences in the use of durational cues for the segmentation of a novel language. Memory and Cognition, 45(5), 863–876. Palmer, S. D., & Mattys, S. L. (2016). Speech segmentation by statisti- cal learning is supported by domain-general processes within working memory. Quarterly Journal of Experimental Psychology (Hove), 69(12), 2390–2401. Ellis, C. T., Skalaban, L. J., Yates, T. S., Bejjanki, V. R., Córdova, N. I., & Turk-Browne, N. B.. Evidence of hippocampal learning in human infants. Current Biology, 31(15), 3358–3364.e4. Farthouat, J., Atas, A., Wens, V., De Tiege, X., & Peigneux, P. (2018). Lack of frequency-tagged magnetic responses suggests statistical regularities remain undetected during NREM sleep. Science Reports, 8(1), 11719. Peña, M., Bonatti, L. L., Nespor, M., & Mehler, J. (2002). Signal-driven computations in speech processing. Science, 298(5593), 604–607. Pothos, E. M., & Juola, P. (2007). Characterizing linguistic structure with mutual information. British Journal of Psychology, 98(2), 291–304. Fernandes, T., Kolinsky, R., & Ventura, P. (2010). The impact of attention load on the use of statistical information and coarticulation as speech segmentation cues. Atten Percept Psychophys, 72(6), 1522–1532. Saffran, J. R., Aslin, R. N., & Newport, E. L. (1996). Statistical learning by 8- month-old infants. Science, 274(5294), 1926–1928. Fernandes, T., Ventura, P., & Kolinsky, R. (2007). Statistical information and coarticulation as cues to word boundaries: A matter of signal quality. Perception and Psychophysics, 69(6), 856–864. Saffran, J. R., Johnson, E. K., Aslin, R. N., & Newport, E. L. (1999). Statistical learning of tone sequences by human infants and adults. Cognition, 70(1), 27–52. Ferry, A. L., Fló, A., Brusini, P., Cattarossi, L., Macagno, F., Nespor, M., & Mehler, J. (2016). On the edge of language acquisition: Inherent constraints on encoding multisyllabic sequences in the neonate brain. Developmental Science, 19(3), 488–503. Saffran, J. R., Newport, E. L., & Aslin, R. N. (1996). Word segmentation: The role of distributional cues. Journal of Memory and Language, 35(4), 606– 621. SUPPORTING INFORMATION Sigurd, B., Eeg-Olofsson, M., & van de Weijer, J. (2004). Word length, sen- tence length and frequency – Zipf revisited, Studia Linguistica, 58(1), 37–52. Additional supporting information can be found online in the Support- ing Information section at the end of this article. Swingley, D. (2005). Statistical clustering and the contents of the infant vocabulary. Cognitive Psychology, 50(1), 86–132. Teinonen, T., Fellman, V., Näätänen, R., Alku, P., & Huotilainen, M. (2009). Statistical language learning in neonates revealed by event-related brain potentials. BMC Neuroscience, 10, 21. How to cite this article: Benjamin, L., Fló, A., Palu, M., Naik, S., Melloni, L., & Dehane-Lambertz, G. (2022). Tracking transitional probabilities and segmenting auditory sequences are dissociable processes in adults and neonates. Developmental Science, e13300. https://doi.org/10.1111/desc.13300 Toro, J. M., Sinnett, S., & Soto-Faraco, S. (2005). Speech segmentation by statistical learning depends on attention. Cognition, 97(2), 25–34. Toro, J. M., & Trobalón, J. B. (2005). Statistical computations over a speech stream in a rodent. Perception and Psychophysics, 67(5), 867–875. Tyler, M. D., & Cutler, A. (2009). Cross-language differences in cue use for speech segmentation. The Journal of the Acoustical Society of America, 126(1), 367–376.
https://openalex.org/W2410182811	http://www.scielo.cl/pdf/rgeong/n63/art03.pdf	Spanish; Castilian	null	Zonas de catástrofe por eventos hidrometeorológicos en Chile y aportes para un índice de riesgo climático	Revista de geografía Norte Grande	2,016	cc-by	9,121	1 Se agradece el financiamiento de los proyectos Fondecyt N° 1130305 y N° 1100657, así como del Centro de Desarrollo Urbano Sustentable (CEDEUS), Conicyt-FONDAP N° 15110020, del Centro Nacio- nal de Investigación para la Gestión Integrada de Desastres Naturales (CIGIDEN), Conicyt-FONDAP N° 15110017 y al Centro de Cambio Global UC. También se agradece el apoyo Ana Rickmers en la compilación de la base de datos y a Natalia Pino en la cartografía y edición de este documento. Artículo recibido el 3 de mayo de 2015, aceptado el 11 de septiembre de 2015 y corregido el 4 de enero de 2016. RESUMEN Se estima que el cambio en los patrones globales del clima así como su dinámica natural pueden ocasionar una alta incidencia en los fenómenos extremos y en con- secuencia aumentar los niveles de riesgos. En este estudio se revisan los principales desastres climáticos e hidrometeorológicos que han afectado el país entre 1984- 2013, a partir de los decretos que declaran zonas de catástrofe (Ley 16.282) por temporales de lluvia, sequías, heladas, nevazones, inundaciones y movimientos en masa desencadenados por temperaturas y precipitaciones extremas. Por otro lado, información de exposición, vulnerabilidad y resiliencia es integrada en un índice de riesgo climático a nivel comunal. De acuerdo a los resultados obtenidos las comu- nas del país que presentan mayor riesgo climático son las ubicadas en el litoral de la zona central. Se concluye sobre la necesidad de usar un enfoque adaptativo y no reactivo en el manejo del riesgo especialmente frente a las amenazas que impone el cambio climático. Palabras clave: Eventos climáticos extremos, zonas de catástrofe, índice de riesgo climático. Zonas de catástrofe por eventos hidrometeorológicos en Chile y aportes para un índice de riesgo climático1 Cristián Henríquez2, Nicolle Aspee3 y Jorge Quense4 Cristián Henríquez2, Nicolle Aspee3 y Jorge Quense4 27 Revista de Geografía Norte Grande, 63: 27-44 (2016) Artículos 27 Revista de Geografía Norte Grande, 63: 27-44 (2016) Artículos 2 Instituto de Geografía, Pontiﬁ cia Universidad Católi- ca de Chile (Chile). E-mail: cghenriq@uc.cl 4 Instituto de Geografía, Pontiﬁ cia Universidad Católi- ca de Chile (Chile). E-mail: jquense@uc.cl 3 Instituto de Geografía, Pontiﬁ cia Universidad Católi- ca de Chile (Chile). E-mail: nvaspee@uc.cl 2 Instituto de Geografía, Pontiﬁ cia Universidad Católi- ca de Chile (Chile). E-mail: cghenriq@uc.cl 3 Instituto de Geografía, Pontiﬁ cia Universidad Católi- ca de Chile (Chile). E-mail: nvaspee@uc.cl 4 Instituto de Geografía, Pontiﬁ cia Universidad Católi- ca de Chile (Chile). E-mail: jquense@uc.cl recibido el 3 de mayo de 2015, aceptado el 11 de septiembre de 2015 y corregido el 4 de enero de 2016. REVISTA DE GEOGRAFÍA NORTE GRANDE 28 declarar zona de catástrofe lo cual permite tomar una serie de medidas y liberar recursos para enfrentar esta situación de crisis en áreas especíﬁ cas afectadas. A nivel internacional existe un am- plio consenso en relación a los impac- tos del cambio climático sobre el medio humanizado. Se estima que el cambio en los patrones globales del clima así como su dinámica natural pueden ocasionar una alta incidencia en el aumento e intensidad de las amenazas naturales como inundaciones, sequías, incendios, movimientos en masa, olas de calor, entre otros efectos. Si bien el cambio climático se asocia a las alteraciones en las condiciones medias del clima y a la variabilidad de sus propiedades que per- sisten por un periodo largo de tiempo (IPCC, 2012), cada vez más se asocia a la intensiﬁ - cación de los eventos climáticos extremos. En este sentido, el último informe del Panel Intergubernamental de Cambio Climáti- co (IPCC, 2014) destaca que las ciudades recibirán el mayor impacto relacionado a desastres por fenómenos extremos. Estos incluyen aumento en el nivel del mar, del oleaje por tormentas, de estrés por calor, de precipitaciones extremas, de inundaciones, de sequías, deslizamientos, de aridez y de contaminación del aire (IPCC, 2014). De esta manera podemos entender como riesgo, a una función que conjuga factores de amenaza y vulnerabilidad (Arenas et al., 2010), y que cuando se materializa se transforma en desastre. Esto sucede generalmente cuando se rompe el umbral de tolerancia del funciona- miento normal de un sistema socioecológico (Gallopin, 2006), como por ejemplo cuando una lluvia intensa no puede ser contenida por los sistemas de evacuación respectivos y en consecuencia inunda sectores ocupados por viviendas. Los efectos pueden ser más inten- sos cuando la población es más vulnerable, por ejemplo cuando afecta a los habitantes de menores recursos económicos, y cuando las condiciones de adaptación y recuperación son débiles, es decir presenta una baja resiliencia. Esta última entendida como la capacidad de los sistemas sociales y económicos para reponerse de la tensión y el impacto de una crisis (Klein et al., 1998; Walker et al., 2002). En Chile, los modelos climáticos regionales estiman para las próximas décadas un aumen- to de las temperaturas y una disminución de las precipitaciones, especialmente en Chile central, mientras que para el zona norte se pronostica una mayor incertidumbre (Comisión Nacional del Medio Ambiente, 2006). ABSTRACT It is estimated that changes in global weather patterns and natural dynamics can cause a high incidence of extreme events, and therefore increase risk levels. In this study, we review the major climatic and hydrometeorological disasters that have affected Chile bet- ween 1984 and 2013, based on ordinances that establish catastrophe zones (Law 16.282) for natural events such as, rainstorms, drought, frost, snowstorms, ﬂ oods, and mass move- ments, caused by extreme temperature and rainfall. Information on exposure, vulnerability and resilience is integrated into a climate risk index at the municipal level. Based on the municipal results for Chile, those areas with increased climate risks are located along the central coast. We conclude that it is necessary to consider an adaptive rather than reactive approach to risk management, especially when coping with threats due to climate change. Key words: Extreme climatic events, catastrophe zones, climate risk index 2 Instituto de Geografía, Pontiﬁ cia Universidad Católi- ca de Chile (Chile). E-mail: cghenriq@uc.cl REVISTA DE GEOGRAFÍA NORTE GRANDE Determinación del índice de riesgo climático Una vez sistematizada la información de catástrofes climáticas, se propone un índice de riesgo climático a escala comunal, que integra indicadores de amenaza, exposición, vulnera- bilidad y resiliencia, usando variables proxy de riesgo, mediante la siguiente fórmula: Por otro lado, interesa además relacionar lo anterior con información de exposición, vulnerabilidad y resiliencia de la población, mediante la propuesta de un índice de ries- go climático, con el objeto de identificar aquellas comunas de Chile con mayor nivel de riesgo climático. Finalmente, es de es- pecial interés proyectar las tendencias de cambio climático sobre el comportamiento de los eventos climáticos extremos que pu- diesen afectar al país en los próximos años. REVISTA DE GEOGRAFÍA NORTE GRANDE Otras modelaciones orientadas a la proyección de índices climáticos extremos (Villarroel, 2013), tales como temperaturas máximas, mínimas y precipitaciones, reaﬁ rman tales tendencias. La forma más efectiva para hacer frente a estos eventos tanto presentes como futuros es la reducción de riesgo de desastre (RRD), deﬁ nida como: “el proceso sistemático de utilizar decisio- nes administrativas, organizaciones, destre- zas y capacidades operativas para ejecutar políticas y fortalecer las capacidades de afrontamiento, con el ﬁ n de reducir el im- pacto adverso de las amenazas naturales y la posibilidad de que ocurra un desastre” (UNISDR, 2009; ECHO, 2012). La importancia de considerar estos eventos extremos es que cuando estos ocurren produ- cen severas alteraciones en el normal funcio- namiento de una sociedad y la comunidad. En situaciones críticas estos episodios pueden des- encadenar un desastre o catástrofe, en donde se producen importantes daños humanos, materi- ales, económicos o ambientales que requieren de una respuesta de emergencia inmediata para satisfacer las necesidades humanas y que pueden requerir ayuda externa para su recu- peración (Wilches-Chaux, 1989; IPCC, 2012). En el caso chileno cuando un desastre alcanza niveles superlativos, el Poder Ejecutivo puede En este contexto, el propósito de esta co- municación consiste, por un lado, analizar la distribución de los eventos climáticos ex- tremos, que hayan involucrado la declaración de zona de catástrofe por parte del Estado de Chile, entre los años 1984-2013. Para tal efecto se entenderá como evento climático extremo, de acuerdo a la clasiﬁ cación de Ayala-Carcedo y Olcina (2002), a fenómenos como graniza- das, heladas, lluvias, neblinas, nevadas, olas de calor, sequías, tempestades, tormentas eléctri- cas y vendavales. Además se incluyen eventos Donde: El área de estudio está conformada por 346 comunas del país. Debido a la carencia de da- tos necesarios para la construcción del índice de riesgo climático, no fueron consideradas las siguientes comunas: Colchane, Ollagüe, Hualañé, Alto Hospicio, Alto del Carmen, Alto Biobío, Cholchol, Hualpén, Isla de Pascua, Juan Fernández, Chaitén Lago Verde, Laguna Blanca, O’Higgins, Guaitecas, Río Verde, Ti- maukel, Torres del Paine, Tortel, Palena, Prima- vera, Cabo de Hornos y Antártica. Ri: índice de riesgo climático A: indicador normalizado de amenaza E: indicador normalizado de exposición V: indicador normalizado de vulnerabilidad Re: indicador normalizado de resiliencia Ri: índice de riesgo climático A: indicador normalizado de amenaza E: indicador normalizado de exposición V: indicador normalizado de vulnerabilidad Re: indicador normalizado de resiliencia En primer lugar, la amenaza fue calculada a través de la frecuencia de zonas de catástrofe declaradas por comuna, entre 1984 y 2013, de acuerdo al paso descrito en la fase anterior. Estos eventos son considerados como el peor escenario climático, de acuerdo a su presencia y extensión espacial. Ri= A+((E+V+Re)/3) 2 2 ZONAS DE CATÁSTROFE POR EVENTOS HIDROMETEOROLÓGICOS EN CHILE Y APORTES PARA UN ÍNDICE DE RIESGO CLIMÁTICO 29 de tipo hidrometeorológicos, correspondientes a crecidas e inundaciones producidas por pre- cipitaciones extremas, fusión de nieve o hielo, desbordamiento de canales, rotura de presas o combinación de varios factores. Por último, se incluyen también eventos como los aluviones y avenidas torrenciales desencadenados por lluvias intensas. principales del evento. Este periodo de análisis responde a la disponibilidad de los decretos catastrados. REVISTA DE GEOGRAFÍA NORTE GRANDE Donde: Pi: puntaje normalizado p j Xi: valor de la observación i de la variable Xi: valor de la observación i de la variable X: promedio de la variable original X: promedio de la variable original Xmin: valor mínimo de la variable l d l bl Xmax: valor máximo de la variable Rsup: razón de puntaje que se asigna a cada unidad de X sobre el promedio Rsup: razón de puntaje que se asigna a cada unidad de X sobre el promedio Rinf: razón de puntaje que se asigna a cada unidad de X bajo el promedio Rinf: razón de puntaje que se asigna a cada unidad de X bajo el promedio Finalmente, el indicador de resiliencia se ha estimado usando el valor del índice de desarrollo humano (IDH), año 2003, a nivel comunal de acuerdo a la información del Pro- grama de Naciones Unidas para el Desarrollo (MIDEPLAN y PNUD, 2005) y el porcentaje de población perteneciente a organizaciones co- munitarias a través de la base de datos de la en- cuesta CASEN año 2009 (MIDEPLAN, 2009). El primer índice se ha escogido en función de de- terminar aquellas comunas que tienen mejores condiciones de ingreso, salud y educación para enfrentar una crisis. Mientras que el segundo se asocia a identiﬁ car comunas donde hay comu- nidades con mayor nivel de participación social, en instituciones como juntas de vecinos, clubes deportivos y otras organizaciones sociales, que en principio pueden enfrentar de mejor mane- ra un desastre natural y también recuperarse después de una perturbación. Pmed: valor del puntaje asignado al pro- medio Pmed: valor del puntaje asignado al pro- medio Pmin: valor del puntaje asignado al valor mínimo de la variable Pmin: valor del puntaje asignado al valor mínimo de la variable Pmax: valor del puntaje asignado al valor máximo de la variable Pmax: valor del puntaje asignado al valor máximo de la variable Para el proceso de normalización se usaron como parámetros de transformación las medias, mínimas y máximas nacionales de cada variable. Para la pobreza y enfermedades respiratorias se usó como unidad de medida los porcentajes de la variable en relación al total comunal. REVISTA DE GEOGRAFÍA NORTE GRANDE 30 porcentaje de población con una o más enfer- medades respiratorias a nivel comunal. Para el cálculo de las enfermedades respiratorias, se utilizaron las respuestas positivas de las perso- nas que declararon haber estado en tratamiento por alguna de las siguientes enfermedades: neu- monía, asma, enfermedad pulmonar obstructiva crónica (EPOC) o infección respiratoria aguda, de acuerdo a la información proporcionada por el Departamento de Estadísticas e Información de Salud del Ministerio de Salud (DEIS, 2012). Se trabaja sobre el supuesto que estas enfer- medades se maniﬁ estan con mayor frecuencia después de un evento extremo, como heladas o lluvias intensas (D’Amato et al., 2012). mínimo valor de la variable y 1 al máximo, considerando una fórmula que aplica dos funciones de asignación de puntaje; una para los puntajes sobre el promedio y otra para los puntajes bajo el promedio, debido a que no necesariamente los recorridos de los pun- tajes a ambos lados de la media son simétri- cos (Carvacho, 2011). La fórmula utilizada corresponde a: Pmax – Pmed x – Xmin Rinf = Pmax – Pmed Xmax – x Rsup = Xi ≥ x – Pmed + (Xi – x) x Rsup Xi < x – Pmin + (Xi – xmin) x Rinf Pi= Xi ≥ x – Pmed + (Xi – x) x Rsup Xi < x – Pmin + (Xi – xmin) x Rinf Pi= Para el caso de la variable de pobreza, se usó información de la encuesta CASEN, a partir del documento “Incidencia de la Pobre- za a nivel Comunal, segú n Metodologí a de Estimació n para Á reas Pequeñas”, que incluye estimaciones de pobreza a nivel comunal que corrigen el sesgo muestral a ese nivel (Ministe- rio de Desarrollo Social, 2013). Evolución de las zonas de catástrofes Para determinar la evolución de los even- tos climáticos extremos, se generó una base de datos a través de la revisión retrospectiva de los decretos de declaración de zonas de catástrofe entre los años 1984 y 2013, promul- gados por el Ministerio del Interior según el Decreto Supremo N° 104, del 25 de junio de 1977, que coordina y sistematiza el Título I de la Ley N° 16.282 (1965), sobre disposiciones permanentes para casos de sismos o catástrofes y sus modiﬁ caciones posteriores (1986, 1991, 2003, 2010 y 2012). Para ello se consultó la base de datos de leyes de la Biblioteca del Congreso Nacional (BCN, 2014) de Chile, para luego sistematizar la información por región, comuna, fecha, tipo de evento (lluvias, sequías, heladas, nevadas y aluviones) y observaciones La exposición fue calculada mediante el porcentaje de población infantil (0 a 15 años) y adulta mayor (sobre 65 años) a nivel comunal, extraídas del Censo de Población y Vivienda año 2002 del Instituto Nacional de Estadística (INE). El supuesto es que esta población se encuentra más expuesta ante un evento extremo de origen climático que la población adulta (IPCC, 2014). Es importante aclarar que no se usaron los datos del censo de población y vivienda de 2012, ya que estos se encuentran objetados por graves deﬁ ciencias en el levantamiento de datos. La vulnerabilidad se ha abordado a través de las características socioeconómicas y de salud de la población, analizando el porcen- taje de población en situación de pobreza y el REVISTA DE GEOGRAFÍA NORTE GRANDE Proyección del cambio climático y la exposición Finalmente, para estimar las tendencias de cambio climático y exposición, se han usado las modelaciones de precipitaciones y tempe- raturas para los años 2010-2100 desarrolladas por el Departamento de Geofísica de la Fa- cultad de Ciencias Físicas y Matemáticas de la Universidad de Chile (DGF/CONAMA, 2006) y las proyecciones de población del Instituto Nacional de Estadística (INE, 2005) para el año 2020. Los resultados se presentan para las cin- co macrozonas del país: Norte Grande, Norte Chico, Zona Central, Zona Sur y Zona Austral. A través de la declaración de un estado de excepción, dependiendo de sus característi- cas, puede verse afectado el ejercicio de los derechos y garantías de las personas, con el ﬁ n de su protección. Estos estados pueden ser “Estado de asamblea” en caso de guerra exte- rior, “Estado de sitio” en caso de guerra inte- rior, “Estado de emergencia” en caso de grave alteración del orden público, daño o peligro para la seguridad de la Nación por motivos internos o externos, y ﬁ nalmente “Estado de catástrofe” para situaciones de calamidad pública. ZONAS DE CATÁSTROFE POR EVENTOS HIDROMETEOROLÓGICOS EN CHILE Y APORTES PARA UN ÍNDICE DE RIESGO CLIMÁTICO 31 Cuadro Nº 1 Valores de referencia para la normalización de las variables a nivel comunal Variables Valor mínimo Valor máximo Valor promedio Fuente Indica- dor Zonas de catástrofe (frecuen- cia) 0 9 3,3 BCN (2014) Amena- za Población infantil y adulto mayor expuesta (%) 0 3,24 0,3 INE (2002) Ex- posición Pobreza (%) 0 100 16,9 Ministerio de Desarrollo Social (2013) Vulnera- bilidad Enfermedades respiratorias (%) 0,27 4,95 1,94 DEIS (2012) Índice de desarrollo humano (IDH) 0 1 0,725 MIDEPLAN y PNUD (2005) Resilien- cia Participación comunitaria (%) 0 100 25,2 MIDEPLAN (2009) Fuente: Elaboración propia. Cuadro Nº 1 Cuadro Nº 1 Valores de referencia para la normalización de las variables a nivel comunal En los casos de los indicadores compues- tos por dos variables, como vulnerabilidad y resiliencia, fueron integrados, previa normali- zación, usando igual peso entre las variables. de Excepción Constitucional, Ley Nº 18.415 (1985), en donde se establecen una serie de normas particulares para cada situación. Los estados de excepción, según el artículo 39 de la Constitución, corresponden a situaciones en las que ciertos eventos afecten gravemente el normal funcionamiento del Estado, tales como guerras, conmoción interior, emergen- cia y calamidad pública. REVISTA DE GEOGRAFÍA NORTE GRANDE Para el caso de la población expuesta se usó un criterio distinto de normalización, ya que se empleó como valor de entrada el porcentaje de la población infantil y adulta de cada comuna en relación al total nacional: en este caso las comunas que tienen mayor cantidad bruta de población en relación al país son las más expuestas. En el caso del IDH y población que participa en organizaciones comunitarias se ha invertido el sentido de los puntajes, de modo que las comunas con mayores porcentajes representen el mejor nivel de resiliencia (valores cercano a 0) y los porcentajes más bajos la peor situación de resiliencia (valores cercano a 1). El Cuadro Nº 1 muestra los valores de referencia para la normalización de cada variable. Una vez levantada la información base se procedió a una fase de normalización, para lo cual se usó el método de designación de unidades valorativas propuesto por Carvacho (2011). Este método permite transformar los atributos de las variables medidas en unidades heterogéneas en unidades homogéneas, en una escala de 0 a 1, donde 0 corresponde al REVISTA DE GEOGRAFÍA NORTE GRANDE REVISTA DE GEOGRAFÍA NORTE GRANDE 32 Ley Nº 16.282 (1974). Además las medidas que se adopten en cada declaración dependerán de la Ley de Presupuestos para el sector público según el período que corresponda. A partir de la declaración de zona de desastre, la normati- va establece excepciones en cuanto a cambios en los presupuestos públicos para facilitar la gestión de daños y de ayuda a damniﬁ cados, en este sentido se establece un marco para generar préstamos con garantías especiales, recibir y canalizar donaciones con facilidades tales como exención de impuestos y tarifas de carga o descarga, movilización, almacenaje entre otros cargos, con el ﬁ n de agilizar la ayuda recibida. En este caso la gestión de la ONEMI se cen- tró en funciones de captura y administración de información sobre daños, entrega de infor- mación oﬁ cial a autoridades y prensa, apoyo en terreno de un equipo de profesionales, ca- nalización de la ayuda del Gobierno hacia las regiones y zonas afectadas por los temporales y labores de coordinación en general. Dentro de la ayuda realizada se puede mencionar la entrega de un total de 8.620 cajas de ali- mentos, 9.615 colchonetas, 18.794 frazadas, 5.500 planchas de zinc, 13.350 planchas de pizarreño, 22.275 pañales desechables y 1.870 viviendas de emergencia, entre otras (ONEMI, 2000). En términos administrativos, el Presiden- te de la República y los encargados de los organismos públicos designados por el Presi- dente se deben reunir en la Oﬁ cina Nacional de Emergencias del Ministerio del Interior y Seguridad Pública (ONEMI) para constituir el Comité de Emergencia (COE) y evaluar los daños y determinar las medidas a seguir (ONEMI, 2013). Por último, es importante mencionar que luego del terremoto del 2010 y como parte de una de las recomendaciones de la misión de las Naciones Unidad que visitó el país para dar cum- plimiento al Marco de Acción de Hyogo, ﬁ rmado por Chile el 2005, se constituyó una plataforma nacional para la reducción del riesgo de desas- tres, liderada por la ONEMI. Uno de los resulta- dos de este trabajo es la Política Nacional para la Gestión del Riesgo de Desastres (ONEMI, 2014). REVISTA DE GEOGRAFÍA NORTE GRANDE Esta política nacional deﬁ ne un marco de acción para cinco ejes estratégicos: forta- lecimiento institucional, fortalecimiento de los sistemas de monitoreo y alerta temprana, fomento de la cultura de la prevención y au- toaseguramiento, reducción de los factores subyacentes del riesgo y fortalecimiento de la preparación ante los desastres. Respecto a la forma de operar de las zo- nas de catástrofe, se puede señalar el evento de junio del año 2000 donde cinco sistemas frontales y un núcleo frío en altura produjeron anegamientos, crecidas de ríos e inundaciones, que resultaron en graves daños a viviendas, servicios y muerte de personas, desencadenan- do una declaratoria de zona de catástrofe para las comunas de Loncoche, Gorbea, Valdivia, San José de La Mariquina, La Unión y Pan- guipulli, mediante Decreto Supremo 3.094 del 5 de junio, ﬁ rmado por el Presidente de la República en su visita a la zona. Poste- riormente, con fecha 12 de junio, mediante Decreto Supremo Nº 3.111, se amplía la zona a las comunas de Los Lagos, Lanco y San Pablo. El miércoles 14 de junio, el Presidente se constituyó en la ONEMI, junto al Ministro de Obras Públicas y Telecomunicaciones, los Subsecretarios del Interior, Obras Públicas y Educación, Intendentes Metropolitanos, Direc- tor y técnicos de ONEMI, para evaluar la emer- gencia producto de los temporales. Además, se decretó mediante el Decreto Supremo (D.S.) Nº 3.120 zonas afectadas por catástrofe en las regiones de Valparaíso y Metropolitana y se de- terminó la suspensión de clases para los niveles prebásico, básico y medio de establecimientos públicos y privados de estas regiones más las regiones VI y VII (ONEMI, 2000). Propuesta de un índice de riesgo climático En este apartado se presenta la propuesta de índice de riesgo climático, compuesta por los indicadores parciales de amenaza, expo- sición, vulnerabilidad y resilencia. Evolución de desastres y catástrofes en Chile Como se ha mencionado anteriormente, en el caso de zonas afectadas por eventos naturales estas pueden ser decretadas como zonas de catástrofe a través de un Decreto Supremo promulgado por el Ministerio del In- terior, según lo dispuesto en la Constitución y la La determinación de zonas afectadas por eventos extremos obedece a las disposiciones establecidas en la Constitución Política de la República de Chile (1980) y en particular a la Ley Orgánica Constitucional de los Estados ZONAS DE CATÁSTROFE POR EVENTOS HIDROMETEOROLÓGICOS EN CHILE Y APORTES PARA UN ÍNDICE DE RIESGO CLIMÁTICO 33 y Austral con 3,9%. Las comunas que presen- tan mayor frecuencia son: La Ligua, Cabildo y Petorca con nueve eventos catastróﬁ cos. sido la Zona Central del país con el 38,5% de los eventos, seguida de la Zona Sur, con 32,1%, Norte Chico 18%, Norte Grande 7,7% y Austral con 3,9%. Las comunas que presen- tan mayor frecuencia son: La Ligua, Cabildo y Petorca con nueve eventos catastróﬁ cos. sido la Zona Central del país con el 38,5% de los eventos, seguida de la Zona Sur, con 32,1%, Norte Chico 18%, Norte Grande 7,7% Figura Nº 1 Evolución de zonas de catástrofes por causas climáticas e hidrometeorológicas en Chile 1984- 2013 Fuente: Elaboración propia en base a datos recolectados de www.leychile.cl (BCN, 2014). Figura Nº 1 Evolución de zonas de catástrofes por causas climáticas e hidrometeorológicas en Chile 1984- 2013 Fuente: Elaboración propia en base a datos recolectados de www.leychile.cl (BCN, 2014). de Camiña y Huara (D.S. 289) en 2012, y en la comuna de La Florida el año 1993 (D.S. 765). Con respecto a la naturaleza de estos eventos, los temporales de lluvia han sido el tipo de evento más recurrente con un 61%, siendo el año 1997 el más importante, ya que concentró la mayor cantidad de eventos afec- tando 10 regiones. Es interesante notar que la tendencia es hacia un aumento en el número de eventos climáticos extremos con un peak el año 2007, año que se caracterizó por la concurrencia de nevadas y heladas en las macrozonas del Norte Chico y Central. La segunda tipología de evento más re- currente son las sequías con un 20% para el periodo, seguida de las nevazones con un 9%, las heladas con un 7% y ﬁ nalmente los aluviones con solo 3 eventos. Estos últimos, constituyen eventos extremos puntuales des- encadenados por las lluvias intensas, los más importantes se han localizado en la región de Norte Grande, en la ciudad de Antofa- gasta en el año 1991 (D.S. 513), comunas Zonas de catástrofes en Chile como indicador de amenaza En función de la revisión de los decretos se han registrado 78 eventos asociados a zonas de catástrofes y desastres en el perio- do comprendido desde el año 1984 al 2013 (Figura Nº 1). La macrozona más afectada ha REVISTA DE GEOGRAFÍA NORTE GRANDE REVISTA DE GEOGRAFÍA NORTE GRANDE 34 años y mayor a 65 años), equivalente a 33,8% de la población del país. Estas se concentran preferentemente en el Área Metropolitana de Santiago (AMS) en la macrozona Central. Las comunas más expuestas son Puente Alto, con un 3,2% de la población expuesta, le siguen las comunas de Maipú y La Florida con un 2,83% y un 2,06% respectivamente. Otras comunas del AMS que presentan alto nivel de exposición son San Bernardo, Las Condes y Peñalolén. Es intere- sante señalar que las comunas que se encuentran en el piedemonte de la cordillera de los Andes son mayormente afectadas por eventos climáticos extremos, tales como deslizamientos de terreno y aluviones, por lo que su alto nivel de exposición constituye un punto clave en la gestión del riesgo. Otras comunas con un alto nivel de ex- posición en la macrozona Central del país son Quintero, Valparaíso y Rancagua. En el Norte Grande ﬁ guran las comunas de Antofagasta y Ari- ca y en la Zona Sur las comunas mayormente ex- puestas son Temuco y Puerto Montt (Figura Nº 2). Figura Nº 2 Mapa de exposición ante amenazas climáticas 2002 Figura Nº 2 g Mapa de exposición ante amenazas climáticas 2002 g Mapa de exposición ante amenazas climáticas 2002 Fuente: Elaboración propia en base a datos Censo de Población y Vivienda (INE, 2002). Fuente: Elaboración propia en base a datos Censo de Población y Vivienda (INE, 2002). al promedio de países OCDE que alcanza a 11,3% al año 2010 (OCDE, 2014). Indicador de exposición Respecto al segundo componente del índice, existen 5.100.458 personas que están expuestas a una amenaza climática (población menor a 15 ZONAS DE CATÁSTROFE POR EVENTOS HIDROMETEOROLÓGICOS EN CHILE Y APORTES PARA UN ÍNDICE DE RIESGO CLIMÁTICO 35 registros se observa una concentración de po- breza en macrozona Sur, especialmente en la Región de la Araucanía. tidad de enfermedades son las regiones de Araucanía y Los Ríos. Al integrar estas dos variables se observa que las comunas con mayor indicador de vulnerabilidad son San Ramón, Chiguayante y Lampa, con un valor de 0,74. Estas comu- nas presentan altos valores de vulnerabilidad explicado por los altos valores las variables parciales; San Ramón posee un 25,8% de po- blación pobre y 4,5% de enfermedades, Chi- guayante 18,4% y 4,7%, y Lampa un 16,1% y 4,9% respectivamente. La distribución espa- cial se presenta en la Figura Nº 3. Por su parte, al año 2009 el país presenta una morbilidad promedio asociada a enfer- medades respiratorias de 1,9%. Las comunas que presentan mayores valores corresponden a Lampa (4,9%), Chiguayante (4,7%) y San Ramón (4,5%). Al comparar estos datos con la tasa de egresos hospitalarios por enferme- dades respiratorias (DEIS, 2012), se observa que las regiones que concentran mayor can- Figura Nº 3 Mapa de vulnerabilidad ante amenazas climáticas 2009-2012 Fuente: Elaboración propia en base a datos del Ministerio de Desarrollo Social (2013) y DEIS (2012). Fuente: Elaboración propia en base a datos del Ministerio de Desarrollo Social (2013) y DEIS (2012). Indicador de vulnerabilidad al promedio de países OCDE que alcanza a 11,3% al año 2010 (OCDE, 2014). Como se mencionó en la metodología, este indicador se compone en dos variables, la pobreza y las enfermedades respiratorias. En el primer caso, Chile ha mejorado sus in- dicadores sociales, pasando el año 1990 de 38,4% de su población en situación de po- breza a 16,9% de pobres el 2009 (Ministerio de Desarrollo Social, 2012; 2013). De todas formas el porcentaje de pobreza es superior De acuerdo a los datos de la encuesta CASEN 2009 las comunas más pobres son: Angol (37,6%), Lumaco (36,8%), Loncoche (36,5%) y Purén (36,4%), mientras que cifras más recientes de la encuesta CASEN 2011 señalan un cambio en la posición: Ercilla (48,8%), Los Á lamos (41,3%), Puerto Saave- dra (37,3%) y Lonquimay (36,1%). En ambos REVISTA DE GEOGRAFÍA NORTE GRANDE REVISTA DE GEOGRAFÍA NORTE GRANDE 36 de 0,295, seguida de Camarones (0,38) en el Norte Grande y San Gregorio (0,39) en la macrozona Austral. Llama la atención que entre las comunas con mejores índices se encuentran comunas del extremo norte y sur del país, lo que se puede explicar por el alto nivel de participación en organizaciones sociales (52% y 32,5%, respectivamente), ya que estos valores son muy superiores a la me- dia nacional (25,2%). Las comunas que presentan peores niveles de resiliencia pertenecen a la Zona Centro- Sur del país: Paredones (0,72), Yerbas Buenas (0,68) y Colina (0,66), en general estas comu- nas presentan bajos niveles de participación en organizaciones sociales y un bajo índice de desarrollo humano (Figura Nº 4). de 0,295, seguida de Camarones (0,38) en el Norte Grande y San Gregorio (0,39) en la macrozona Austral. Llama la atención que entre las comunas con mejores índices se encuentran comunas del extremo norte y sur del país, lo que se puede explicar por el alto nivel de participación en organizaciones sociales (52% y 32,5%, respectivamente), ya que estos valores son muy superiores a la me- dia nacional (25,2%). Figura Nº 4 g Mapa de resiliencia ante amenazas climáticas 2003-2009 g Mapa de resiliencia ante amenazas climáticas 2003-2009 g Mapa de resiliencia ante amenazas climáticas 2003-2009 Fuente: Elaboración propia en base a datos de MIDEPLAN y PNUD (2005) y MIDEPLAN (2009). Fuente: Elaboración propia en base a datos de MIDEPLAN y PNUD (2005) y MIDEPLAN (2009). Indicador de resiliencia porcentaje de población perteneciente a or- ganizaciones comunitarias son Alto del Car- men (60,4%), Currarehue (56,2%) y Camiña (54,6%); y las comunas con menor porcentaje son: Paredones (4,8%), Yerbas Buenas (7,5%) y San Ramón (8,4%). El IDH, primer componente del indi- cador de resilencia, muestra que las co- munas con mayor desarrollo humano son Vitacura (0,949), Las Condes (0,933) y Lo Barnechea (0,912); mientras que las comunas con menor valor son: San Juan de La Costa (0,510), Treguaco (0,562) y Ninhue (0,569). En segundo término, las comunas con mayor De la combinación de ambas variables se obtiene que las comunas más resilientes del país resultaron: Vitacura, con un índice Índice de riesgo climático en Chile de riesgo climático. Las comunas con mayores índices de riesgo climático son La Ligua (0,73), Cabildo (0,69) y Petorca (0,69), de la provincia de Petorca, V Región de Valparaíso. Estas co- munas se caracterizan por presentar altos nive- les de amenaza, con valores sobre la media nacional (3,3 eventos). Finalmente, una vez normalizados los datos usados en los indicadores, se procedió a integrarlos en el índice de riesgo climático. De esta forma, las zonas del país que presen- taron mayores niveles de riesgo climático son el área costera de la macrozona Central y del Norte Chico, en donde se presentan la mayor cantidad de comunas con un índice elevado Estas comunas registran nueve eventos climáticos extremos asociados a cuatro REVISTA DE GEOGRAFÍA NORTE GRANDE REVISTA DE GEOGRAFÍA NORTE GRANDE catástrofe, mediante D. S. 1.251. En junio del mismo año fuertes temporales de viento y lluvia afectaron gran parte del país con da- ños considerables a la población y viviendas, e infraestructura vial, social, urbana y rural (D.S. 1543). A ﬁ nes de agosto de 1997 se de- clara zona de catástrofe (D.S. 849) por bajas temperaturas que provocaron la destrucción de cultivos agrícolas, afectando a pequeños agricultores y a empresas, disminuyendo el empleo. Al mes siguiente ocurrieron nuevos temporales con similares efectos a los del evento de junio (D.S. 2.149). nuevamente a las tres comunas con mayor índice de riesgo anteriormente señaladas. Por otro lado, estas comunas presentan niveles de resiliencia cercanos a la media na- cional: La Ligua 0,51, Cabildo 0,5 y Petorca 0,47. En cuanto a los niveles de exposición, estas comunas poseen cifras menores a la me- dia nacional (0,29) con excepción de la co- muna de La Ligua (0,35). Finalmente, respec- to a vulnerabilidad Petorca (0,57), La Ligua (0,49) y Cabildo (0,44) son las comunas que se encuentran más vulnerables dentro de la provincia, junto con Papudo (0,49), mientras que Zapallar alcanza el mejor nivel (0,39). El ciclo de sequías y temporales de lluvia que afectó a la zona central del país continuó en 1999 con una sequía que afectó gravemente la agricultura, ganadería y disponibilidad de agua declarándose como zona de catástrofe la provincia de Petorca y sus comunas integrantes (D.S. 3.144). El año 2000 se caracterizó por presentar intensas lluvias que provocaron el anegamiento de vías de circulación y paralización del transporte, además de daños en infraestructura pública y privada, en junio de ese año se declaró como zona de catástrofe a las regiones de Valparaíso y Metropolitana de Santiago (D.S. 3.120). Finalmente, las comunas que presentan menor nivel de riesgo son María Elena, Quin- chao, y Puqueldón, con valores bajo 0,15. Si bien todos los indicadores son bajos, el común denominador es que son comunas donde no se ha registrado eventos climáticos catastróﬁ cos. ZONAS DE CATÁSTROFE POR EVENTOS HIDROMETEOROLÓGICOS EN CHILE Y APORTES PARA UN ÍNDICE DE RIESGO CLIMÁTICO 37 y vías férreas, suspensión de servicios públi- cos, daños a propiedad pública y privada, y muerte de personas. Con motivo de este evento se declaró zona de catástrofe a las re- giones Norte Chico y Central (D.S. 741). sequías, tres temporales y dos heladas. El primer evento corresponde a los temporales de lluvia que afectaron a gran parte del país el año 1984, estos temporales generaron aludes de nieve, inundaciones, irrupción de caminos y vías férreas, suspensión de servicios públi- cos, daños a propiedad pública y privada, y muerte de personas. Con motivo de este evento se declaró zona de catástrofe a las re- giones Norte Chico y Central (D.S. 741). sequías, tres temporales y dos heladas. El primer evento corresponde a los temporales de lluvia que afectaron a gran parte del país el año 1984, estos temporales generaron aludes de nieve, inundaciones, irrupción de caminos Figura Nº 5 Fuente: Elaboración propia. Fuente: Elaboración propia. Fuente: Elaboración propia. afectación en cuanto a la disponibilidad de alimentos y de agua. Posteriormente, en 1990, una prolon- gada sequía (D.S. 750) afectó al país con especial intensidad en algunas comunas de las regiones administrativas de Atacama, de Coquimbo y de Valparaíso (parte sur del Norte Chico y zona costera de la macro- zona Central), encontrándose Cabildo, La Ligua, Petorca y Zapallar como las áreas más afectadas dentro de esta última región. Entre los efectos de este fenómeno se encontraron grandes pérdidas en las ramas económicas de agricultura, ganadería, minería, y también En 1997 se registraron tres eventos que afectaron a estas comunas; una sequía en marzo que afectó a comunas de las regiones de Atacama, Coquimbo y Valparaíso (D.S. 866), produciendo daños en la agricultura, ganadería y disponibilidad de agua para el riego y consumo humano. En este decreto se excluyen sectores urbanos y luego se in- corporan nuevas comunas afectadas por la 38 ZONAS DE CATÁSTROFE POR EVENTOS HIDROMETEOROLÓGICOS EN CHILE Y APORTES PARA UN ÍNDICE DE RIESGO CLIMÁTICO ZONAS DE CATÁSTROFE POR EVENTOS HIDROMETEOROLÓGICOS EN CHILE Y APORTES PARA UN ÍNDICE DE RIESGO CLIMÁTICO 39 Figura Nº 6 Proyección del cambio climático para el período 2010-2100 y estimación de población para el año 2020 Fuente: Elaboración propia con datos de CEPAL (2012) e INE (2015). Figura Nº 6 Figura N 6 Proyección del cambio climático para el período 2010-2100 y estimación de población para el año 2020 Fuente: Elaboración propia con datos de CEPAL (2012) e INE (2015). Proyección de variables climáticas y exposición ante el cambio climático A partir de las modelaciones realizadas por el modelo PRECIS (Comisión Nacional del Me- dio Ambiente, 2006), para los próximos años (2010-2040, 2040-2070 y 2070-2100), se pro- yecta que en el país existirá un aumento de las temperaturas y una disminución de las preci- pitaciones (Figura Nº 6). Una excepción a esta proyección corresponde al comportamiento del Norte Grande donde no hay una tendencia clara, en términos que se podría esperar una disminución de temperaturas en la zona coste- ra y un aumento de precipitaciones en el sector altiplánico (Falvey & Garreud, 2009). En agosto de 2007 (D.S. 849) fuertes he- ladas impactaron las comunas de Cabildo, La Ligua y Petorca, las que destruyeron los cul- tivos de la zona, afectando tanto a pequeños agricultores como a empresarios agrícolas. Finalmente, el evento más reciente es del año 2012 (D.S. 234), en el cual una intensa y pro- longada sequía, afectó los sectores agrícola, productivo, ganadero, de la pequeña minería, de riego y de energía eléctrica debido a la reducción del caudal en las cuencas de los ríos Petorca, La Ligua y Aconcagua, afectando REVISTA DE GEOGRAFÍA NORTE GRANDE REVISTA DE GEOGRAFÍA NORTE GRANDE 40 reducción de la vulnerabilidad en esta comu- na debe estar enfocada en el mejoramiento integral de las cifras comunales. ción y ﬁ nanciamiento de un proyecto de Cen- tro de Salud Familiar (CESFAM) para 30.000 habitantes, iniciativa muy positiva para la re- ducción de su vulnerabilidad y de las comu- nas vecinas, ya que de esta manera se puede descongestionar la demanda sobre el hospital comunal San Agustín, que no da abasto para la población, especialmente en la estación de invierno, donde el hospital generalmente colapsa debido a la falta de espacios para los enfermos (Díaz, 2012). Es importante señalar que las comunas de la provincia de Petorca: La Ligua, Cabildo y Petorca, son comunas con una marcada vocación agrícola. El 85,5% de la superﬁ cie de Petorca es cultivada, 54,6% en Cabildo y 31,4% en La Ligua (INE, 2008). Uno de los principales cultivos es la palta con el 90% de su superﬁ cie destinada a este cultivo. Estas comunas son muy sensibles a los impactos del clima por la sobreexplotación del recurso agua y la asignación de derechos de aprove- chamiento de aguas más allá de la capacidad física de la cuenca y de los acuíferos. La sequía que ha afectado a esta provincia “ha provocado la pauperización de más de 7.000 pequeños agricultores, más de 50.000 perso- nas padecen de la carencia de agua para be- ber y servicios higiénicos básicos, a pesar de las innumerables denuncias realizadas” (Insti- tuto Nacional de Derechos Humanos, 2010: 142). En este caso, la gestión debería apuntar a lo que O’Hare y Rivas (2005) describen como medidas de adaptación soft, o no estructurales, en donde los recursos se enfo- can en acciones para reducir la vulnerabilidad de la población en el corto y mediano plazo. Sin embargo, la reducción de vulnera- bilidad de la comuna de La Ligua no solo debe estar enfocada en el mejoramiento de la salud pública, puesto que también presenta cifras desfavorables en participación en or- ganizaciones sociales, ya que se queda atrás respecto a las otras comunas de la provincia con un 23%, cifra inferior al promedio nacio- nal de 25%. La participación de la población en este tipo de organizaciones es muy rele- vante para las fases de prevención, emergen- cia y recuperación ante un evento climático extremo. Revisión de comunas peor evaluadas De todas formas los impactos más latentes se verían en el Norte Chico y Zona Central, donde se estima una disminución de las precipitaciones y un aumento de las tem- peraturas, lo que incidiría fuertemente en los desastres asociados a la sequía. Si bien no se ha registrado ninguna zona de catástrofe por olas de calor, es probable que a futuro este fenómeno vaya surgiendo especialmente en comunas ubicadas en los valles interiores. Para mejorar la situación de las comunas peor evaluadas mediante el índice de riesgo climático aplicado, la gestión del riesgo de- biese enfocarse en aquellos aspectos más dé- biles tanto en temas de vulnerabilidad como de resiliencia. Por ejemplo, la comuna de La Ligua debiese enfocar su gestión en mejorar su sistema de salud ya que presenta un ele- vado porcentaje de población expuesta que presentó enfermedades respiratorias el año 2009, esto es 2,2%, cifra que supera la me- dia nacional. Este dato es relevante dada la cantidad y tipología de amenazas climáticas, como por ejemplo heladas y temporales. No obstante, es importante mencionar que esta comuna logró hace poco (2013) la aproba- Con respecto a la población proyectada para el año 2020, se observa que la macrozona Central tendrá la mayor cantidad de población y junto con ello una eventual mayor vulnerabili- dad, explicado por la presencia de la metrópolis de Santiago, principal polo económico y de- mográﬁ co del país, la conurbación de Valparaí- so-Viña del Mar y otras ciudades intermedias. Consideraciones ﬁ nales En relación al índice de riesgo climático propuesto se puede concluir que no nece- sariamente las comunas con mayor nivel socioeconómico tienen mejores niveles de resiliencia. Esto es algo que debe resaltarse, ya que es muy importante contar con una comunidad comprometida y organizada que fortalezcan la capacidad de respuesta ante eventos extremos. Finalmente, es importante centrar los esfuerzos en aquellas regiones que se verán a futuro mayormente afectadas por los im- pactos del cambio climático y variabilidad natural del clima, de manera de cambiar el enfoque reactivo, como es la declaratoria de zona de desastres, por un enfoque proactivo y estratégico como la gestión efectiva del riesgo de desastre. Las regiones y comunas más vul- nerables y riesgosas frente al cambio climáti- co corresponden a la zona de Chile central, especialmente en la costa y valles interiores, donde se concentrará mayormente la po- blación y se pronostica una agudización de los problemas climáticos e hidrometeorológi- cos observados como sequías, inundaciones, heladas, así como marejadas, incendios, olas de calor, entre otros y que requieren de una mayor preparación. Por otro lado, es importante tener pre- sente la importancia de espacializar el riesgo climático para poder focalizar eﬁ cientemente los recursos y las políticas públicas. Del mismo modo, existen varias indi- caciones para mejorar el índice propuesto: en primer lugar se debe considerar que el indicador de amenaza está basado en una visión retrospectiva de las zonas de catástro- fes y no en las dinámicas naturales propia- mente tales; al mismo tiempo según la base de datos consultada no fue posible acceder a las declaraciones de zonas de catástrofes anteriores al año 1984; se pueden incluir otras fuentes de información, actualizadas y del mismo período de tiempo; o bien incluir nuevos indicadores que sean relevantes para la toma de decisiones, como por ejemplo las capacidades de gestión instaladas. Se enfatiza la importancia de seguir realizando este tipo de estudios a distintas escalas geográficas, que permitan evaluar el panorama actual y futuro para poder proponer medidas de adaptación adecuadas y anticiparse a crisis socionaturales. ZONAS DE CATÁSTROFE POR EVENTOS HIDROMETEOROLÓGICOS EN CHILE Y APORTES PARA UN ÍNDICE DE RIESGO CLIMÁTICO 41 2007 se observa una escasa cantidad de pre- cipitación total anual para la zona central de Chile, determinando que la década 2000- 2010 es una de las más secas de los últimos 60 años, siendo el año 2002, el último año lluvioso observado (Dirección Meteorológica de Chile, 2013). conclusión tiene que ver con la necesidad de mejorar la política pública que enfrente los desafíos del cambio climático, pasando por mejorar nuestros instrumentos de planiﬁ cación territorial y de coordinación de emergencias como también la materialización de obras de infraestructura que permitan disminuir los niveles de vulnerabilidad y aumentar los de resiliencia. Infraestructura para enfrentar la sequía, diseño de obras hidraúlicas para periodos de retornos mayores, planiﬁ cación de áreas verdes que actúen como buffers frente a amenazas hidrometeorológicas son algunos ejemplos que se pueden implementar para prepararse ante eventos extremos. REVISTA DE GEOGRAFÍA NORTE GRANDE Mejorar este aspecto es clave para que la comuna aumente su resiliencia y dis- minuya su vulnerabilidad ante eventos de manifestación inmediata o de largo plazo. Como referencia se puede indicar que el nivel de participación en organizaciones de nivel comunal en países como Brasil asciende a 38,3% (Bizberg, 2010). Fuera del ámbito del índice aplicado, el año 2013 la macrozona Central del país fue testigo de los problemas derivados de inten- sas lluvias estivales durante el mes de enero y febrero que sumadas a las altas temperaturas y deshielos propios de la temporada de vera- no, generaron aluviones en la zona cordille- rana de la ciudad de Santiago lo cual provocó el colapso masivo del servicio de agua pota- ble en 22 comunas del AMS, produciéndose cortes del suministro (Diario El Mercurio, 2013). Esto es especialmente relevante, puesto que no todos los eventos extremos desecandenan una declaratoria de zona de catástrofe, sin embargo estos eventos pueden ocasionar pérdidas y perjuicios tanto como los ligados a la zonas de catátrofes y por lo tanto, deben enfrentarse de una manera más preventiva que reactiva. Por otra parte, la comuna de Petorca es la más vulnerable en temas de pobreza, con un 16,8% de población en situación de pobreza al año 2009, esta situación podría ser explica- da por sus bajos niveles de ingreso (US$ 625 promedio mensual de ingreso autónomo por hogar a nivel comunal versus un promedio de US$1.164 de ingreso autónomo en el país) y sus altos niveles de analfabetización (8%) comparados con la realidad nacional (3,9%) (BCN, 2013). A pesar de esta desfavorable situación, la comuna de Petorca presenta un alto porcentaje de participación en organi- zaciones sociales (37,2%), lo que si bien en términos del índice de riesgo ayuda a aumen- tar la resiliencia, no compensa el mal desem- peño en las otras dimensiones. Por su parte, la comuna de Cabildo también tiene un alto porcentaje comparativo de participación en organizaciones sociales (29,7%), pero presen- ta el índice más bajo de desarrollo humano de las comunas analizadas (0,671), por lo que la Finalmente, cabe destacar que hay cier- to tipo de riesgos que son de manifestación más lenta, como por ejemplo las sequías o el proceso de desertiﬁ cación. Desde el año Referencias bibliográﬁ cas ARENAS, F.; HIDALGO, R. y LAGOS, M. Los riesgos naturales en la planiﬁ cacion territorial. Santiago de Chile: Centro de Políticas Públicas UC, 2010. AYALA-CARCEDO, F.J. y OLCINA, J. Riesgos Naturales. Barcelona: Editorial Ariel, 2002. AYALA-CARCEDO, F.J. y OLCINA, J. Riesgos Naturales. Barcelona: Editorial Ariel, 2002. En términos humanos, económicos y políticos resulta mucho más lógico aplicar medidas de adaptación climática en lugar que medidas reactivas como la declaración de zonas de catástrofe, que solo están dirigidas a atender la emergencia y no abordar toda la complejidad del problema. Por tal razón, otra BIBLIOTECA DEL CONGRESO NACIONAL DE CHILE (BCN). Reportes Estadísticos y Co- munales, 2013. Disponible en Internet: http:// reportescomunales.bcn.cl/ REVISTA DE GEOGRAFÍA NORTE GRANDE 42 de Chile: Diario El Mercurio, Sección Economía, 2013. Disponible en Internet: http://www.emol.com BIBLIOTECA DEL CONGRESO NACIONAL DE CHILE (BCN). Base de datos de Leyes chile- nas. Disponible en Internet: http://www.leychile. cl/ DÍAZ, J. Aprueban recursos para diseño de construcción de centro de salud familiar de La Ligua. La Ligua: Ilustre Municipalidad de La Li- gua, 2012. Disponible en Internet: http://www. laligua.cl/noti01.php?id=437 BIZBERG, I. Una Democracia Vacía. Socie- dad Civil, Movimientos Sociales y Democracia. En: BIZBERG, I. y ZAPATA, F. (coordinadores). Movimientos sociales. México: El Colegio de México, 2010. DIRECCIÓN METEOROLÓGICA DE CHILE. Boletín N°1 Monitoreo de Sequía Meteorológi- ca. Santiago de Chile, 2013. CARVACHO, L. Asigcon, propuesta de un método para la comparación de unidades espa- ciales utilizando variables normalizadas sobre una base conceptual. Anales del XXXII Congreso Nacional y XVII Internacional de Geografía: Conciencia Geográfica en el Tercer Milenio, 2012, p. 448-455. FALVEY, M. & GARREAUD, R. Regional cooling in a warming world: Recent tempera- ture trends in the southeast Paciﬁ c and along the west coast of subtropical South Ameri- ca (1979-2006). Journal of Geophysical Re- search Atmospheres, 2009, Vol. 114, D04102, doi:10.1029/2008JD010519 CEPAL. La Economía del Cambio Climático en Chile. Síntesis. Santiago de Chile: Comisión Económica para América Latina, Naciones Uni- das, 2012. GALLOPÍ N, G. Linkages between vulnera- bility, resilience, and adaptative capacity. Global Environmental Change, 2006, 16, p. 293-303. CONSTITUCIÓN POLÍTICA DE LA REPÚBLICA DE CHILE 1980. Ministerio Secre- taría General de la Presidencia. Diario Oﬁ cial de la República de Chile. Santiago de Chile, 17 de septiembre de 2005 (Decreto 100). INSTITUTO NACIONAL DE DERECHOS HUMANOS (INDH). Mapa de Conﬂ ictos Socio- ambientales en Chile. Santiago de Chile: Unidad de Estudio, INDH, 2012. D’AMATO, G.; CECCHI, L.; D’AMATO, M. & ANNESI-MAESANO, I. Climate change and respiratory diseases. European Respiratory Re- view, 2014, Nº 23, p. 161-169. INSTITUTO NACIONAL DE ESTADÍSTICAS (INE). Censo de Población y Vivienda 2002. San- tiago de Chile: INE, 2002. DECRETO SUPREMO N° 104. Chile. Fija el texto refundido, coordinado y sistematizado del Título I de la Ley N° 16.282. Diario Oﬁ cial de la República de Chile, Santiago de Chile, 25 de junio de 1977. INSTITUTO NACIONAL DE ESTADÍSTICAS (INE). Chile: proyecciones y estimaciones de población. 1990-2020. Santiago de Chile: INE, 2005. INSTITUTO NACIONAL DE ESTADÍSTICAS (INE). BIBLIOTECA DEL CONGRESO NACIONAL VII Censo Nacional Agropecuario año agrícola 2006-2007. Santiago de Chile: INE, 2008. DEIS. Base de datos egresos hospitalarios 2001-2012. Santiago de Chile: Departamento de Estadísticas e Información de Salud, Ministe- rio de Salud [base de datos], 2012. INTERGOVERNMENTAL PANEL ON CLI- MATE CHANGE (IPCC). Managing the Risks of Extreme Events and Disasters to Advance Climate Change Adaptation. Special Report of the Intergovernmental Panel on Climate Change. New York: Cambridge University Press, 2012. DEPARTAMENTO DE GEOFÍSICA DE LA UNIVERSIDAD DE CHILE/ Comisión Nacional del Medio Ambiente (DGF/ CONAMA). Estudio de la variabilidad climática en Chile para el siglo XXI. Santiago de Chile, 2006. INTERGOVERNMENTAL PANEL ON CLI- MATE CHANGE (IPCC). Climate Change 2014: Impacts, Adaptation, and Vulnerability. Part A: DIARIO EL MERCURIO. Aguas Andinas compensará con hasta $4.000 a clientes afectados por cortes de agua en la RM. Santiago ZONAS DE CATÁSTROFE POR EVENTOS HIDROMETEOROLÓGICOS EN CHILE Y APORTES PARA UN ÍNDICE DE RIESGO CLIMÁTICO 43 Global and sectoral Aspects. Working Group II Contribution to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge/New York: Cambridge University Press, 2014. OFICINA NACIONAL DE EMERGENCIA (ONEMI). Política Nacional para la Gestión de Riesgo de Desastres. Santiago de Chile: ONEMI, 2014. O’HARE, G. & RIVAS, S. The landslide ha- zard and human vulnerability in La Paz, Bolivia. The Geographical Journal, 2005, Vol. 171, Nº 3, p. 239-258. KLEIN, R.J.; SMITH, M.J.; GOOSEN, H. HULSBERGEN, C.H. Resilience and Vulnera- bility: Coastal Dynamics or Dutch Dikes? The Geographical Journal, 1998, Vol. 164, Nº 3, p. 259-268. MINISTERIO DE PLANIFICACIÓN Y COOPERACIÓN (MIDEPLAN) y PROGRAMA DE LAS NACIONES UNIDAS PARA EL DESARROLLO (PNUD). Las trayectorias del desarrollo humano en las comunas de Chile, 1994-2003. Santiago de Chile: División Social del Ministerio de Planiﬁ cación y Cooperación (MIDEPLAN) y Equipo de Desarrollo Humano del Programa de las Naciones Unidas para el Desarrollo (PNUD), 2005. LEY N° 16.282. Chile. Fija Disposiciones para Casos de Sismos o Catástrofes. Establece Normas para la Reconstruccion de la Zona Afectada por el Sismo de 28 de Marzo de 1965 y Modiﬁ ca la Ley N° 16.250. Ministerio de Ha- ci enda. Diario Oﬁ cial de la República de Chile, Santiago de Chile, 28 de julio de 1965. LEY N° 18.415. Chile. Ley Orgánica Consti- tucional de los Estados de Excepción. Ministerio del Interior. Diario Oﬁ cial de la República de Chile, Santiago de Chile, 14 de junio de 1985. ECHO. Documento País. Análisis de riesgos de desastres en Chile. VII Plan de Acción DIPECHO en Sudamérica 2011-2012. Santiago de Chile: Organización de las Naciones Unidas para la Educación la Ciencia y la Cultura (UNESCO), PNUD, Cruz Roja Chilena y DG ECHO, 2012. MINISTERIO DE DESARROLLO SOCIAL MINISTERIO DE DESARROLLO SOCIAL. Incidencia de la Pobreza a nivel Comunal, segú n Metodologí a de Estimació n para Á reas Pequeñ as. Chile 2009 y 2011. Santiago de Chile: Observatorio Social, Serie Informes Co- munales Nº 1, 6 de febrero de 2013. UNISDR. Terminología sobre Reducción del Riesgo de Desastres. Estrategia Internacional para la Reducción de Riesgos de las Naciones Unidas (UNISDR). Disponible en Internet: http://www.unisdr.org/les/7817_UNISDRTermi- nologySpanish.pdf. MINISTERIO DE PLANIFICACIÓN Y COOPERACIÓN (MIDEPLAN). Encuesta de caracterización socioeconómica nacional CASEN 2009. Módulo comunal [Base de datos]. Santiago de Chile: Departamento de Información Social, División Social, 2009. VILLARROEL, C. Eventos extremos de pre- cipitación y temperatura en Chile: proyecciones para ﬁ nes del siglo XXI. Santiago de Chile: Tesis (Magister en Meteorología y Climatología), Uni- versidad de Chile, Facultad de Ciencias Físicas y Matemáticas, Departamento de Geofísica, 2013. ORGANIZACIÓN PARA LA COOPERACIÓN Y EL DESARROLLO ECONÓMICO (OCDE). Society at a Glance 2014: OECD: Social Indicators. OECD Publishing, 2014. Disponible en Internet: http://dx.doi.org/10.1787/soc_glance-2014-en WALKER, B.; CARPENTER, S.; ANDERIES, J.; ABEL, N.; CUMMING, G.; JANSSEN, M.; LEBEL, L.; NORBERG, J.; PETERSON, G.D. & PRITCHARD, R. Resilience Management in Social-ecological Systems: a Working Hypothe- sis for a Participatory Approach. Ecology and society, 2002, Vol. 6, Nº 1, Art. 14. Disponible en Internet: http://www.consecol.org/vol6/iss1/art14/ OFICINA NACIONAL DE EMERGENCIAS (ONEMI). Web institucional. Disponible en In- ternet: http://www.onemi.cl/ http://www.consecol.org/vol6/iss1/art14/ OFICINA NACIONAL DE EMERGENCIA (ONEMI). Informe consolidado temporales 1 junio - 2 de julio 2000. Santiago de Chile: Departamento de Protección Civil, ONEMI, 2000. WILCHES-CHAUX, G. Herramientas para la Crisis: Desastres, Ecologismo y Formación Profe- sional. Popayán: Servicio Nacional de Aprendi- zaje (SENA), 1989.
https://openalex.org/W4362394326	https://figshare.com/articles/journal_contribution/Supplementary_Figure_S3_from_Epigenetic_Inactivation_of_microRNA-34b_c_Predicts_Poor_Disease-Free_Survival_in_Early-Stage_Lung_Adenocarcinoma/22446297/1/files/39897345.pdf	English	null	Supplementary Figure S2 from Epigenetic Inactivation of microRNA-34b/c Predicts Poor Disease-Free Survival in Early-Stage Lung Adenocarcinoma	null	2,023	cc-by	291	A Supplementary Figure S3. A Supplementary Figure S3. A Supplementary Figure S3. A B 5 n (log2) P = 0.002 P 4 n (log2) 4 3 2 relative expression 3 2 1 relative expression 0 1 miR-34b Unmethylated Methylated (n=19) (n=30) Unmethylated Me (n=19) ( 0 1 miR-34b B 5 n (log2) P = 0.002 4 3 2 relative expression 0 1 miR-34b Unmethylated Methylated (n=19) (n=30) B B 5 n (log2) P = 0.002 P = 0.003 4 n (log2) 4 3 2 relative expression 3 2 1 relative expression 0 1 miR-34b Unmethylated Methylated (n=19) (n=30) Unmethylated Methylated (n=19) (n=30) 0 1 miR-34b Supplementary Figure S3. Frequency of miR-34b/c promoter methylation and miR- 34b/c expression in early stage lung AC primary tumors. A 5% cutoff for estimated DNA methylation was used for classifying tumors as either methylated or unmethylated. (A) Bar plot depicts the estimated percent of DNA methylation based upon the AUC values obtained by MCA in a cohort of 140 lung AC tumors. (B) Box plots show the expression of miR-34b/c according to the miR-34b/c methylation status in a subset of 49 lung AC tumors. P = 0.003 4 n (log2) 3 2 1 relative expression Unmethylated Methylated (n=19) (n=30) 0 1 miR-34b Supplementary Figure S3. Frequency of miR-34b/c promoter methylation and miR- 34b/c expression in early stage lung AC primary tumors. A 5% cutoff for estimated DNA methylation was used for classifying tumors as either methylated or unmethylated. (A) Bar plot depicts the estimated percent of DNA methylation based upon the AUC values obtained by MCA in a cohort of 140 lung AC tumors. (B) Box plots show the expression of miR-34b/c according to the miR-34b/c methylation status in a subset of 49 lung AC tumors.
W2945447892.txt	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/CC724F9381BEEE90965EC09966308350/S0884291419001699a.pdf/div-class-title-microtensile-creep-testing-of-freestanding-mcraly-bond-coats-div.pdf	en		Microtensile creep testing of freestanding MCrAlY bond coats	Journal of materials research/Pratt's guide to venture capital sources	2,019	cc-by	5,715	Article DOI: 10.1557/jmr.2019.169 Microtensile creep testing of freestanding MCrAlY bond coats Sven Giese1,a), Steffen Neumeier1, Doris Amberger-Matschkal1, Jan Bergholz2, Robert Vaßen2, Mathias Göken1,b) 1 Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Materials Science & Engineering, Institute I, Erlangen 91058, Germany IEK-1: Materials Synthesis and Processing, Forschungszentrum Jülich GmbH, Institute of Energy and Climate Research, Jülich 52425, Germany a) Address all correspondence to this author. e-mail: sven.giese@fau.de b) This author was an editor of this journal during the review and decision stage. For the JMR policy on review and publication of manuscripts authored by editors, please refer to http://www.mrs.org/editor-manuscripts/. 2 Received: 25 February 2019; accepted: 23 April 2019 https://doi.org/10.1557/jmr.2019.169 Published online by Cambridge University Press Introduction The continuous improvement of aero engines and land-based gas turbines leads to increasing gas inlet temperatures and the demands for new materials. Such increased temperatures are possible with thermal barrier coating (TBC) systems consisting of Al- and Cr-containing bond coatings for enhanced oxidation- and corrosion resistance and ceramic TBCs, which are essential for thermal isolation [1]. The difference in thermal expansion coefﬁcients between the metal substrate and the TBC would lead to delamination. A metallic bond coat can improve the bonding between substrate and TBC and can reduce the risk of spallation of the ceramic top coat. Therefore, MCrAlY overlay coatings are applied to superalloy components, tailored to provide protection against high-temperature oxidation and hot corrosion [2, 3, 4, 5, 6, 7]. Studies have investigated hardness, Young’s modulus, diffusion or the inﬂuence of bond coats with different chemical compositions on the mechanical properties of the superalloys so far [8, 9, 10, 11]. However, less information about the mechanical properties of bond coats themselves exists [12]. This is certainly disadvantageous, as the mechanical properties of the bond coats determine, to a certain extent, the relaxation and the local stress state, especially on rough bond coats within in the system [13]. With respect to the importance of relaxation at high temperatures, creep-resistant bond coats (e.g., by the addition of oxide phases) might be beneﬁcial. In addition, it was also observed that ODS bond coats show a superior oxidation and thermal cyclic performance [14, 15]. It is known that ODS-NiAl shows a remarkable creep resistance at temperatures up to 1000 °C. The creep behavior of ﬁne-grained ODS-NiAl is described by Arzt et al. for materials with undissociated dislocations, like in NiAl (Göhring-Arzt model) [16]. A complex creep behavior as a mixture of diffusional and detachment controlled creep mechanisms was observed [16, 17, 18, 19]. TEM investigations on ODS-NiAl, -FeAl and -Ni3Al reveal that the size of the ODS particles has a large inﬂuence on the interaction between the ª Materials Research Society 2019. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. cambridge.org/JMR j Journal of Materials Research j www.mrs.org/jmr Bond coats are essential in gas turbine technology for oxidation protection. Freestanding MCrAlY (M = Ni, Co) bond coats were investigated with respect to their creep strength at elevated temperatures. Three types of MCrAlY, a Ni-based bond coat Amdry 386, a Co-based bond coat Amdry 9954 and Amdry 9954 + 2 wt% Al2O3 (ODS = oxide dispersion strengthened) produced by low pressure plasma spraying, were analyzed. The two phase microstructure of the bond coats consists of a fcc c-Ni solid solution and a B2 b-NiAl phase. Constant load experiments were performed in a thermomechanical analyzer at temperatures between 900 and 950 °C. Microtensile test specimens with a diameter of 450 lm were produced by a high-precision grinding and polishing process. Creep rupture was mainly due to void nucleation along the b–c interfaces and grain boundaries. The time to failure is larger in Ni-based Amdry 386 compared to that in Co-based Amdry 9954 due to a higher fraction of the high-strength b-NiAl phase at test temperatures. The addition of ODS-particles in the Co-based bond coat Amdry 9954 resulted in a better creep resistance but lower ductility in comparison to ODSparticle-free Amdry 9954. 1 particle and the dislocations. If the particle is twice as large as the dissociation width of the dislocation, the leading particle is strongly attached to the ODS particle and this force must be overcome before the dislocation can fully detach [20]. The mechanical properties of bond coats and the inﬂuence of ODSparticles on the creep behavior of freestanding bond coats are analyzed. For this, microtensile samples are used with a diameter of a few hundred micrometers. The thickness of the tested samples is in the range of the coating thickness and therefore allows testing of the bond coats independent of any substrate inﬂuence. Studies of microtensile testing of small samples under uniaxial loading are a reliable technique to evaluate the mechanical properties at the microscale [8, 21, 22, 23]. As known from the literature, the mechanical properties obtained by microtensile tests agree well with macroscopic tests [24]. Dog-bone–shaped miniaturized specimens made from Nimonic-75 samples veriﬁed by Luan and Riesch-Oppermann show that the minimum creep rates of the miniaturized specimens are in agreement with the steady-state creep strain rates of conventional specimens [24]. However, microtensile creep experiments of freestanding bond coat have not been investigated in detail so far. Alam et al. developed a hightemperature tensile device for micromechanical characterization of metallic alloys and ceramic coatings [21]. With this device, creep experiments on small ﬂat tensile specimens of Nibase superalloys Ni-625 and René 88DT were performed [21]. Kumar et al. investigated the inﬂuence of reducing the sample thickness of miniaturized samples on the mechanical properties compared to macroscopic tensile tests [25]. Tensile tests and FEM simulations showed good agreement relating to yield stress, ultimate tensile strength and uniform elongation for three steel alloys down to a thickness of 0.25 mm. However, due to the smaller sample volumes, the strain to failure decreased with thinner sample thickness due to earlier necking and micro-void coalescence [25, 26]. A sample processing procedure for cylindrical submillimeter tensile specimens with a homogeneous thickness over the whole gauge length, similar to those used in this work, was described by Rathmayr et al. [27]. By using circular cross sections of the tested samples, stress concentrations due to the geometric shape are avoided and surface inhomogenities are minimized during polishing. Nevertheless, achieving a homogeneous sample thickness and prevention of plastic deformation during sample preparation are quite challenging. A precise temperature and load control during testing is required. Therefore, the creep experiments on the microtensile samples were conducted in a thermomechanical analyzer (TMA) with excellent temperature control. The aim of the work was to demonstrate that the TMA setup using microtensile creep specimens provides a suitable method for determining the mechanical properties of freestanding MCrAlY bond coats with different Ni and Co contents at their operating ª Materials Research Society 2019 temperature and thickness. The different creep behavior of Niand Co-based bond coats depending on their chemical composition as well as the inﬂuence of the strengthening effect of ODS-particles is discussed to design improved bond coats for future applications. Results Microstructure of the initial state The two-phase microstructure of Amdry 386 and 9954, after the ﬁrst annealing step of 2 h at 1100 °C, consists of b-NiAl (dark), c-Ni/Co-solid solution (bright), and an additional third phase (Al2O3) in the case of the 9954 1 ODS coating, as shown in Fig. 1. Due to its higher content of Ni and Al, Amdry 386 shows a higher fraction of the b-NiAl phase with 60 6 0.5% compared to Amdry 9954 with 31 6 1.2%, see Fig. 2(a). The addition of 2 wt% of Al2O3 particles does not have a signiﬁcant inﬂuence on the phase fractions. The distribution of the ODSparticles is even and larger accumulations could rarely be observed, see Fig. 1(f). The additional heat treatment of 72 h at 1100 °C does not have a signiﬁcant inﬂuence on the area and the phase fractions of all bond coats, Figs. 2(a) and 2(b). Furthermore, the grain sizes after the ﬁrst (2 h) and the second heat treatment (72 h) were analyzed, Fig. 2(c). For Amdry 386 and especially for the ODS-containing bond coat, the grain size distribution is broader after a thermal exposure of 2 h compared to the additional second heat treatment of 72 h. After the second heat treatment, all three bond coats show a more homogeneous grain size distribution, resulting in a smaller standard deviation. After the ﬁrst heat treatment, some unmelted particles, marked with a black dashed line, due to the coating process are still visible, Fig. 2(d). After the second heat treatment, the amount of these unmelted particles is reduced, narrowing the grain size distribution. Furthermore, the coarsening of the smaller grains led to a more homogeneous distribution after the additional heat treatment of 72 h. A few unmelted particles were still observed and were often surrounded by a semicontinuous b-phase layer acting as preferred regions for cracking [28]. Chen et al. investigated the inﬂuence of these unmelted particles on the mechanical properties with a similar bond coat Amdry 9951 in compression tests and determined a coarsening rate coefﬁcient for the b-phase up to 1100 °C of 6.49 105 lm3/s [28, 29]. Microtensile creep experiments Microtensile creep curves were measured for Amdry 386, 9954, and 9954 1 ODS at temperatures between 900 and 950 °C, Fig. 3. The Ni-based bond coat Amdry 386 shows a higher creep strength than the Co-based bond coat Amdry 9954 at all cambridge.org/JMR j Journal of Materials Research j www.mrs.org/jmr https://doi.org/10.1557/jmr.2019.169 Published online by Cambridge University Press Article 2 Article Figure 2: (a) Area fraction of phases and (b) phase size (c) grain sizes of Amdry 386, Amdry 9954 and Amdry 9954 1 ODS after a heat treatment of 2 and 72 h at 1100 °C in vacuum before testing and (d) an EBSD mapping of Amdry 9954 with a large unmelted particle due to insufﬁcient homogenization during thermal exposure. ª Materials Research Society 2019 cambridge.org/JMR j Journal of Materials Research j www.mrs.org/jmr https://doi.org/10.1557/jmr.2019.169 Published online by Cambridge University Press Figure 1: BSE images of the microstructures of Amdry 386 (a 1 b), Amdry 9954 (c 1 d) and Amdry 9954 1 ODS (e + f) after a heat treatment of 2 h in vacuum at 1100 °C. 3 Figure 3: True plastic strain over time plot (a–c) and strain rate over true plastic strain plot (d–f) of Amdry 386, Amdry 9954 and Amdry 9954 1 ODS after aging at 1100 °C for 2 h (solid lines) and 72 h (dashed lines) at testing temperatures of 900–950 °C with 15 MPa. temperatures, due to the higher amount of b-NiAl in Amdry 386 compared to Amdry 9954. However, Amdry 9954 with Al2O3-particles reveals the highest creep resistance because the addition of Al2O3-particles to Amdry 9954 outweighs the lower b-NiAl content. The creep minima occurred at all temperatures at a plastic strain of about 0.05–0.1. The slope of the creep curves after the creep minima increases, whereas the strain to failure of the ODS-free bond coats increases with increasing temperature. The long-term heat-treated conditions (1100 °C/ 72 h) of all bond coats showed a higher creep strength than the ª Materials Research Society 2019 short-term heat-treated conditions (1100 °C/2 h). A comparison of the creep minima of the three bond coats at all testing temperatures is shown in Fig. 4(a). The differences of the creep minima after different durations of thermal exposure are more pronounced for Amdry 386 compared to Amdry 9954. The lower amount of b-NiAl led to smaller differences for the Co-based bond coat. The equal creep minima for Amdry 386 tested at 950 °C and Amdry 9954 tested at 900 °C are quite similar, which shows the signiﬁcant inﬂuence of the composition and the content of phases in the cambridge.org/JMR j Journal of Materials Research j www.mrs.org/jmr https://doi.org/10.1557/jmr.2019.169 Published online by Cambridge University Press Article 4 Article bond coats. The activation energy of creep for the three bond coats determined by tensile tests between 900 and 950 °C after short- and long-term heat treatment reveals that the activation energy after the additional heat treatment is higher than for the experiments after the short-term heat treatment, Fig. 4(b). Q ¼ 2:304 R d ðlg e_ Þ d T1 ; ð1Þ Q is the activation energy, which can be calculated as the slope of the minimum creep rate against the inverse temperature and the gas constant R. Amdry 386 shows the highest activation energy followed by Amdry 9954 1 ODS. Amdry 9954 showed the strongest creep deformation at all testing temperatures, which corresponds to the low activation energy of creep. https://doi.org/10.1557/jmr.2019.169 Published online by Cambridge University Press Microstructure after creep experiments The microstructure of the tensile specimens of all three bond coats after 72 h of thermal exposure and creep testing at 950 °C at a constant force of 2.35 N until fracture can be seen in Fig. 5. Due to the large plastic deformation, Amdry 386 and especially Amdry 9954 show very ductile fracture behavior. In contrast to 9954, the oxide dispersion strengthened bond coat 9954 1 ODS failed more brittle at an earlier state according to a larger diameter of the fracture surface, based on the higher strength and lower ductility of the alloy. Cracks could be observed at phase and grain boundaries between b-NiAl and c-solid solution inside the samples as well as starting at the surface of the bond coats. The ODS-particles are equally distributed in the b-NiAl phase and the c-solid solution for Amdry 9954 1 ODS. The grain size of all bond coats was analyzed by EBSD after heat treatment of 72 h at 1100 °C and after the same heat ª Materials Research Society 2019 treatment and microtensile tests at 950 °C, Fig. 6(a). All bond coats show an increase in their grain size after tensile testing. This increase occurred due to normal grain coarsening during the creep test. The ODS bond coat showed smaller grain sizes before and after tensile tests compared to the ODS-free coatings but did not prevent grain coarsening. In addition to the grain size, Fig. 6(b) shows the size of the c-Ni phase and b-NiAl phase for all bond coats after the creep experiments at 950 °C after the short-term (1100 °C/2 h) and the long-term (1100 °C/72 h) heat treatment. The additional long-term heat treatment leads to a much stronger increase of the phase sizes of all three bond coats compared to the initial state, see Fig. 2(b). It is also noticeable that the addition of ODS-particles does not lead to a signiﬁcant prevention of phase coarsening. To investigate the arrangement of the grains depending on their size before and after microtensile tests, grain sizes smaller than the median grain sizes of Amdry 386 and Amdry 9954 1 ODS were pigmented in red, Fig. 7. Therefore, the microstructures of these samples were analyzed along the loading axis by EBSD measurements. After the additional heat treatment of 72 h and before the creep experiment, the median grain size was 0.6 lm for Amdry 386 and 0.45 lm for the ODS-containing bond coat and grains smaller than the median were found all over the specimen. Accumulation of areas with smaller grains, especially for Amdry 386, could be observed. Figure 7(b) shows the coarsening of the grains due to the time and temperature during the creep test. The median grain size after the creep tests was about 1.55 lm for Amdry 386 and 0.8 lm for the ODS-containing bond coat. It can clearly be seen that for Amdry 386, the smaller grains are mainly elongated along the loading axis, separated by layers of larger grains. In contrast to that, no preferential cambridge.org/JMR j Journal of Materials Research j www.mrs.org/jmr Figure 4: (a) Minima creep rates of Amdry 386, Amdry 9954 and Amdry 9954 1 ODS at testing temperatures of 900–950 °C and an applied stress of 15 MPa after a ﬁrst (2 h) and an additional second (72 h) heat treatment and (b) activation energies of all three bond coats at all testing temperatures. 5 Article Figure 5: BSE images of the microstructures of Amdry 386 (a 1 b), 9954 (c 1 d) and 9954 1 ODS (e 1 f) after 72 h of thermal exposure and microtensile creep tests at 950 °C. accumulation of smaller grains could be observed for the ODS-containing bond coat, Fig. 7(d). less b-NiAl and therefore more c-solid-solution phase. This is due to the higher creep strength of NiAl, as compared to Ni at temperatures above 900 °C, which was investigated in previous Discussion studies [30, 31, 32, 33]. Moreover, the higher yttrium content in In this work, microtensile tests on three different MCrAlY-bond coats after heat treatments of 2 and 72 h at 1100 °C were performed in the temperature range between 900 and 950 °C. The second heat treatment led to grain coarsening, resulting in lower creep rate minima during all experiments. The higher b-NiAl amount of Amdry 386 is responsible for the superior creep properties in comparison to Amdry 9954, which contains Amdry 386 leads to a higher amount of small Y2O3-particles ª Materials Research Society 2019 during plasma spraying. These nanoscale oxides resulted in an additional hindering effect for deformation. After the tensile creep experiments, an increase of the size of the c-solid-solution phase, b-NiAl phase and the ODS-particles was observed. The grain size of all tested conditions increased slightly. However, an inhomogeneous grain coarsening of the grains took place in the cambridge.org/JMR j Journal of Materials Research j www.mrs.org/jmr https://doi.org/10.1557/jmr.2019.169 Published online by Cambridge University Press Figure 6: Grain sizes of Amdry 386, Amdry 9954 and Amdry 9954 1 ODS (a) after 72 h of thermal exposure and after the same heat treatment and microtensile tests at 950 °C and (b) phase sizes after microtensile creep tests at 950 °C heat treated for 2 and 72 h at 1100 °C in vacuum. 6 Article ODS-free bond coat. After testing, EBSD measurements revealed layers of grains smaller and larger than the median grain size which were aligned along the loading axis of Amdry 386 and Amdry 9954. This alternately layered structure occurred presumably due to the formation of small oxides during the manufacturing process. The deformation mechanism in this stress temperature regime should be grain boundary sliding, as described in former studies by Hebsur [34]. However, due to the high activation energies of the bond coats, determined at this work, dislocation creep is suggested to be the dominant deformation mechanism. The superior creep resistance of Amdry 9954 1 ODS in comparison to the dispersoid-free Amdry 9954 can be explained by the detailed investigations of Arzt et al. on the addition of 2 at.% of yttria particles in NiAl with similar ﬁne grain size compared to our bond coats [16, 17]. It is assumed that the nanoscaled oxides Al2O3 particles provide a similar beneﬁcial effect of dispersion strengthening and hinder the dislocation motion. This results in a higher activation energy, but also leads to a much lower plastic strain to failure. independent of the substrate, small cylindrical tensile specimens with a precise and reproducible shape were fabricated by grinding and polishing to a diameter of 450 lm. Forcecontrolled creep experiments were performed at temperatures between 900 and 950 °C under constant Ar-ﬂow at a constant load of 2.35 N. The higher amount of b-NiAl in the Ni-based bond coat Amdry 386 led to superior creep properties in comparison to the Co-based bond coat Amdry 9954. A longer annealing heat treatment of 72 h compared to 2 h at 1100 °C led to lower creep rates for all three bond coats at all testing temperatures due to grain coarsening of both phases. The main deformation mechanism for Amdry 386 and Amdry 9954 could be determined as dislocation creep. The addition of ODS particles in Amdry 9954 led to a signiﬁcant decrease of the creep rate due to the additional interaction between the dislocation and the oxide particles. The strain to failure decreases from around 0.6 for Amdry 9954 to 0.14 for Amdry 9954 1 ODS. Methodology Conclusion Material Small-scale tensile creep experiments have been performed on three freestanding MCrAlY bond coats using a TMA. To investigate the mechanical properties of the bond coats Three MCrAlY bond coats were investigated in this study. An Nibased NiCoCrAlY (Amdry 386) and two Co-based CoNiCrAlY (Amdry 9954) bond coats. One of the Co-based bond coats ª Materials Research Society 2019 cambridge.org/JMR j Journal of Materials Research j www.mrs.org/jmr https://doi.org/10.1557/jmr.2019.169 Published online by Cambridge University Press Figure 7: EBSD measurements after thermal exposure of 72 h of (a) Amdry 386 and (b) Amdry 9954 1 ODS. EBSD measurements after the same heat treatment and microtensile tests at 950 °C for (c) Amdry 386 and (d) Amdry 9954 1 ODS. Grains smaller than the median grain sizes (red) and grains larger than the medium grain size (purple). 7 Article At.% Amdry 386 Amdry 9954 Amdry 9954 1 ODS Ni Co Al Cr Si Y Hf 40.35 28.68 29.59 18.97 34.18 35.01 23.04 15.57 16.52 16.49 21.25 18.64 0.79 0.13 0.08 0.36 0.32 0.16 0.08 ... ... contains additional 2 wt% of Al2O3 for oxide dispersion strengthening (ODS) (Amdry 9954 1 ODS) produced by mechanical alloying [36]. The chemical compositions are listed in Table I. Coatings with a thickness of approximately 3 mm were produced by low-pressure plasma spraying using a F4 torch in an Oerlikon Metco (Wohlen, Switzerland) facility on a steel substrate. Afterwards, plates of 20 10 2 mm in size were spark eroded out of the coating layer and heat treated in argon atmosphere at 1100 °C for 2 h. Then, half of the samples got an additional heat treatment for 72 h under equal conditions to investigate the inﬂuence of thermal exposure. The porosity of the samples in the as-sprayed conditions was about 0.55 6 0.14% and decreases after the ﬁrst heat treatment of 2 h at 1100 °C to 0.2 6 0.08%. No signiﬁcant change of the porosity was to be observed after the additional heat treatment. https://doi.org/10.1557/jmr.2019.169 Published online by Cambridge University Press Sample preparation The specimens were cut with a precision wet abrasive cutting machine Brillant 220 from ATM to a width of about 550 lm. For the preparation of a small circular microtensile specimen out of the rectangular shape, a further step was necessary. The challenge of producing microtensile specimens, especially of dimensions below 1 mm, is that notches across the gauge length and an inhomogeneous sample thickness can have a much larger inﬂuence on the experimental results, as with standard tensile specimens on the macroscale. This means that the cross section, no matter if it is rectangular or circular, must be well known and reproducible for all samples. Common fabrication techniques for rectangular tensile specimens are mechanical milling or electrical discharge machining methods [37, 38]. Samples with rectangular cross sections are commonly used for macroscopic testing. In the submillimeter range, the sample edges of rectangular-shaped samples are rounded. This results in inaccurate determinations of the cross section and edges that are preferential sites for notches because of insufﬁcient grinding and polishing. To eliminate the inﬂuence of varying geometries and to prove the reliability and reproducibly of the tensile tests on the submillimeter scale, the use of samples with circular cross sections is more suitable. To produce specimens with circular cross sections without notches and high accuracy, the sliced samples were prepared by a highprecision grinding and polishing process at Microsample GmbH, specialized to produce circular samples with diameters smaller than 1 mm. The specimen is ﬁxed in a specimen holder ª Materials Research Society 2019 with two grips. A liquid cooling system prevents any heat inﬂuence during milling with a diamond wire wheel. The desired gauge length can be adjusted before tailoring the sample. During grinding, the sample holder rotates synchronic to prevent any torsion effect in the sample and is mounted perpendicular to the rotation axis of the grinding wheel. The measurement of the sample diameter is monitored at high speed and high precision with a 2D optical micrometer system. After grinding, further polishing steps are performed to smoothen the sample surface, removing the thin mechanically affected zone of the grinding process and reducing notches [27, 39]. The cross section was determined to be 450 6 2 lm in diameter, over the entire gauge length of 2500 lm. The surface roughness of 2 lm was determined by a VHX-1000-laser microscope from Keyence, Osaka, Japan. Microstructural analysis before and after testing was carried out with a Zeiss Crossbeam 540 (Oberkochen, Germany) using back-scattered electron imaging (BSE). The area fraction, as well as the phase and grain size of the individual phases, was determined by electron backscatter diffraction (EBSD) with the software ATZEC from Oxford Instruments, Abingdon, U.K. Longitudinally polished sections of the tested specimens after the tensile creep experiments were analyzed by the software ImageJ in horizontal direction 250 lm away from the crack tip. TMA The microtensile creep tests were performed in a SiC furnace of a thermomechanical analyzer type 402 F3 Hyperion from Netzsch, Selb, Germany. The advantage of this setup is a very accurate, controllable force and length resolution during the creep tests. The force sensor has a digital resolution smaller than 0.01 mN and a force range from 1 mN to a maximum of 3 N. Therefore, testing of small samples or thin coatings in the conditions, which are commonly used during service, can be investigated. The change in length of the sample is measured accurately by an inductive linear variable displacement transducer (LVDT) with a digital resolution of 1.25 nm and a maximum change in length of 4 mm. During the tests, the measuring system is thermally stabilized by water cooling. Hebsur et al. studied NiCoCrAlY bond coats, which were tested in vacuum and air between 660 and 850 °C [8]. The negative effect of oxygen on NiCoCrAlY bond coats led to blunting of initial surface pores during testing. Hence, in this work, a constant argon gas ﬂow was applied to prevent oxidation of the samples. Creep tests were performed at 900, 925, and 950 °C under a constant argon ﬂow of about 200 mL/min. Before testing, the samples were heated up with 5 K/min under a constant load of 50 mN to ﬁx the specimen until the testing temperature is achieved. A holding period of 2 h was used to cambridge.org/JMR j Journal of Materials Research j www.mrs.org/jmr TABLE I: Composition of Amdry 386 and Amdry 9954 (1ODS) in at.%. 8 Article 5. A. Gil, V. Shemet, R. Vassen, M. Subanovic, J. Toscano, D. Naumenko, L. Singheiser, and W.J. Quadakkers: Effect of surface condition on the oxidation behaviour of MCrAlY coatings. Surf. Coat. Technol. 201, 3824 (2006). 6. D. Naumenko, R. Pillai, A. Chyrkin, and W.J. Quadakkers: Overview on recent developments of bondcoats for plasma-sprayed thermal barrier coatings. J. Therm. Spray Technol. 26, 1743 (2017). 7. R. Webler, M. Ziener, S. Neumeier, P.J. Terberger, R. Vaßen, and M. Göken: Evolution of microstructure and mechanical properties of coated Co-base superalloys during heat treatment and thermal exposure. Mater. Sci. Eng., A 628, 374 (2015). 8. M.G. Hebsur and R.V. Miner: Stress rupture and creep behavior Figure 8: (a) TMA setup with fused silica tension holder, pushrod and clamping jaw made of alumina for mounting the (b) polished tensile specimen before creep testing with a diameter of 450 6 2 lm over the entire gauge length of 2500 lm. of a low pressure plasma-sprayed NiCoCrAlY coating alloy in air and vacuum. Thin Solid Films 147, 143 (1987). 9. M. Funk, K. Ma, C. Eberl, J.M. Schönung, M. Göken, and K.J. Hemker: High-temperature mechanical behavior of end-of- achieve thermal equilibrium of the whole setup. The TMA is a load controlled setup in which the load was increased within 1 min from 50 mN to a constant load of 2.35 N over the whole test duration after the holding period. In respect to the sample’s diameter of 450 lm, this led to a stress of 15 MPa at the beginning of the tests. The sample holder and the pushrod are made of fused silica, which is thermally stable up to 1100 °C. The clamping jaws with the hexagonal bolts and nuts, as well as the upper parts of the clamp with a ground cutting edge for mounting the sample, are made of alumina. The TMA setup and a microtensile specimen before testing are shown in Fig. 8. life cryomilled NiCrAlY bond coat materials. Metall. Mater. Trans. A 42, 2233 (2011). 10. O. Franke, K. Durst, and M. Göken: Microstructure and local mechanical properties of Pt-modiﬁed nickel aluminides on nickelbase superalloys after thermo-mechanical fatigue. Mater. Sci. Eng., A 467, 15 (2007). 11. S. Wöllmer, S. Zaefferer, M. Göken, T. Mack, and U. Glatzel: Characterization of phases of aluminized nickel base superalloys. Surf. Coat. Technol. 167, 83 (2003). 12. K. Schneider and H.W. Grünling: Mechanical aspects of high temperature coatings. Thin Solid Films 107, 395 (1983). 13. R. Vaßen, S. Giesen, and D. Stöver: Lifetime of plasma-sprayed The authors would like to acknowledge Dr. Georg Rathmayr from Microsample GmbH for manufacturing the microtensile specimens. The authors acknowledge funding by the Deutsche Forschungsgemeinschaft (DFG) through projects A6 and B6 of the collaborative research center SFB/TR 103 “From Atoms to Turbine Blades-a Scientiﬁc Approach for Developing the Next Generation of Single Crystal Superalloys.” thermal barrier coatings: Comparison of numerical and experimental results. J. Therm. Spray Technol. 18, 835 (2009). 14. K.A. Unocic, J. Bergholz, T. Huang, D. Naumenko, B.A. Pint, R. Vaßen, and W.J. Quadakkers: High-temperature behavior of oxide dispersion strengthening CoNiCrAlY. Mater. High Temp. 35, 108 (2018). 15. C. Vorkötter, D.E. Mack, O. Guillon, and R. Vaßen: Superior cyclic life of thermal barrier coatings with advanced bond coats on single-crystal superalloys. Surf. Coat. Technol. 361, 150 (2019). References 1. D.R. Clarke and S.R. Phillpot: Thermal barrier coating materials. Mater. Today 8, 22 (2005). 2. G.W. Goward: Progress in coatings for gas turbine airfoils. Surf. Coat. Technol. 108, 73 (1998). 3. J.R. Nicholls: Advances in coating design for high-performance gas turbines. MRS Bull. 28, 659 (2003). 4. E. Hejrani, D. Sebold, W.J. Nowak, G. Mauer, D. Naumenko, R. Vaßen, and W.J. Quadakkers: Isothermal and cyclic oxidation 16. E. Arzt and P. Grahle: High temperature creep behavior of oxide dispersion strengthened NiAl intermetallics. Acta Mater. 46, 2717 (1998). 17. E. Arzt, R. Behr, E. Göhring, P. Grahle, and R.P. Mason: Dispersion strengthening of intermetallics. Mater. Sci. Eng., A 234, 22 (1997). 18. P. Grahle and E. Arzt: Microstructural development in dispersion strengthened NiAl produced by mechanical alloying and secondary recrystallization. Acta Mater. 45, 201 (1997). 19. U.E. Klotz, R.P. Mason, E. Göhring, and E. Arzt: High- behavior of free standing MCrAlY coatings manufactured by high-velocity temperature creep in a coarse-grained oxide-dispersion atmospheric plasma spraying. Surf. Coat. Technol. 313, 191 (2017). strengthened Ni3Al alloy. Mater. Sci. Eng., A 231, 198 (1997). ª Materials Research Society 2019 cambridge.org/JMR j Journal of Materials Research j www.mrs.org/jmr https://doi.org/10.1557/jmr.2019.169 Published online by Cambridge University Press Acknowledgments 9 Article 20. R. Behr, J. Mayer, and E. Arzt: TEM investigations of the superdislocations and their interaction with particles in dispersion strengthened intermetallics. Intermetallics 7, 423 (1999). 21. Z. Alam, D. Eastman, M. Jo, and K. Hemker: Development of a high-temperature tensile tester for micromechanical characterization of materials supporting meso-scale ICME models. JOM 68, 2754 (2016). 22. K.J. Hemker, B.G. Mendis, and C. Eberl: Characterizing the microstructure and mechanical behavior of a two-phase NiCoCrAlY bond coat for thermal barrier systems. Mater. Sci. Eng., A 483–484, 727 (2008). 23. N.B. Jaya and M.Z. Alam: Small-scale mechanical testing of materials. Curr. Sci. 105, 1073 (2013). 24. L. Luan, H. Riesch-Oppermann, and M. Heilmaier: Tensile creep of miniaturized specimens. J. Mater. Res. 32, 4563–4572(2017). 25. K. Kumar, A. Pooleery, K. Madhusoodanan, R.N. Singh, J.K. Chakravartty, B.K. Dutta, and R.K. Sinha: Use of miniature tensile specimen for measurement of mechanical properties. Procedia Eng. 86, 899 (2014). 26. K. Kumar, A. Pooleery, K. Madhusoodanan, R.N. Singh, A. Chatterjee, B.K. Dutta, and R.K. Sinha: Optimisation of thickness of miniature tensile specimens for evaluation of mechanical properties. Mater. Sci. Eng., A 675, 32 (2016). 27. G.B. Rathmayr, A. Bachmaier, and R. Pippan: Development of a new testing procedure for performing tensile tests on specimens with sub-millimetre dimensions. J. Test. Eval. 41, 20120175 (2013). 28. H. Chen: Microstructure characterisation of un-melted particles in a plasma sprayed CoNiCrAlY coating. Mater. Charact. 136, 444 (2018). Mater. Sci. Eng., A 356, 283 (2003). 31. S. Karashima, H. Oikawa, and T. Motomiya: Steady-state creep characteristics of polycrystalline nickel in the temperature range 500 to 1000 °C. Trans. Jpn. Inst. Met. 10, 205 (1969). 32. S.V. Raj: Tensile creep fracture of polycrystalline nearstoichiometric NiAl. Mater. Sci. Eng., A 381, 154 (2004). 33. H. ur Rehman, K. Durst, S. Neumeier, A. Sato, R. Reed, and M. Göken: On the temperature dependent strengthening of nickel by transition metal solutes. Acta Mater. 137, 54 (2017). 34. M.G. Hebsur and R.V. Miner: High temperature tensile and creep behaviour of low pressure plasma-sprayed Ni–Co–Cr–Al–Y coating alloy. Mater. Sci. Eng. 83, 239 (1986). 35. E. Arzt and D.S. Wilkinson: Threshold stresses for dislocation climb over hard particles: The effect of an attractive interaction. Acta Metall. 34, 1893 (1986). 36. J. Bergholz, B.A. Pint, K.A. Unocic, and R. Vaßen: Fabrication of oxide dispersion strengthened bond coats with low Al2O3 content. J. Therm. Spray Technol. 26, 868 (2017). 37. S. Kumar, R. Singh, T.P. Singh, and B.L. Sethi: Surface modiﬁcation by electrical discharge machining: A review. J. Mater. Process. Technol. 209, 3675 (2009). 38. L.C. Lee, L.C. Lim, V. Narayanan, and V.C. Venkatesh: Quantiﬁcation of surface damage of tool steels after EDM. Int. J. Mach. Tool Manuf. 28, 359 (1988). 39. G.B. Rathmayr, A. Hohenwarter, and R. Pippan: Inﬂuence of grain shape and orientation on the mechanical properties of high the b-phase coarsening behaviour in a thermally sprayed pressure torsion deformed nickel. Mater. Sci. Eng., A 560, 224 CoNiCrAlY bond coat alloy. J. Alloys Compd. 704, 359 (2017). (2013). j Journal of Materials Research j www.mrs.org/jmr https://doi.org/10.1557/jmr.2019.169 Published online by Cambridge University Press 29. H. Chen, Y.Q. Si, and D.G. McCartney: An analytical approach to 30. S.V. Raj: Tensile creep of polycrystalline near-stoichiometric NiAl. ª Materials Research Society 2019 cambridge.org/JMR 10
https://openalex.org/W3182031095	https://mdsoar.org/bitstream/11603/25347/1/ICRC2021_957.pdf	English	null	Gamma-ray burst observation & gravitational wave event follow-up with CALET on the International Space Station	Proceedings of 37th International Cosmic Ray Conference — PoS(ICRC2021)	2,021	public-domain	6,653	This work was written as part of one of the author's official duties as an Employee of the United States Government and is therefore a work of the United States Government. In accordance with 17 U.S.C. 105, no copyright protection is available for such works under U.S. Law. This work was written as part of one of the author's official duties as an Employee of the United States Government and is therefore a work of the United States Government. In accordance with 17 U.S.C. 105, no copyright protection is available for such works under U.S. Law. Public Domain Mark 1.0 https://creativecommons.org/publicdomain/mark/1.0/ Access to this work was provided by the University of Maryland, Baltimore County (UMBC) ScholarWorks@UMBC digital repository on the Maryland Shared Open Access (MD-SOAR) platform. Access to this work was provided by the University of Maryland, Baltimore County (UMBC) ScholarWorks@UMBC digital repository on the Maryland Shared Open Access (MD-SOAR) platform. 37th International Cosmic Ray Conference (ICRC 2021) July 12th – 23rd, 2021 Online – Berlin, Germany © Copyright owned by the author(s) under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND 4.0). ∗Presenter Please provide feedback Please support the ScholarWorks@UMBC repository by emailing scholarworks-group@umbc.edu and telling us what having access to this work means to you and why it’s important to you. Thank you. https://pos.sissa.it/ Gamma-ray burst observation & gravitational wave event follow-up with CALET on the International Space Station Gamma-ray burst observation & gravitational wave event follow-up with CALET on the International Space Station PoS(ICRC2021)957 Yuta Kawakuboa,∗on behalf of the CALET Collaboration (a complete list of authors can be found at the end of the proceedings) aDepartment Physics & Astronomy, Louisiana State University, 202 Nicholson Hall, Baton Rouge, LA 70803, USA E-mail: kawakubo1@lsu.edu Yuta Kawakuboa,∗on behalf of the CALET Collaboration (a complete list of authors can be found at the end of the proceedings aDepartment Physics & Astronomy, Louisiana State University, 202 Nicholson Hall, Baton Rouge, LA 70803, USA E-mail: kawakubo1@lsu.edu The CALorimetric Electron Telescope (CALET) has been observing high-energy cosmic rays and gamma-rays on the International Space Station since October 2015. The CALET gamma-ray burst monitor (CGBM), mounted on CALET to observe prompt emissions of gamma-ray bursts (GRBs) in the hard X-ray and soft gamma-ray band, has been monitoring all-sky with ∼60 % duty cycle without any problems since October 2015. As of end May 2021, CGBM has detected 254 GRBs, including 31 short GRBs, thanks to the onboard trigger system. The Calorimeter (CAL), the primary instrument of CALET, has also collected gamma-ray data in the energy range from 1 GeV to 10 TeV while maintaining both instruments in good condition. We continue searching for high- energy gamma-rays from GRBs detected by CGBM, and have found two possible gamma-rays from GRBs. As described above, CALET can detect prompt emissions and high energy gamma- ray emission of GRBs. Therefore, we also have actively participated in the follow-up campaign for electromagnetic counterparts of the gravitational wave events observed by LIGO/Virgo since the operation start of the CALET. Although we have found no candidates of electromagnetic counterparts of the gravitational wave events, we have derived upper limits of the high-energy gamma-ray ﬂux for 26 events in the LIGO/Virgo third observation run. © Copyright owned by the author(s) under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND 4.0). https://pos.sissa.it/ https://pos.sissa.it/ GRB observation & GW event follow-up with CALET Yuta Kawakubo 1. Introduction Gamma-ray bursts (GRB) have been observed by many telescopes in space and on the ground in various wavelengths since the discovery by the Vela satellite [1][2]. In recent years, GRBs, especially short GRBs, have taken on increased importance because a short GRB is a plausible candidate for gravitational wave events’ electromagnetic counterparts. Fermi-GBM and INTEGRAL SPI-ACS detected GRB 170817A in association with binary neutron star merger GW 170817 [4][5]. Since GRB 170817A is the only GRB observed and associated with a gravitational wave event, further detection of GRBs associated with gravitational wave events is essential to understand the association between binary neutron star mergers and short GRBs, and the short GRB itself. PoS(ICRC2021)957 Since the coverage by a single GRB instrument is limited, observing GRBs with multiple instruments increases the validity of the observation and the chance of detecting GRBs associated with gravitational wave events. The CALorimetric Electron Telescope (CALET) is a payload on the International Space Station [7]. CALET consists of the Calorimeter (CAL) and CALET Gamma- ray burst monitor (CGBM). CALET has been monitoring all-sky with X-ray and gamma-rays. In particular, CGBM has been detecting GRBs with a rate of ∼45 GRBs / year. In this paper, we will present an overview of GRB observations with CALET in ﬁve years. Also, we will present a summary of follow-up observations for electromagnetic counterparts of gravitational wave events in the LIGO/Virgo third observation run. Detailed gamma-ray observations of CAL will be presented in diﬀerent papers in ICRC 2021 [8][9]. 2. GRB observation with CALET CGBM count rates increase at the high latitude and around the South Atlantic Anomaly (SAA). CGBM high voltages are turned oﬀat high latitude and around the SAA to avoid high count rates and false triggers due to charged particles. As a result, the duty cycle of CGBM is ∼60 %. Since background count rates vary depending on time, CGBM is triggered by high count rates due to charged particles sometimes, even if the CGBM high voltages are turned oﬀat the high count rate region. Since HXM has a sensitivity to X-ray below 10 keV, bright X-ray sources in the HXM ﬁeld of view cause increased HXM count rates. Figure 2 shows background spectra observed by each CGBM detector on October 5 in 2015 and April 5 in 2021. CGBM has been collecting X-ray and gamma-rays data without any problem for more than ﬁve years since the operation start. All CGBM detectors can see the 511 keV line due to annihilation. Two internal background lines can be seen around 35 keV and 1470 keV in the HXM background spectra [12]. The 2.2 MeV line originating from activation can be seen in the SGM background spectra [13]. These background lines can be used for energy calibration. PoS(ICRC2021)957 At the end of May 2021, CGBM has detected 254 GRBs thanks to the onboard trigger system. The total observation interval is 2066 days, and the GRB detection rate is 44.9 GRBs / year. Figure 3 shows the T90 distribution of GRBs detected by CGBM. T90 was measured by SGM in the energy range 40 ∼1000 keV using ‘battblocks’, which is software for Swift-BAT data to measure the duration using the Bayesian block method [14]. Although TH data were used for T90 calculation, event data were used if calculated T90 was less than 2 s. Since 5 out of 254 GRBs were low signiﬁcance or not seen in SGM data, 249 GRBs were included in Figure 3. The T90 distribution by CGBM is bimodal in logarithmic scale like those by other instruments, and well ﬁtted with two logarithmic normal distributions. The means of the two logarithmic normal distributions were 0.51 s and 16.98 s. The intersection of the two logarithmic normal distributions was 1.44 s. If we classify GRBs by 1.44 s, the number of long bursts and short bursts were 218 and 31, respectively. 2. GRB observation with CALET In GRB observations with CALET, CGBM is primarily responsible for observing prompt emission of GRBs. CGBM consists of two Hard X-ray Monitors (HXMs) and a Soft Gamma-ray Monitor (SGM) [10][11]. Both HXM and SGM are scintillation detectors that have LaBr3(Ce) and BGO for each. CGBM covers the energy range from 7 keV to 20 MeV thanks to HXM (7 keV - 1 MeV) and SGM (40 keV - 20 MeV). CGBM collects two types of monitor data continuously: Time History (TH) data, which have eight energy channels and are collected every 1/8 s, and Pulse Height (PH) data, which have 512 energy channels, are collected every 4 s. CGBM has an onboard trigger system to detect GRBs and other X-ray/gamma-ray transients. The onboard trigger system calculates the signal-to-noise ratio (SNR) according to (1) SNR = Ntot −NBG ∆tBG ∆t q NBG ∆tBG ∆t (1) (1) where ∆t is the integration time of the foreground (1/4 s, 1/2 s, 1 s, and 4 s); ∆tBG is integration time of the background (8, 16, 32, 64 s). Ntot is integrated counts over ∆t in the selected energy range, and NBG is integrated counts over ∆tBG in the selected energy range. In the ﬂight operation, trigger thresholds (σ) are selected from 4, 5.5, 7, 8.5, 10, 11, and 13 for each ∆t. ∆tBG is selected from 8 s, 16 s, 32 s, 64 s and used for all ∆t conditions. ∆tBG is taken from a time interval before ∆t. Since CGBM has three sensors, and there are four ∆t conditions, SNRs are calculated every 1/4 s in the twelve trigger conditions in parallel. If any SNRs exceed trigger thresholds, CGBM captures event 2 GRB observation & GW event follow-up with CALET Yuta Kawakubo data that have higher time and energy resolution than those of monitor data. The onboard CGBM buﬀer is able to store four events. If four triggers occur before downlink, the onboard trigger is disabled until the event data is transferred and deleted from the buﬀer. Also, the ground analysis server analyzes real-time TH data and sends an alert as a GCN notice when a CGBM onboard trigger occurs. The trigger settings as of May 31, 2021, are shown in Table 1. Figure 1 shows averaged count rate maps of each CGBM detector. Background count rates due to charged particles highly depend on geomagnetic position. 2. GRB observation with CALET Also, the ﬁve GRBs for which SGM did not measure T90 were long GRBs judging from HXM data. Therefore, 12.4 % of GRBs seen by CGBM were short GRBs. Figure 4 shows the GRB position in SGM coordinates with regions obstructed by ﬁxed structures. Since CGBM has no capability of GRB localization, we used GRB positions that were reported to GCN by other GRB instruments [15]. 182 out of 254 GRBs were localized by other GRB instruments and included in Figure 4. Table 1: Settings for CGBM onboard trigger HXM SGM Trigger threshold σ 8.5 7.0 ∆tBG 16 s 16 s Energy range 25 ∼100 keV 50 ∼300 keV Table 1: Settings for CGBM onboard trigger 3 3 GRB observation & GW event follow-up with CALET Yuta Kawakubo Figure 1: Background count rate maps measured by CGBM. The top, middle, and bottom panels are av- eraged count rate maps for each geographic position for HXM1, HXM2 and SGM, respectively. The count rates were calculated using PH data for September 2020 and averaged in each 5 deg. pixel. 10−3 10−2 10−1 100 101 Rate [counts/s/channel] 2015/10/05 HXM1 HXM2 SGM 101 102 103 104 Energy [keV] 10 3 10 2 10 1 100 101 Rate [counts/s/channel] 2021/04/05 HXM1 HXM2 SGM Figure 2: Background spectra of CGBM. The top and bottom panels shows time averaged back- ground spectra for 1000 s on October 5 2015 and April 5 2021. HXM and SGM gains were cor- rected by the position of 1.4 MeV and 2.2 MeV lines for each. Gray dotted lines show the position of 511 keV. 10−3 10−2 10−1 100 101 Rate [counts/s/channel] 2015/10/05 HXM1 HXM2 SGM 101 102 103 104 Energy [keV] 10 3 10 2 10 1 100 101 Rate [counts/s/channel] 2021/04/05 HXM1 HXM2 SGM Figure 2: Background spectra of CGBM. The top and bottom panels shows time averaged back- ground spectra for 1000 s on October 5 2015 and April 5 2021. HXM and SGM gains were cor- rected by the position of 1.4 MeV and 2.2 MeV lines for each. Gray dotted lines show the position of 511 keV. 10−3 10−2 10−1 100 101 Rate [counts/s/channel] 2015/10/05 HXM1 HXM2 SGM 101 102 103 104 Energy [keV] 10 3 10 2 10 1 100 101 Rate [counts/s/channel] 2021/04/05 HXM1 HXM2 SGM Figure 2: Background spectra of CGBM. 2. GRB observation with CALET There was no gamma-ray event near the GRB positions for 59 GRBs, even if the GRB positions were in the CAL ﬁeld of view. Gamma-ray events were found near the position of GRB 180526A, GRB 200101A, and GRB 200613A. In the case of GRB 200613A, we found the robotic arm obstructed the CAL ﬁeld of view on June 13, 2020, and we concluded the gamma-ray event was a secondary gamma-ray event from the obstructions. CGBM detected GRB 180526A at T0 = 2018/05/26 11:03:36.20 UT. A 3.4 GeV gamma-ray event was found at 1.3 deg. away from the reported position (R.A., Dec.) = (108.48 deg., 3.64 deg.) by Fermi-LAT at T0 + 244 s [24] . The central position of GRB 180526A was within the 99 % PSF region of the candidate. CGBM also detected GRB 200101A at T0 = 2020/01/01 20:39:30.40 UT. A 4.9 GeV gamma-ray event was found at 0.6 deg. away from the reported position (R.A., Dec.) = (258.995 deg., −32.304 deg.) by IPN at T0 + 105 s [15] (#26635). The central position of GRB 200101A was within the 90 % PSF region period when a CGBM onboard trigger occurs [22]. We have continued to search for high-energy gamma-rays using CAL data [23]. We searched for high-energy gamma-rays from GRBs detected by CGBM using CAL data up to September 30 in 2020. We searched for gamma-ray events in 1 GeV ∼10 GeV for 99 GRBs which were well localized by Swift-BAT, XRT, UVOT, Fermi-LAT, MAXI-GSC, and IPN, using LEG data from T0 −60 s to T0 + 7200 s within 2 deg from the reported GRB central position, where T0 is the trigger time of CGBM. The gamma-ray identiﬁcation was performed according to the method described in [16]. The directions obstructed by ﬁxed structures and moving structures except transient obstruction (e.g., robotic arms), were masked to exclude secondary gamma-rays from the structures. In the case of gamma-ray candidates from GRBs were found, we checked the eﬀects of transient obstruction for each candidate by making scatter plots of arrival directions of gamma-ray candidates on the detector coordinate. As a result, the GRB positions were outside of the CAL ﬁeld of view, or there was no available LEG data for 37 GRBs. There was no gamma-ray event near the GRB positions for 59 GRBs, even if the GRB positions were in the CAL ﬁeld of view. 2. GRB observation with CALET A gray shaded region is the ISS ﬁxed structure viewed from CALET. Figure 3: T90 distribution of CGBM GRBs. Energy ranges of CGBM, BATSE, Fermi-GBM, and Swift-BAT are 40 ∼ 1000 keV, 25 ∼2000 keV, 50 ∼300 keV, and 15 ∼350 keV, respectively [17–21]. Blue and red dashed lines are two optimized logarithmic normal distributions. Blue and red dotted lines show mean of the two distributions. Gray dotted line shows the intersection of the two distributions. Figure 4: Incident angle distribution of GRBs in the SGM ﬁeld of view. Black points are GRB positions in the SGM co- ordinate. A gray shaded region is the ISS ﬁxed structure viewed from CALET. PoS(ICRC2021)957 Figure 3: T90 distribution of CGBM GRBs. Energy ranges of CGBM, BATSE, Fermi-GBM, and Swift-BAT are 40 ∼ 1000 keV, 25 ∼2000 keV, 50 ∼300 keV, and 15 ∼350 keV, respectively [17–21]. Blue and red dashed lines are two optimized logarithmic normal distributions. Blue and red dotted lines show mean of the two distributions. Gray dotted line shows the intersection of the two distributions. Figure 4: Incident angle distribution of GRBs in the SGM ﬁeld of view. Black points are GRB positions in the SGM co- ordinate. A gray shaded region is the ISS ﬁxed structure viewed from CALET. period when a CGBM onboard trigger occurs [22]. We have continued to search for high-energy gamma-rays using CAL data [23]. We searched for high-energy gamma-rays from GRBs detected by CGBM using CAL data up to September 30 in 2020. We searched for gamma-ray events in 1 GeV ∼10 GeV for 99 GRBs which were well localized by Swift-BAT, XRT, UVOT, Fermi-LAT, MAXI-GSC, and IPN, using LEG data from T0 −60 s to T0 + 7200 s within 2 deg from the reported GRB central position, where T0 is the trigger time of CGBM. The gamma-ray identiﬁcation was performed according to the method described in [16]. The directions obstructed by ﬁxed structures and moving structures except transient obstruction (e.g., robotic arms), were masked to exclude secondary gamma-rays from the structures. In the case of gamma-ray candidates from GRBs were found, we checked the eﬀects of transient obstruction for each candidate by making scatter plots of arrival directions of gamma-ray candidates on the detector coordinate. As a result, the GRB positions were outside of the CAL ﬁeld of view, or there was no available LEG data for 37 GRBs. 2. GRB observation with CALET The top and bottom panels shows time averaged back- ground spectra for 1000 s on October 5 2015 and April 5 2021. HXM and SGM gains were cor- rected by the position of 1.4 MeV and 2.2 MeV 10−3 10−2 10−1 100 101 Rate [counts/s/channel] 2015/10/05 HXM1 HXM2 SGM 101 102 103 104 Energy [keV] 10 3 10 2 10 1 100 101 Rate [counts/s/channel] 2021/04/05 HXM1 HXM2 SGM PoS(ICRC2021)957 Figure 2: Background spectra of CGBM. The top and bottom panels shows time averaged back- ground spectra for 1000 s on October 5 2015 and April 5 2021. HXM and SGM gains were cor- rected by the position of 1.4 MeV and 2.2 MeV lines for each. Gray dotted lines show the position of 511 keV. Figure 1: Background count rate maps measured by CGBM. The top, middle, and bottom panels are av- eraged count rate maps for each geographic position for HXM1, HXM2 and SGM, respectively. The count rates were calculated using PH data for September 2020 and averaged in each 5 deg. pixel. Since CALET is not a satellite but a payload on the ISS, ISS structures obstruct the CAL and CGBM ﬁelds of view. In addition to ﬁxed structures, there are both regularly transient structures (e.g. solar panels and radiators) and irregularly transient structures (e.g. robotic arms). Most GRBs detected by CGBM arrived from the direction not obstructed by the ﬁxed structures. However, some GRBs arrived from the obstructed region. Although localization errors were ignored, there is 5 −15 deg uncertainty for each point. Also, there is a possibility that ISS structures might have gaps. Full eﬀects of the ISS structures on CGBM data are unclear, and we continue to investigate this issues. In the gamma-ray analysis with CAL, data collected in the high energy trigger (HE) mode and low energy gamma-ray (LEG) mode are used for the analysis above 10 GeV and 1 GeV, respectively [16]. The HE mode is the primary trigger mode of CAL. The HE mode is available anytime except when CAL is collecting pedestal data. LEG mode is enabled only at low latitude, or for a short 4 GRB observation & GW event follow-up with CALET Yuta Kawakubo Figure 4: Incident angle distribution of GRBs in the SGM ﬁeld of view. Black points are GRB positions in the SGM co- ordinate. 2. GRB observation with CALET Gamma-ray events were found near the position of GRB 180526A, GRB 200101A, and GRB 200613A. In the case of GRB 200613A, we found the robotic arm obstructed the CAL ﬁeld of view on June 13, 2020, and we concluded the gamma-ray event was a secondary gamma-ray event from the obstructions. CGBM detected GRB 180526A at T0 = 2018/05/26 11:03:36.20 UT. A 3.4 GeV gamma-ray event was found at 1.3 deg. away from the reported position (R.A., Dec.) = (108.48 deg., 3.64 deg.) by Fermi-LAT at T0 + 244 s [24] . The central position of GRB 180526A was within the 99 % PSF region of the candidate. CGBM also detected GRB 200101A at T0 = 2020/01/01 20:39:30.40 UT. A 4.9 GeV gamma-ray event was found at 0.6 deg. away from the reported position (R.A., Dec.) = (258.995 deg., −32.304 deg.) by IPN at T0 + 105 s [15] (#26635). 2. GRB observation with CALET No excess can be seen in CGBM at the arrival time of the candidates for GRB 180526A and GRB 200101A. A detailed analysis is still underway. of the candidate. No excess can be seen in CGBM at the arrival time of the candidates for GRB 180526A and GRB 200101A. A detailed analysis is still underway. 2. GRB observation with CALET The central position of GRB 200101A was within the 90 % PSF region 5 GRB observation & GW event follow-up with CALET Yuta Kawakubo Table 2: Summary of follow-up observation for gravitational wave events in O3 Event name Possible source Event time (T0) CGBM trigger Ph Pcal Run mode 90 % Upper limit GCN Circular# [erg s−1 cm−2] S190408an BBH (>99 %) 2019/04/08 18:18:02.288180 No trigger 96 % 80 % LEG 2.3 ×10−6 24088 S190412m BBH (>99 %) 2019/04/12 05:30:44.165622 Disabled - - - - - S190421ar BBH (97 %) 2019/04/21 21:38:56.250977 No trigger 3 % 0 % - - - S190425z BNS (>99 %) 2019/04/25 08:18:05.017147 Disabled - 5 % HE 1.0 ×10−4 24218 S190426c BNS (49 %) 2019/04/26 15:21:55.336540 Disabled - 10 % HE 2.5 ×10−5 24276 S190503bf BBH (96 %) 2019/05/03 18:54:04.294490 Disabled - 10 % HE 4.2 ×10−5 24403 S190510g Terrestrial (58 %) 2019/05/10 02:59:39.291636 No trigger 16 % 0 % - - 24495 S190512at BBH (99 %) 2019/05/12 18:07:14.422363 No trigger 100 % 10 % HE 1.9 ×10−5 24531 S190513bm BBH (94 %) 2019/05/13 20:54:28.747089 No trigger 100 % 5 % LEG 6.0 ×10−5 24548 S190517h BBH (98 %) 2019/05/17 05:51:01.830582 No trigger 89 % 0 % - - 24593 S190519bj BBH (96 %) 2019/05/19 15:35:44.397949 No trigger 100 % 0 % - - 24617 S190521g BBH (97 %) 2019/05/21 03:02:29.447266 Disabled - 30 % HE 6.0 ×10−6 24648 S190521r BBH (>99 %) 2019/05/21 07:43:59.463379 Disabled - 0 % - - 24649 S190602aq BBH (99 %) 2019/06/02 17:59:27.089355 No trigger 99 % 5 % HE 2.9 ×10−4 24735 S190630ag BBH (94 %) 2019/06/30 18:52:05.179550 Disabled - 25 % HE 1.2 ×10−5 24960 S190701ah BBH (93 %) 2019/07/01 20:33:06.577637 No trigger 30 % 0 % - - 24970 S190706ai BBH (99 %) 2019/07/06 22:26:41.344727 Disabled - 0 % - - 25027 S190707q BBH (>99 %) 2019/07/07 09:33:26.181226 No trigger 76 % 20 % LEG 2.1 ×10−6 25033 S190718y Terrestrial (98 %) 2019/07/18 14:35:12.067865 No trigger 22 % 5 % LEG 1.7 ×10−6 25099 S190720a BBH (99 %) 2019/07/20 00:08:36.704102 Disabled - 25 % HE 3.0 ×10−5 25134 S190727h BBH (92 %) 2019/07/27 06:03:33.985887 No trigger 35 % 0 % - - 25184 S190728q MassGap (52 %) 2019/07/28 06:45:10.529205 No trigger 0 % 0 % - - 25214 S190814bv NSBH (>99 %) 2019/08/14 21:10:39.012957 Disabled - 0 % - - 25390 Fermi GBM-190816 sub-threshold 2019/08/16 21:22:13.027 No trigger 66 % 25 % HE 2.1 ×10−4 - S190828j BBH (>99 %) 2019/08/28 06:34:05.756472 No trigger 42 % 0 % - - 25536 S190828l BBH (>99 %) 2019/08/28 06:55:09.886557 No trigger 79 % 0 % - - 25537 S190901ap BNS (86 %) 2019/09/01 23:31:01.837767 Disabled 82 % 5 % LEG 6.3 ×10−5 25647 S190910d NSBH (98 %) 2019/09/10 01:26:19.242676 No trigger 77 % 0 % - - 25734 S190910h BNS (61 %) 2019/09/10 08:29:58.544448 No trigger 75 % 10 % LEG 9.4 ×10−6 25735 S190915ak BBH (99 %) 2019/09/15 23:57:02.690891 No trigger 100 % 0 % - - 25770 S190923y NSBH (68 %) 2019/09/23 12:55:59.645508 No trigger 68 % 10 % HE 1.2 ×10−5 25830 S190924h MassGap (> 99 %) 2019/09/24 02:18:46.846654 Disabled - 0 % - - 25844 S190930s MassGap (95 %) 2019/09/30 13:35:41.246810 No trigger 100 % 5 % HE 3.5 ×10−5 25891 S190930t NSBH (74 %) 2019/09/30 14:34:07.685342 No trigger 74 % 5 % HE 1.7 ×10−5 25892 S191105e BBH (95 %) 2019/11/05 14:35:21.933105 Disabled - 0 % - - 26195 S191109d BBH (>99 %) 2019/11/09 01:07:17.220703 Disabled - 0 % - - 26236 S191129u BBH (>99 %) 2019/11/29 13:40:29.197372 No trigger 68 % 0% - - 26321 S191204r BBH (>99 %) 2019/12/04 17:15:26.091822 No trigger 4 % 0 % - - 26358 S191205ah NSBH (93 %) 2019/12/05 21:52:08.568738 Disabled - 0% - - 26377 S191213g BNS (77 %) 2019/12/13 04:34:08.142224 No trigger 33 % 0 % - - 26419 S191215w BBH (>99 %) 2019/12/15 22:30:52.333152 No trigger 51 % 0 % - - 26465 S191216ap BBH (99 %) 2019/12/16 21:33:38.472999 No trigger 26 % 0 % - - 26481 S191222n BBH (>99 %) 2019/12/22 03:35:37.119478 No trigger 60 % 0 % - - 26602 S200105ae Terrestrial (97 %) 2020/01/05 16:24:26.057208 No trigger 84 % 60 % HE 6.5 ×10−6 26664 S200112r BBH (>99 %) 2020/01/12 15:58:38.093931 No trigger 70 % 5 % HE 1.1 ×10−6 26740 S200114f - 2020/01/14 02:08:18.239300 Disabled - 80 % HE 4.7 ×10−6 26761 S200115j MassGap (>99 %) 2020/01/15 04:23:09.742047 Disabled - 20 % HE 1.7 ×10−6 26797 S200128d BBH (97 %) 2020/01/28 02:20:11.903320 No trigger 65 % 10 % HE 4.6 ×10−6 26924 S200129m BBH (>99 %) 2020/01/29 06:54:58.435104 Disabled - 5 % HE 5.7 ×10−5 26941 S200208q BBH (>99 %) 2020/02/08 13:01:17.991118 Disabled - 0 % - - 27030 S200213t BNS (63 %) 2020/02/13 04:10:40.327981 No trigger 31 % 0 % - - 27084 S200219ac BBH (96 %) 2020/02/19 09:44:15.195312 No trigger 73 % 0 % - - 27149 S200224ca BBH (>99 %) 2020/02/24 22:22:34.405762 Disabled - 95 % HE 5.0 ×10−7 27231 S200225q BBH (96 %) 2020/02/25 06:04:21.396973 Disabled - 0 % - - 27232 S200302c BBH (89 %) 2020/03/02 01:58:11.519119 No trigger 81 % 0 % - - 27299 S200311bg BBH (>99 %) 2020/03/11 11:58:53.397788 Disabled - 0 % - - 27372 S200316bj MassGap (>99 %) 2020/03/16 21:57:56.157221 No trigger 82 % 35 % HE 2.8 ×10−6 27405 Table 2: Summary of follow-up observation for gravitational wave events in O3 PoS(ICRC2021)957 of the candidate. 3. Follow-up for gravitational wave events in LIGO/Virgo O3 CALET participated in the follow-up campaign for the LIGO/Virgo ﬁrst and second observation runs [25][26][6]. Also, we performed a follow-up observation of electromagnetic counterparts of the gravitational wave using both CAL and CGBM data in the LIGO/Virgo third observation run 6 GRB observation & GW event follow-up with CALET Yuta Kawakubo (O3) [27] [11]. Table 2 shows a summary of CALET follow-up observations. There are 56 events reported by the LIGO/Virgo collaboration (LVC) and one sub-threshold event reported by LVC and Fermi-GBM team[28] [15]. ‘Event name,’ ‘Possible source,’ and ‘Event time (T0)’ are based on GraceDB and GCN circulars reported by the LVC and Fermi-GBM team [28][15](#25406). The ‘Possible source’ column shows just the highest probability source in the GCN circulars. ‘CGBM trigger’ shows the status of the CGBM onboard trigger at T0. ‘No trigger’ means that CGBM onboard trigger was enabled; however, no trigger occurred in T0-60 s∼T0+ 60 s. ‘Disabled’ means the CGBM onboard trigger was disabled due to CGBM high voltages were oﬀ, or the CGBM event data storage was full. There were no onboard triggers associated with any gravitational wave events. ‘Ph’ shows the summed LIGO/Virgo localization probability above the horizon. If the CGBM high voltages were oﬀ, the column was ﬁlled by ‘-.’ We also searched for electromagnetic signals in TH data for 36 events that occurred when the CGBM high voltage was on. We calculated the SNRs using (1) with extended conditions and searched for signiﬁcant signals according to the method described in [11] using TH data for T0-60 s∼T0+ 60 s, where T0 is the trigger time of the gravitational wave event. As a result, there was no signiﬁcant signal associated with the gravitational wave events in the CGBM data. PoS(ICRC2021)957 ‘Pcal’ shows the summed LIGO/Virgo localization probability in the CAL ﬁeld of view for T0-60 s∼T0+ 60 s. Although CAL’s high voltages are typically always on, CALET was oﬀdue to a special activity on ISS when S190412 occurred. ‘Run mode’ shows the CAL run mode at T0. If Pcal is zero, the column was ﬁlled by ‘-.’ In the case where Pcal was 5 % or greater, we searched for gamma-ray events from the LIGO/Virgo localization high probability region in HE (10 GeV ∼ 100 GeV) or LEG (1 GeV ∼10 GeV) data for T0-60 s∼T0+ 60 s. 3. Follow-up for gravitational wave events in LIGO/Virgo O3 Although there was no high energy gamma-ray candidate associated with the gravitational wave events, we estimated 90 % upper limits of gamma-ray ﬂux for each direction according to the method described in [26][27]. The 90 % upper limits were calculated for the energy range 10 GeV ∼100 GeV and 1 GeV ∼10 GeV in the case of HE and LEG data, respectively. As examples of the analysis, Figure 5 shows the 90% upper limits maps for S190408an and S200316bj. Since eﬀective areas for small incident angles are larger than that for large incident angles, stricter upper limits are derived near the CALET zenith than near the edge of the CAL ﬁeld of view. The dented structures around the edge of the CAL ﬁeld of view were masked due to the ﬁxed ISS structures. ‘90 % Upper limit ’ shows the highest 90 % upper limits of gamma-ray ﬂux when the summed LIGO/Virgo localization probability reached Pcal. 4. Summary CALET has been in in-orbit operation since October 2015 without any problems. CGBM has been continuing all-sky monitoring of GRBs with a ∼60 % duty cycle and observed 254 GRBs, including 31 short GRBs, by the end of May 2021. As the result of high energy gamma- ray search from GRBs detected by CGBM using CAL, two gamma-ray candidates were found from GRB 180526A and GRB 200101A. CALET also participated in the follow-up campaign for electromagnetic counterparts of gravitational wave events in O3. Although there was no candidate of the electromagnetic counterparts, we estimated 90 % upper limits of gamma-ray ﬂux for 26 gravitational wave events using CAL data. 7 GRB observation & GW event follow-up with CALET Yuta Kawakubo Figure 5: The 90 % upper limits for S190408an (left) and S200316bj (right). The color maps show the 90 % upper limits of gamma-ray ﬂux. In the case of S190408A, the energy range is 1 GeV ∼10 GeV. In the case of S200316bj, energy range is 10 GeV ∼100 GeV. Green contours are the LIGO/Virgo localization high probability region. Black bold cross is the CAL zenith at T0. Cyan bold lines are tracks of the CAL zenith for T0-60 s∼T0+ 60 s. Figure 5: The 90 % upper limits for S190408an (left) and S200316bj (right). The color maps show the 90 % upper limits of gamma-ray ﬂux. In the case of S190408A, the energy range is 1 GeV ∼10 GeV. In the case of S200316bj, energy range is 10 GeV ∼100 GeV. Green contours are the LIGO/Virgo localization high probability region. Black bold cross is the CAL zenith at T0. Cyan bold lines are tracks of the CAL zenith for T0-60 s∼T0+ 60 s. PoS(ICRC2021)957 References [1] R. Y. Klebesadel et al., OBSERVATIONS OF GAMMA-RAY BURSTS OF COSMIC ORIGIN, THE ASTROPHYSICAL JOURNAL, 182, L85, (1973) [2] P. Kumar, and B. Zhang, The physics of gamma-ray bursts & relativistic jets, Physics Report, 561, 1, (2015). [3] C. Kouveliotou et al., IDENTIFICATION OF TWO CLASSES OF GAMMA-RAY BURSTS, THE ASTROPHYSICAL JOURNAL, 413, L101, (1993). [ ] , , , , , ( ) [4] B. P. Abbott et al., GW170817: Observation of Gravitational Waves from a Binary Neutron Star Inspiral, PHYSICAL REVIEW LETTERS, 119, 161101, (2017). [4] B. P. Abbott et al., GW170817: Observation of Gravitational Waves from a Binary Neutron Star Inspiral, PHYSICAL REVIEW LETTERS, 119, 16 [5] B. P. Abbott et al., Gravitational Waves and Gamma-Rays from a Binary Neutron Star Merger: GW170817 and GRB 170817A, THE ASTROPHYSICAL JOURNAL LETTERS, 848, L13, (2017). [6] B. P. Abbott et al., Multi-messenger Observations of a Binary Neutron Star Merger, The Astrophysical Journal Letters, 848, L12, (2017). 6] B. P. Abbott et al., Multi-messenger Observations of a Binary Neutron Star Merger, T [7] Y Asaoka for the CALET Collaboration, The CALorimetric Electron Telescope (CALET) on the International Space Station, Proc.36th ICRC (Madison, USA, 2019), POS(ICRC2019), 001, (2019). 8] N. Cannady on behalf of the CALET Collaboration, Low-energy gamma-ray observat [9] M. Mori on behalf of the CALET Collaboration., High-energy gamma-ray observations above 10 GeV with CALET on the International Space Station, in this conference. [9] M. Mori on behalf of the CALET Collaboration., High-energy gamma-ray observations above 10 GeV with CALET on the International Space Station, in this conference. 10] K. Yamaoka et al., The CALET Gamma-ray Burst Monitor (CGBM)) Proc. 7th Huntsville Gamma-Ray Burst Symposium (Nashville, USA, 2013), paper 41 in eConf Proceedings C1304143, (2013). [11] Y. Kawakubo et al., Search for electromagnetic counterparts of gravitational wave sources with CALET, Proc. GAMMA-RAY BURSTS IN THE GRAVITATIONAL WAVE ERA, 31, (2020). [12] F.G.A. Quarati et al., Study of 138La radioactive decays using LaBr3 scintillators, Nuclear Instruments and Methods in Physics Research A, 683, 46, (2012) [13] P.R. Truscott et al., Activation of Space-Borne Bismuth Germanate γ-ray Detectors, IEEE TRANSACTIONS ON NUCLEAR SCIENCE, 42, No.4, (1995) 3] P.R. Truscott et al., Activation of Space-Borne Bismuth Germanate γ-ray Detectors, [14] NASA HEASARC battblocks https://heasarc.gsfc.nasa.gov/ftools/caldb/help/battblocks.html [14] NASA HEASARC battblocks https://heasarc.gsfc.nasa.gov/ftools/c [15] The Gamma-ray Coordinates Network https://gcn.gsfc.nasa.gov [15] The Gamma-ray Coordinates Network https://gcn.gsfc.nasa.gov [16] N. Full Authors List: CALET Collaboration O. Adriani1,2, Y. Akaike3,4, K. Asano5, Y. Asaoka5, E. Berti1,2, G. Bigongiari6,7, W. R. Binns8, M. Bongi1,2, P. Brogi6,7, A. Bruno9,10, J. H. Buckley8, N. Cannady11,12,13, G. Castellini14, C. Checchia6, M. L. Cherry15, G. Collazuol16,17, K. Ebisawa18, A. W. Ficklin15, H. Fuke18, S. Gonzi1,2, T. G. Guzik15, T. Hams11, K. Hibino19, M. Ichimura20, K. Ioka21, W. Ishizaki5, M. H. Israel8, K. Kasahara22, J. Kataoka23, R. Kataoka24, Y. Katayose25, C. Kato26, N. Kawanaka27,28, Y. Kawakubo15, K. Kobayashi3,4, K. Kohri29, H. S. Krawczynski8, J. F. Krizmanic11,12,13, P. Maestro6,7, P. S. Marrocchesi6,7, A. M. Messineo30,7, J.W. Mitchell12, S. Miyake32, A. A. Moiseev33,12,13, M. Mori34, N. Mori2, H. M. Motz35, K. Munakata26, S. Nakahira18, J. Nishimura18, G. A. de Nolfo9, S. Okuno19, J. F. Ormes36, N. Ospina16,17, S. Ozawa37, L. Pacini1,14,2, P. Papini2, B. F. Rauch8, S. B. Ricciarini14,2, K. Sakai11,12,13, T. Sakamoto38, M. Sasaki33,12,13, Y. Shimizu19, A. Shiomi39, P. Spillantini1, F. Stolzi6,7, S. Sugita38, A. Sulaj6,7, M. Takita5, T. Tamura19, T. Terasawa40, S. Torii3, Y. Tsunesada41, Y. Uchihori42, E. Vannuccini2, J. P. Wefel15, K. Yamaoka43, S. Yanagita44, A. Yoshida38, K. Yoshida22, and W. V. Zober8 PoS(ICRC2021)957 g 1Department of Physics, University of Florence, Via Sansone, 1, 50019 Sesto, Fiorentino, Italy, 2INFN Sezione di Florence, Via Sansone, 1, 50019 Sesto, Fiorentino, Italy, 3Waseda Research Institute for Science and Engineering, Waseda University, 17 Kikuicho, Shinjuku, Tokyo 162-0044, Japan, 4JEM Utilization Center, Human Spaceﬂight Technology Directorate, Japan Aerospace Exploration Agency, 2-1-1 Sengen, Tsukuba, Ibaraki 305-8505, Japan, 5Institute for Cosmic Ray Research, The University of Tokyo, 5-1-5 Kashiwa-no-Ha, Kashiwa, Chiba 277-8582, Japan, 6Department of Physical Sciences, Earth and Environment, University of Siena, via Roma 56, 53100 Siena, Italy, 7INFN Sezione di Pisa, Polo Fibonacci, Largo B. Pontecorvo, 3, 56127 Pisa, Italy, 8Department of Physics and McDonnell Center for the Space Sciences, Washington University, One Brookings Drive, St. Full Authors List: CALET Collaboration Louis, Missouri 63130-4899, USA, 9Heliospheric Physics Laboratory, NASA/GSFC, Greenbelt, Maryland 20771, USA, 10Department of Physics, Catholic University of America, Wash- ington, DC 20064, USA, 11Center for Space Sciences and Technology, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250, USA, 12Astroparticle Physics Laboratory, NASA/GSFC, Greenbelt, Maryland 20771, USA, 13Center for Research and Exploration in Space Sciences and Technology, NASA/GSFC, Greenbelt, Maryland 20771, USA, 14Institute of Applied Physics (IFAC), National Research Council (CNR), Via Madonna del Piano, 10, 50019 Sesto, Fiorentino, Italy, 15Department of Physics and Astronomy, Louisiana State University, 202 Nicholson Hall, Baton Rouge, Louisiana 70803, USA, 16Department of Physics and Astronomy, University of Padova, Via Marzolo, 8, 35131 Padova, Italy, 17INFN Sezione di Padova, Via Marzolo, 8, 35131 Padova, Italy, 18Institute of Space and Astronautical Science, Japan Aerospace Exploration Agency, 3-1-1 Yoshinodai, Chuo, Sagamihara, Kanagawa 252-5210, Japan, 19Kanagawa University, 3-27-1 Rokkakubashi, Kanagawa, Yokohama, Kanagawa 221-8686, Japan, 20Faculty of Sci- ence and Technology, Graduate School of Science and Technology„ Hirosaki University, 3, Bunkyo, Hirosaki, Aomori 036-8561, Japan, 21Yukawa Institute for Theoretical Physics, Kyoto University, Kitashirakawa Oiwakecho, Sakyo, Kyoto 606-8502, Japan, 22Department of Electronic Information Systems, Shibaura Institute of Technology, 307 Fukasaku, Minuma, Saitama 337-8570, Japan, 23School of Advanced Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku, Tokyo 169-8555, Japan, 24National Institute of Polar Research, 10-3, Midori-cho, Tachikawa, Tokyo 190-8518, Japan, 25Faculty of Engineering, Division of Intelligent Systems Engineer- ing, Yokohama National University, 79-5 Tokiwadai, Hodogaya, Yokohama 240-8501, Japan, 26Faculty of Science, Shinshu University, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan, 27Hakubi Center, Kyoto University, Yoshida Honmachi, Sakyo-ku, Kyoto 606-8501, Japan, 28Department of Astronomy, Graduate School of Science, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606- 8502, Japan, 29Institute of Particle and Nuclear Studies, High Energy Accelerator Research Organization, 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan, 30University of Pisa, Polo Fibonacci, Largo B. References Cannady et al., Characteristics and Performance of the CALorimetric Electron Telescope (CALET) Calorimeter for Gamma-Ray Observations, The Astrophys Journal Supplement Series, 238, 5, (2018) 17] D. Gruber et al., THE FERMI GBM GAMMA-RAY BURST SPECTRAL CATALOG: FOUR YEARS OF DATA, THE ASTROPHYSICAL JOURNAL SUPPLEMENT SERIES, 211, 12, (2014). 18] A. von Kienlin et al., THE SECOND FERMI GBM GAMMA-RAY BURST CATALOG: THE FIRST FOUR YEARS, THE ASTROPHYSICAL JOURNAL SUPPLEMENT SERIES, 211, 13, (2014). [19] P. N. Bhat et al., THE THIRD FERMI GBM GAMMA-RAY BURST CATALOG: THE FIRST SIX YEARS, THE ASTROPHYSICAL JOURNAL SUPPLEMENT SERIES, 28, (2016). [20] The BATSE Current Gamma-Ray Burst Catalog, the BATSE GRB Team, https://gammaray.msfc.nasa.gov/batse/grb/catalog/current/ [21] The Swift/BAT Gamma-Ray Burst Catalog, https://swift.gsfc.nasa.gov/results/batgrbcat/ [22] Y. Asaoka et al., On-orbit operations and oﬄine data processing of CALET onboard the ISS, Astroparticle Physics, 100, 29, (2018). [23] N. Cannady for the CALET collaboration, CALET upper limits on GeV-energy gamma-ray burst emission, Proc.36th ICRC (Madison, USA, 2019), POS(ICRC2019), (2019). [24] Ajello, et al., A Decade of Gamma-Ray Bursts Observed by Fermi-LAT: The Second GRB Catalog, The Astrophysical Journal, 878, 52, (2019). [25] O. Adriani et al., CALET UPPER LIMITS ON X-RAY AND GAMMA-RAY COUNTERPARTS OF GW151226, The Astrophysical Journal Letters, 829, L20, (2016 driani et al., Search for GeV Gamma-Ray Counterparts of Gravitational Wave Events by 27] M. Mori & Y. Asaoka for the CALET Collaboration, High-Energy Gamma-ray Observations Using the CALorimetric Electron Telescope (CALET) on the ISS, Proc.36th ICRC (Madison, USA, 2019), POS(ICRC2019), 586, (2019). [28] GraceDB, https://gracedb.ligo.org/superevents/public/O3/ 8 Yuta Kawakubo GRB observation & GW event follow-up with CALET Full Authors List: CALET Collaboration Pontecorvo, 3, 56127 Pisa, Italy, 31Astroparticle Physics Laboratory, NASA/GSFC, Greenbelt, Maryland 20771, USA, 32Department of Electrical and Electronic Systems Engineering, National Institute of Technology, Ibaraki College, 866 Nakane, Hitachinaka, Ibaraki 312-8508, Japan 33Department of Astronomy, University of Maryland, College Park, Maryland 20742, USA, 34Department of Physical Sciences, College of Science and Engineering, Ritsumeikan University, Shiga 525-8577, Japan, 35Faculty of Science and Engineering, Global Center for Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku, Tokyo 169-8555, Japan, 36Department of Physics and Astronomy, University of Denver, Physics Building, Room 211, 2112 East Wesley Avenue, Denver, Colorado 80208-6900, USA, 37Quantum ICT Advanced Development Center, National Institute of Information and Communications Technology, 4-2-1 Nukui-Kitamachi, Koganei, Tokyo 184-8795, Japan, 38College of Science and Engineering, Department of Physics and Mathematics, Aoyama Gakuin University, 5-10-1 Fuchinobe, Chuo, Sagamihara, Kanagawa 252-5258, Japan, 39College of Industrial Technology, Nihon University, 1-2-1 Izumi, Narashino, Chiba 275-8575, Japan 40RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan, 41Division of Mathematics and Physics, Graduate School of Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi, Osaka 558-8585, Japan, 42National Institutes for Quantum and Radiation Science and Technology, 4-9-1 Anagawa, Inage, Chiba 263-8555, Japan, 43Nagoya University, Furo, Chikusa, Nagoya 464-8601, Japan, 44College of Science, Ibaraki University, 2-1-1 Bunkyo, Mito, Ibaraki 310-8512, Japan 9 9
https://openalex.org/W2137103411	https://api.research-repository.uwa.edu.au/ws/files/9180065/J._Exp._Bot._2015_Jayakannan_1865_75.pdf	English	null	The NPR1-dependent salicylic acid signalling pathway is pivotal for enhanced salt and oxidative stress tolerance in Arabidopsis	Journal of experimental botany	2,015	cc-by	11,981	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 reuse, distribution, and reproduction in any medium, provided the original work is properly cited. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. Abbreviations: AtNUDT, Arabidopsis Nudix hydrolase; NPR1, non-expresser of pathogenesis related gene1; SA, salicylic acid. Abstract The role of endogenous salicylic acid (SA) signalling cascades in plant responses to salt and oxidative stresses is unclear. Arabidopsis SA signalling mutants, namely npr1-5 (non-expresser of pathogenesis related gene1), which lacks NPR1-dependent SA signalling, and nudt7 (nudix hydrolase7), which has both constitutively expressed NPR1- dependent and NPR1-independent SA signalling pathways, were compared with the wild type (Col-0) during salt or oxidative stresses. Growth and viability staining showed that, compared with wild type, the npr1-5 mutant was sensi- tive to either salt or oxidative stress, whereas the nudt7 mutant was tolerant. Acute salt stress caused the strong- est membrane potential depolarization, highest sodium and proton influx, and potassium loss from npr1-5 roots in comparison with the wild type and nudt7 mutant. Though salt stress-induced hydrogen peroxide production was low- est in the npr1-5 mutant, the reactive oxygen species (ROS) stress (induced by 1 mM of hydroxyl-radical-generating copper-ascorbate mix, or either 1 or 10 mM hydrogen peroxide) caused a higher potassium loss from the roots of the npr1-5 mutant than the wild type and nudt7 mutant. Long-term salt exposure resulted in the highest sodium and the lowest potassium concentration in the shoots of npr1-5 mutant in comparison with the wild type and nudt7 mutant. The above results demonstrate that NPR1-dependent SA signalling is pivotal to (i) controlling Na+ entry into the root tissue and its subsequent long-distance transport into the shoot, and (ii) preventing a potassium loss through depo- larization-activated outward-rectifying potassium and ROS-activated non-selective cation channels. In conclusion, NPR1-dependent SA signalling is central to the salt and oxidative stress tolerance in Arabidopsis. Key words: ROS, membrane potential, oxidative stress, potassium fluxes, proton fluxes, salinity, salicylic acid, sodium fluxes, viability staining Key words: ROS, membrane potential, oxidative stress, potassium fluxes, proton fluxes, salinity, salicylic acid, sodium fluxes, viability staining. Maheswari Jayakannan1,2,3, Jayakumar Bose2, Olga Babourina1, Sergey Shabala2, ndine Massart4,5, Charlotte Poschenrieder3 and Zed Rengel1,* * To whom correspondence should be addressed. E-mail: zed.rengel@uwa.edu.au * To whom correspondence should be addressed. E-mail: zed.rengel@uwa.edu.au Received 31 July 2014; Revised 6 December 2014; Accepted 9 December 2014 The NPR1-dependent salicylic acid signalling pathway is pivotal for enhanced salt and oxidative stress tolerance in Arabidopsis eswari Jayakannan1,2,3, Jayakumar Bose2, Olga Babourina1, Sergey Shabala2, Journal of Experimental Botany, Vol. 66, No. 7 pp. 1865–1875, 2015 p y, , pp , doi:10.1093/jxb/eru528 Advance Access publication 22 January 2015 This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details) p y, , pp , doi:10.1093/jxb/eru528 Advance Access publication 22 January 2015 This paper is available online free of all access charges (see http://jxb oxfordjournals org/open access doi:10.1093/jxb/eru528 Advance Access publication 22 January 2015 This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details) This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details) \| Jayakannan et al. An Arabidopsis sid2 (SA induction deficient 2) mutant defective in the expression of the isochorismate synthase (ICS1) gene is hypersensitive to salt stress (Lee et al., 2010; Asensi-Fabado and Munné-Bosch, 2011), implying that this pathway is essential for salinity tolerance in plants. In con- trast, some studies have found that a SA-deficient Arabidopsis mutant exhibited higher salinity stress tolerance compared with the wild type and SA-hyper-accumulating mutants (Borsani et al., 2001; Cao et al., 2009; Hao et al., 2012). However, opposite to the aforementioned results were also reported by some other authors (Asensi-Fabado and Munné- Bosch, 2011; Miura et al., 2011). The reason for such discrep- ancy is due to the use of mutants that were not altered in the isochorismate-synthase-mediated SA synthesis causing subsequent changes in SA accumulation. Instead, the SA levels were altered by SA hydroxylase (NahG) activity, allow- ing for the possibility that SA signalling might be turned on before NahG converts SA into catechol (Borsani et al., 2001). Moreover, among the SA biosynthesis pathways, only the isochorismate-synthase-mediated SA synthesis pathway is stress inducible (see above); hence, it is imperative to evaluate specifically the isochorismate-synthase-mediated SA-hyper- accumulating mutants during salt stress to decipher SA signalling. Recently, an Arabidopsis NPR1 knockout mutant (npr1-1) accumulated SA upon salt stress and showed enhanced salt tolerance (Hao et al., 2012). On the other hand, an NPR1- hyper-accumulating Arabidopsis double mutant (npr3npr4) failed to undergo programmed cell death (Attaran and He, 2012; Fu et al., 2012), suggesting NPR1-mediated preven- tion of programmed cell death may be beneficial for salt tol- erance. Overall, it seems that salt tolerance in plants can be controlled by both NPR1-independent and NPR1-dependent mechanisms. Comparison of a nudt7 mutant (which has both constitutively expressed NPR1-independent and NPR1- dependent SA-mediated pathways) with a NPR1 knockout mutant (without SA-mediated NPR1-dependent pathway) will pave the way for characterizing a SA-mediated defence response against salt stress. Salt stress increases the production of various forms of reac- tive oxygen species (ROS) namely superoxide (O2 −), singlet oxygen (1O2), hydrogen peroxide (H2O2), and hydroxyl radi- cal (˙OH) in plants (reviewed in Parida and Das, 2005). Some of these ROS species (˙OH, O2 −, and H2O2) can induce K+ loss via ROS-activated channels and trigger programmed cell death during salt stress (e.g Shabala et al., 2007; Demidchik et al., 2010; Poór et al., 2012; Tran et al., 2013). \| Jayakannan et al. 1866 responsible for high SA in nudt7 mutant. Hence, characteri- zation of nudt7 mutant under salt stress may be a useful tool to answer whether isochorismate-synthase-mediated SA bio- synthesis and SA accumulation are essential for salt tolerance in plants. (Martinez-Beltran and Manzur, 2005), increasing on average by up to 0.5 M ha each year. Remediation of salt-affected arable lands is very expensive, time consuming, and hard to implement on a large scale. Thus, increasing the salt toler- ance of crop plants through molecular and plant breeding approaches is the most attractive and viable option to sus- tain food production in salt-affected environments (Ondrasek et al., 2011). In this regard, salicylic acid (SA) has gained importance as an important signalling phytohormone that can marshal salt tolerance in plants (Borsani et al., 2001; Horváth et al., 2007). However, the exact SA signalling cas- cades during salt stress remain elusive. p To activate a defence response, SA should bind to some specific receptors. The NPR1 (non-expresser of pathogene- sis-related gene 1) protein was identified as one of these (Wu et al., 2012). Simultaneous studies revealed that SA also binds with NPR1 prologues NPR3 and NPR4, which in turn trig- ger the reduction of inactive oligomeric NPR1 into active monomeric NPR1 (a master regulator of SA-induced defence genes) in the cytoplasm (Fu et al., 2012). The monomeric NPR1 enters the nucleus and functions as a transcriptional co-activator of defence genes (Attaran and He, 2012; Fu et al., 2012). Microarray analysis in Arabidopsis reported that among SA-induced defence genes, more than 90 percent were NPR1-dependent genes (Wang et al., 2006; Blanco et al., 2009). In particular, the Atnudt7 mutant has been reported to mediate both NPR1-dependent and NPR1-independent defence response against pathogens (Ge et al., 2007). Moreover, defence genes that control programmed cell death and osmotic and oxidative stress tolerance (all important for salt tolerance) fall under either pathway (Blanco et al., 2009). g Endogenous SA is synthesised from a primary metabo- lite, chorismate, by two distinct pathways: the phenylalanine ammonia-lyase pathway in the cytoplasm, and the isochoris- mate pathway in the chloroplast (reviewed in Dempsey et al., 2011; Rivas-San Vicente and Plasencia, 2011). The latter pathway is responsible for the bulk of the pathogen-induced SA synthesis in diverse plant species (reviewed in Vlot et al., 2009). Introduction (Rengasamy, 2006). In addition, secondary salinization, resulting from poor irrigation and/or drainage practices, affects more than 50% of productive irrigated land globally Soil salinity is one of the major abiotic stresses that threaten sustainable food production worldwide. About 831 mil- lion ha of land is affected by natural salinization worldwide \| Jayakannan et al. Several inde- pendent studies confirmed that Atnudt7 mutant participated in redox homeostasis maintenance (Ge et al., 2007; Ishikawa et al., 2009; Jambunathan et al., 2010; Straus et al., 2010) and delayed programmed cell death (Straus et al., 2010). However, it needs to be tested whether delayed programmed cell death in the nudt7 mutant is due to prevention of K+ loss through ROS-activated channels. Exploring this issue was one of the aims of this study. g g The Arabidopsis genome contains 25–32 Nudix (nucleoside diphosphates linked to moiety X) hydrolases (AtNUDTs) that hydrolyse nucleoside derivatives (Kraszewska, 2008); however, the work on estimating the number of Nudix genes is ongoing. Among the members, AtNUDT7 (At4g12720) was identified as a gene induced by multiple stresses, includ- ing salinity (Jambunathan and Mahalingam, 2006), and its knockout mutant, nudt7-1 (SALK_046441; formerly known as growth factor gene 1; hereafter described as nudt7) was found to have three- to four-fold higher concentration of SA than the wild type under control growth conditions (Bartsch et al., 2006; Straus et al., 2010; Wang et al., 2012). This SA concentration increase is absent in the double mutant nudt7 sid2-1 (Bartsch et al., 2006; Straus et al., 2010), suggesting that isochorismate-synthase-mediated SA biosynthesis is The present study hypothesized that the elevated SA con- centration may mediate adaptive responses against salt and oxidative stresses through both NPR1-independent and Salicylic acid signalling during salinity stress \| 1867 1867 grow down along the agar surface without penetrating it, but being anchored in it via root hairs. The 4- to 5-day-old seedlings were used for all the short-term experiments (measurements of ion fluxes, a c f a b e a d g 0 200 400 600 800 1000 1200 Control 50 mM NaCl 100 mM NaCl Col-0 nudt7 npr1-5 Fresh weight (mg pot-1) Control 50 mM NaCl 100 mM NaCl (i) (ii) (iii) Radicle emergence at 150 mM NaCl b a c 0 10 20 30 40 50 60 col-0 nudt7 npr1-5 Col-0 (i) (ii) (iii) (i) (ii) (iii) Number (% of seeds sown) A B C D E Fig. 1. Growth and radicle emergence of Arabidopsis thaliana grown in full-strength MS medium with 2% w/v phytogel infused with different concentration of salt. (A–C) Photographs of radicle emergence in (i) Col-0, (ii) nudt7, (iii) npr1-5 at the indicated NaCl concentrations 7 d after sowing. Ion flux measurements The Microelectrode Ion Flux Estimation (MIFETM, University of Tasmania, Hobart, Australia) technique was used to measure net fluxes of H+, K+, and Na+. The principles and methods of this MIFETM technique can be found in Newman (2001). The details pertinent to microelectrode fabrication, conditioning, and calibra- tion were detailed in previous publications (Jayakannan et al., 2011; Bose et al., 2013; Jayakannan et al., 2013). \| Jayakannan et al. (D) Quantification of radicle emergence out of 20 seeds shown under 150 mM NaCl treatment at 7 d after sowing. (E) Fresh weight of the three genotypes under indicated NaCl concentrations 2 weeks after sowing. Each bar in the graphs represents mean±SEM. Different letters in bar graphs indicate significant differences. (This figure is available in colour at JXB online.) NPR1-dependent pathways. This hypothesis was tested by characterizing roots of Arabidopsis mutants, namely nudt7, and npr1-5 under saline and oxidative stresses. The nudt7 contains the constitutively expressed SA-mediated NPR1- independent and NPR1-dependent defence genes, whereas npr1-5 (formerly known as sai1, salicylic acid-insensitive1), is a NPR1-knockout mutant without the SA-mediated NPR1- dependent defence response (Shah et al., 1997; Shah et al., 1999). The reported results confirm that SA-mediated salt and oxidative stress tolerance is NPR1-dependent. Particularly, NPR1-dependent SA signalling helps plants to (i) prevent Na+ loading into root tissue and its subsequent transport into shoots, and (ii) retain K+ both in the roots and shoots by controlling K+ loss through depolarization-activated out- ward-rectifying K+ channels (KOR) and ROS-activated non- selective cation channels (NSCC). 50 mM NaCl (i) (ii) (iii) B D Control (i) (ii) (iii) A B A (i) 50 mM NaCl Control 50 mM NaCl Radicle emergence at 150 mM NaCl b a c 0 10 20 30 40 50 60 col-0 nudt7 npr1-5 Col-0 Number (% of seeds sown) D D Control (i) (ii) (iii) C C Plant material Seeds of Arabidopsis thaliana L. wild type (Col-0) and mutant seeds of loss-of-function of NPR1 gene npr1-5 (Salk_CS3724, Col-0) and NUDT7 gene nudt7 (Salk_046441, Col-0) were obtained from the Arabidopsis Biological Resource Centre (http://www.Arabidopsis. org/abrc/). Arabidopsis seeds were surface sterilized with 1 % v/v sodium hypochlorite (commercial Bleach) plus 0.01 % v/v Triton (wetting agent) for 10 min followed by at least three rinses with ster- ile deionized water. 100 mM NaCl Radicle emergence at 150 mM NaCl Radicle emergence at a c f a b e a d g 0 200 400 600 800 1000 1200 Control 50 mM NaCl 100 mM NaCl Col-0 nudt7 npr1-5 Fresh weight (mg pot-1) g 150 mM NaCl E E Long-term growth experiments (This figure is available in colour at JXB online.) grow down along the agar surface without penetrating it, but being anchored in it via root hairs. The 4- to 5-day-old seedlings were used for all the short-term experiments (measurements of ion fluxes, membrane potential, and root viability). At the end of the experiment, plants were harvested and thor- oughly rinsed with ice-cold 0.5 mM CaSO4 solution; excess water was removed by blotting shoots with paper towels, and fresh weight was measured immediately. Plants were then dried at 65 °C for 2 d in a Unitherm Dryer (Birmingham, UK) and weighed. Shoot water content (%) was calculated as the difference between fresh and dry weight. Long-term growth experiments For genotype comparison, 15 surface-sterilized seeds of each geno- type (Col-0, nudt7, and npr1-5) were sown on the surface of 90-mm Petri dishes containing solid 0.35 % w/v phytogel, full strength Murashige and Skoog medium (MS; Sigma-Aldrich, Castle Hill, NSW, Australia), 1% w/v sucrose, and various concentrations of NaCl (0, 50, 100, or 150 mM). Media pH was adjusted to 5.7 by adding either KOH or HCl. The Petri dishes were divided into three equal parts to accommodate three genotypes per dish (Fig. 1). The Petri dishes containing seeds were sealed with Parafilm strips, kept at 4 °C for 2 d, and then transferred into a growth chamber with 16/8 h day/night photoperiod, 150 µmol m–2 s–1 photon flux density and 23 °C temperature. The Petri dishes were placed in a horizontal position, allowing the roots to grow through the phytogel MS media for 25 d. To assess radicle emergence during salt stress, Arabidopsis seeds were sown on the MS media containing 150 mM NaCl. Seeds were then vernalized (as above), and the germination percentage was assessed after 7 d in the growth chamber. These experiments were repeated at least twice, with four replicates each time. Fig. 1. Growth and radicle emergence of Arabidopsis thaliana grown in full-strength MS medium with 2% w/v phytogel infused with different concentration of salt. (A–C) Photographs of radicle emergence in (i) Col-0, (ii) nudt7, (iii) npr1-5 at the indicated NaCl concentrations 7 d after sowing. (D) Quantification of radicle emergence out of 20 seeds shown under 150 mM NaCl treatment at 7 d after sowing. (E) Fresh weight of the three genotypes under indicated NaCl concentrations 2 weeks after sowing. Each bar in the graphs represents mean±SEM. Different letters in bar graphs indicate significant differences. (This figure is available in colour at JXB online.) Fig. 1. Growth and radicle emergence of Arabidopsis thaliana grown in full-strength MS medium with 2% w/v phytogel infused with different concentration of salt. (A–C) Photographs of radicle emergence in (i) Col-0, (ii) nudt7, (iii) npr1-5 at the indicated NaCl concentrations 7 d after sowing. (D) Quantification of radicle emergence out of 20 seeds shown under 150 mM NaCl treatment at 7 d after sowing. (E) Fresh weight of the three genotypes under indicated NaCl concentrations 2 weeks after sowing. Each bar in the graphs represents mean±SEM. Different letters in bar graphs indicate significant differences. Short-term experiments Surface-sterilized seeds were sown on the surface of 90-mm Petri dishes containing 0.4 % w/v agar, 1.0 mM KCl plus 0.1 mM CaCl2 at pH 5.7 (Jayakannan et al., 2011; Jayakannan et al., 2013). The Petri dishes containing seeds were sealed, vernalized, and grown under controlled conditions as described above. In the short-term experi- ments, the Petri dishes were placed vertically, allowing the roots to Preparation of Arabidopsis seedlings for MIFE measurements In vivo hydrogen peroxide imaging The H2O2 imaging of root tissue was done by following the stand- ard procedure adopted in a previous publication (Bose et al., 2014). The 4- to 5-day-old Arabidopsis seedlings were treated with 100 mM NaCl in BSM background. At 4 h and 24 h after salt treatment, the roots were washed with 10 mM Tris-HCl buffer and incubated in 25 µM 2′,7′-dichlorofluorescein diacetate (DCF-DA, D6883; Sigma) for 30 min at 30 °C. Following DCF-DA incubation, the amount of H2O2 produced in roots was assessed by visualizing fluores- cence intensity using a confocal microscope (Leica TCS SP5, Leica Microsystems). The Argon, visible laser power was set at 20%. Given that the H2O2 fluorescence intensity at 4 h was stronger than at 24 h time point, two different settings (and, hence, two different sets of controls) were used to resolve the signal. The acousto-optic tuneable filter (AOTF-488) was set at 10 % and 40 %, and the hybrid detector (HyD) gain was set at 19 and 120 for 4-h and 24-h time points, respectively. The software Leica Application Suite Advanced Fluorescence (LAS AF, Leica Microsystems) used to acquire images, and ImageJ (National Institutes of Health) was used to calculate the mean fluorescence intensity. Preparation of Arabidopsis seedlings for MIFE measurements Preparation of Arabidopsis seedlings for MIFE measurements The roots of an intact Arabidopsis seedling were immobilized and conditioned in a Petri dish containing 30 ml of BSM (basal salt medium; 1 mM KCl and 0.1 mM CaCl2, pH 5.5) for at least 30 min Jayakannan et al. 1868 Jayakannan et al. before commencing MIFE measurements (Jayakannan et al., 2011; Jayakannan et al., 2013). The Petri dish was then placed on the microscope stage of the MIFE system. Electrodes were positioned at either the distal elongation zone (180–300 µm from the root cap) or mature root zone (>2 mm from the root cap) as described in Bose et al. (2010a, b). Ion fluxes were measured under control conditions for 5 min before treatment application. Treatments (100 mM NaCl; 1 mM copper-ascorbate mix; 1 or 10 mM hydrogen peroxide) were applied by pipetting the required volume of treatment stock solu- tions into the bathing solution in the Petri dish. After addition, the bathing solution was thoroughly mixed by sucking into, and expel- ling from, a pipette approximately five times. The bathing solution was allowed to equilibrate for 1 min before recording ion fluxes under treatment conditions; hence, the time required for the stock addition and the establishment of the diffusion gradients is about 40 s (Shabala and Hariadi, 2005). Accordingly, flux measurements dur- ing the first minute after treatment applications were discarded from the analysis and appear as gaps in the figures. Transient flux kinetics of K+, H+, and Na+ were measured for specified times. The extent of salt-induced loss of cell viability was more severe in npr1-5 than nudt7 roots The roots of an intact Arabidopsis seedling were gently secured in a measuring chamber in a horizontal position using a Parafilm strip and small plastic blocks. The seedling was then placed in a 10-ml Perspex measuring chamber filled with 7 ml of BSM and pre-conditioned as described above. The specific details pertinent to microelectrode preparation, impalement into the epidermal cells of mature root zone, and data recording can be found in previous publications (Bose et al., 2013; Jayakannan et al., 2013). Once a stable membrane potential measurement was obtained for 1 min, salt treatment (100 mM NaCl) was imposed. The transient mem- brane potential kinetics was recorded up to 30 min after treatment commencement. The membrane potential values of eight indi- vidual seedlings were averaged for every genotype and treatment combination. To determine the effect of salinity on root cell viability, 4- to 5-day-old Arabidopsis seedlings were exposed to 100 mM NaCl for 1 or 12 h and then double stained with fluorescein diacetate– propidium iodide (FDA–PI; Fig. 2). Under the fluorescence microscope, viable cells fluoresced bright green, whereas dead/ damaged cells fluoresced bright red (Fig. 2). The Arabidopsis seedlings incubated in BSM alone (control) showed green fluo- rescence even after 12 h, suggesting the control roots were via- ble and healthy in our experimental solutions (Fig. 2). An hour of salt stress severely affected the viability of npr1- 5 root cells in the elongation and meristematic regions, with the wild-type roots also showing a few dead cells in the elon- gation zone (Fig. 2). However, no damage was observed in the roots of nudt7 mutant (Fig. 2). Prolonged salt exposure (12 h) increased the extent of the damage in the following order npr1-5 > Col-0 > nudt7. These results were consistent with the long-term salinity exposure data (Fig. 1 and Supplementary Fig. S1) and imply that roots of npr1-5 were sensitive to salt stress, whereas nudt7 was salt-tolerant. Results nudt7 and npr1-5 plants differ in salt sensitivity Similar to a previous report (Bose et al., 2013), 2 weeks of salt stress had a strong effect on plant growth, with fresh mass, dry mass, and water content all declining significantly and in a dose-dependent manner for all three Arabidopsis genotypes tested (Fig. 1 and Supplementary Fig. S1). This decline was smallest in nudt7 plants, followed by the wild type, and then by npr1-5 (most sensitive to salinity; Fig. 1 and Supplementary Fig. S1). Furthermore, under control conditions (i.e. no salt), the fresh (Fig. 1) and dry mass (Supplementary Fig. S1) were slightly lower in npr1-5 plants than the wild type and nudt7, but the difference was not statistically significant. At 150 mM NaCl, salt-sensitive npr1-5 had fewer radicles emerging than nudt7 and the wild type (Fig. 1). Membrane potential measurements Viability staining Root viability was assessed by fluorescein diacetate/propidium iodide double staining method as described in a previous publica- tion (Bose et al., 2014). NaCl-induced ion flux responses varied between nudt7 and npr1-5 Consistent with our previous observations on Arabidopsis roots (Jayakannan et al., 2011; Bose et al., 2013), salin- ity (100 mM NaCl) caused significant changes in net ion fluxes measured from the elongation and mature zones of Arabidopsis roots (Figs 3, 4 and 5). Acute salt stress caused significant K+ efflux from elonga- tion and mature root zones in all genotypes tested (Fig. 3). The peak K+ efflux was reached within 2 min after imposition of salt stress, followed by gradual recovery and stabilization 20 min later. Nearly a 4-fold difference in peak K+ fluxes was found between the elongation and the mature root zones in each Arabidopsis genotype (Fig. 3), implying the root elon- gation zone is more sensitive to salt stress than the mature root zone. Statistical analysis Statistical analysis The control plants were treated only with BSM; the image shown is the control plant after 12 h in BSM. (This figure is available in colour at JXB online.) Fig. 3. Transient K+ fluxes measured at the root elongation and the mature zones of 4- to 5-day-old Arabidopsis thaliana seedlings exposed to 100 mM salt stress. The insets were average K+ fluxes during 1-h exposure to 100 mM NaCl stress. Each point or bar represents mean±SEM of 8–12 seedlings. Different letters below the bars in the insets indicate significant differences. Among the three genotypes, the highest NaCl-induced K+ efflux was measured from npr1-5 roots in both the elongation and the mature root zones (–9269 ± 574 and –2096 ± 367 nmol m–2 s–1, respectively), whereas nudt7 showed about a 3-fold smaller peak K+ efflux (Fig. 3). The wild type had a peak K+ efflux in between the two mutants. In addition, the average K+ efflux over the first 60 min of salt treatment was about 9-fold (elongation zone) and 6-fold (mature zone) higher in salt-sen- sitive npr1-5 than salt-tolerant nudt7 mutant (Fig. 3 insets). both the elongation and mature root zones was highest in the npr1-5 mutant followed by Col-0 and was least in nudt7 (Fig. 4 insets). Na+ fluxes were measured in the mature root zone of the three Arabidopsis genotypes (Fig. 5A) using an improved Na+-selective resin (Jayakannan et al., 2011). Acute salt stress caused an immediate Na+ influx in Col-0 and npr1-5 (Fig. 5A). The peak Na+ influx was observed within minutes of salt addition and declined thereafter, but remained positive (influx) throughout the measurement period in npr1-5 and the wild type, while hovering around zero in nudt7 (Fig. 5A). The average Na+ flux measured during 1-h salt stress was about 28-fold higher in npr1-5 than nudt7 (Fig. 5A inset). p ( g ) Salinity-induced H+ fluxes also showed genotypic differ- ences, in both the elongation and mature root zones (Fig. 4). Under control conditions (no salt), a significantly higher net H+ influx was observed in the root elongation zone of the npr1-5 mutant in comparison with Col-0 and the nudt7 mutant (Fig. 4 top panel). Addition of 100 mM NaCl caused a significant increase in net H+ influx in the elongation zone of npr1-5 (58 ± 8.5 nmol m–2 s–1) and Col-0 (7.4 ± 4.4 nmol m–2 s–1; Fig. 4 top panel). Statistical analysis Data are reported as means±SEM. Statistical significance of mean values was determined using the standard LSD test at P≤0.05 level. Salicylic acid signalling during salinity stress \| 1869 -10000 -8000 -6000 -4000 -2000 0 0 5 10 15 20 25 30 35 Col-0 nudt7 npr1-5 -2200 -1700 -1200 -700 -200 0 5 10 15 20 25 30 35 Net K+ flux, nmol m-2 s-1 Mature zone Elongation zone Time, min b a c -900 -700 -500 -300 -100 Col-0 nudt7 npr1-5 nudt7 npr1-5 b a c -2000 -1600 -1200 -800 -400 0 Col-0 nudt7 npr1-5 nudt7 npr1-5 A B Fig. 3. Transient K+ fluxes measured at the root elongation and the mature zones of 4- to 5-day-old Arabidopsis thaliana seedlings exposed to 100 mM salt stress. The insets were average K+ fluxes during 1-h exposure to 100 mM NaCl stress. Each point or bar represents mean±SEM of 8–12 seedlings. Different letters below the bars in the insets indicate significant differences. Col-0 npr1-5 nudt7 Fig. 2. Viability staining images of 4- to 5-day-old Arabidopsis thaliana roots exposed to 100 mM salt stress. The seedlings were grown in basal salt medium (BSM) containing 0.4% (w/v) agar for 4–5 d, then pre- treated with 100 mM NaCl in BSM for 1 or 12 h, and double stained with fluorescein diacetate–propidium iodide for imaging under a fluorescence microscope. The control plants were treated only with BSM; the image shown is the control plant after 12 h in BSM. (This figure is available in colour at JXB online.) -10000 -8000 -6000 -4000 -2000 0 0 5 10 15 20 25 30 35 Col-0 nudt7 npr1-5 -2200 -1700 -1200 -700 -200 0 5 10 15 20 25 30 35 Net K+ flux, nmol m-2 s-1 Mature zone Elongation zone Time, min b a c -900 -700 -500 -300 -100 Col-0 nudt7 npr1-5 nudt7 npr1-5 b a c -2000 -1600 -1200 -800 -400 0 Col-0 nudt7 npr1-5 nudt7 npr1-5 A B Col-0 npr1-5 nudt7 35 Fig. 2. Viability staining images of 4- to 5-day-old Arabidopsis thaliana roots exposed to 100 mM salt stress. The seedlings were grown in basal salt medium (BSM) containing 0.4% (w/v) agar for 4–5 d, then pre- treated with 100 mM NaCl in BSM for 1 or 12 h, and double stained with fluorescein diacetate–propidium iodide for imaging under a fluorescence microscope. Statistical analysis By contrast, 100 mM NaCl addi- tion induced an initial H+ efflux in the elongation zone of the nudt7 mutant followed by recovery towards the steady state before salt treatment (Fig. 4 top panel). In the mature root zone (Fig. 4, bottom panel), NaCl increased H+ influx for all three genotypes with the following magnitude npr1-5 > Col-0 > nudt7 (Fig. 4, bottom panel). Similarly, the aver- age H+ influx (over the first 60 min after salt application) at 1870 \| Jayakannan et al. Each point or bar represents mean±SEM of 8–12 seedlings. Different letters above the bars in the insets indicate significant differences. -110 -100 -90 -80 -70 -60 -50 -40 -30 -20 -10 -2 3 8 13 18 23 28 Col-0 (n=8) nudt7 (n=8) npr1-5 (n=7) Time, min Net Na+ flux, nmol m-2 s-1 -5000 0 5000 10000 15000 20000 25000 30000 35000 40000 0 5 10 15 20 25 30 35 Col-0 nudt7 npr1-5 b c a -2000 0 2000 4000 6000 8000 10000 Col-0 nudt7 npr1-5 nudt7 npr1-5 mV A B Mature zone Mature zone -10 0 10 20 30 40 50 60 70 0 5 10 15 20 25 30 35 Col-0 nudt7 npr1-5 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 Col-0 nudt7 npr1-5 Elongation zone Mature zone Time, min Net H+ flux, nmol m-2 s-1 b b a 0 5 10 15 20 25 30 Col-0 nudt7 npr1-5 nudt7 npr1-5 b c a 0 5 10 15 Col-0 nudt7 npr1-5 nudt7 npr1-5 A B 10 Net Na+ flux, nmol m-2 s-1 -5000 0 5000 10000 15000 20000 25000 30000 35000 40000 0 5 10 15 20 25 30 35 Col-0 nudt7 npr1-5 b c a -2000 0 2000 4000 6000 8000 10000 Col-0 nudt7 npr1-5 nudt7 npr1-5 A Mature zone Net H+ flux, nmol m-2 s-1 -110 -100 -90 -80 -70 -60 -50 -40 -30 -20 -10 -2 3 8 13 18 23 28 Col-0 (n=8) nudt7 (n=8) npr1-5 (n=7) Time, min Mature zone B mV Fig. 4. Transient H+ fluxes measured at the root elongation and the mature zones of 4- to 5-day-old Arabidopsis thaliana seedlings exposed to 100 mM salt stress. The insets were average H+ fluxes during 1-h exposure to 100 mM NaCl stress. Each point or bar represents mean±SEM of 8–12 seedlings. Different letters above the bars in the insets indicate significant differences. Fig. 5. Transient (A) Na+ fluxes and (B) membrane potential dynamics measured at the mature root zone of 4- to 5-day-old Arabidopsis thaliana seedlings exposed to 100 mM salt stress. The inset was average Na+ fluxes during 1-h exposure to 100 mM NaCl stress. Each point or bar represents mean±SEM of 8–12 seedlings. Different letters above the bars in the inset indicate significant differences. 1870 \| Jayakannan et al. depolarization observed within minutes of NaCl treatment; approximately at the same time as the peak Na+ influx and K+ efflux (the magnitude of the former being greater than that of the latter) (Figs 3 and 5). Initial depolarization was followed by a substantial (10–20 mV) membrane repolari- zation, with the membrane potential reaching new steady- state values in all three Arabidopsis genotypes 20–30 min after salt application (Fig. 5B). Among the genotypes, the salt-sensitive npr1-5 showed the highest magnitude of mem- brane depolarization (to –15 ± 1 mV), whereas salt-tolerant nudt7 showed the least membrane depolarization (to –30 ± 1 mV) (Fig. 5B). A ≈25mV difference between nudt7 and npr1- 5 plants was maintained throughout the measurement period (Fig. 5B). signalling blockage had lower capacity to increase H2O2 pro- duction under salt stress, whereas nudt7 mutant showed sus- tained elevation in H2O2 production under salt stress at both time points. Shoot Na and K concentrations differed between nudt7 and npr1-5 during long-term salt exposure As expected, 25 d of growth in NaCl-supplemented MS media caused a substantial increase in the shoot Na+ concentration and a decrease in the shoot K+ concentration in all three Arabidopsis genotypes tested (Fig. 7). Under salt stress, nudt7 showed the lowest Na+ concentration in shoots followed by the wild type, whereas the npr1-5 mutant had the highest con- centration (Fig. 7A). In contrast, the shoot K+ concentration was the highest in the nudt7 mutant followed by the wild type and was lowest in the npr1-5 mutant (Fig. 7B) under either 50 or 100 mM NaCl stress. 1870 \| Jayakannan et al. -110 -100 -90 -80 -70 -60 -50 -40 -30 -20 -10 -2 3 8 13 18 23 28 Col-0 (n=8) nudt7 (n=8) npr1-5 (n=7) Time, min Net Na+ flux, nmol m-2 s-1 -5000 0 5000 10000 15000 20000 25000 30000 35000 40000 0 5 10 15 20 25 30 35 Col-0 nudt7 npr1-5 b c a -2000 0 2000 4000 6000 8000 10000 Col-0 nudt7 npr1-5 nudt7 npr1-5 mV A B Mature zone Mature zone Fig. 5. Transient (A) Na+ fluxes and (B) membrane potential dynamics measured at the mature root zone of 4- to 5-day-old Arabidopsis thaliana seedlings exposed to 100 mM salt stress. The inset was average Na+ fluxes during 1-h exposure to 100 mM NaCl stress. Each point or bar represents mean±SEM of 8–12 seedlings. Different letters above the bars in the inset indicate significant differences. -110 -100 -90 -80 -70 -60 -50 -40 -30 -20 -10 -2 3 8 13 18 23 28 Col-0 (n=8) nudt7 (n=8) npr1-5 (n=7) Time, min Net Na+ flux, nmol m-2 s-1 -5000 0 5000 10000 15000 20000 25000 30000 35000 40000 0 5 10 15 20 25 30 35 Col-0 nudt7 npr1-5 b c a -2000 0 2000 4000 6000 8000 10000 Col-0 nudt7 npr1-5 nudt7 npr1-5 mV A B Mature zone Mature zone Fig. 5. Transient (A) Na+ fluxes and (B) membrane potential dynamics measured at the mature root zone of 4- to 5-day-old Arabidopsis thaliana seedlings exposed to 100 mM salt stress. The inset was average Na+ fluxes during 1-h exposure to 100 mM NaCl stress. Each point or bar represents mean±SEM of 8–12 seedlings. Different letters above the bars in the inset indicate significant differences -10 0 10 20 30 40 50 60 70 0 5 10 15 20 25 30 35 Col-0 nudt7 npr1-5 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 Col-0 nudt7 npr1-5 Elongation zone Mature zone Time, min Net H+ flux, nmol m-2 s-1 b b a 0 5 10 15 20 25 30 Col-0 nudt7 npr1-5 nudt7 npr1-5 b c a 0 5 10 15 Col-0 nudt7 npr1-5 nudt7 npr1-5 A B Fig. 4. Transient H+ fluxes measured at the root elongation and the mature zones of 4- to 5-day-old Arabidopsis thaliana seedlings exposed to 100 mM salt stress. The insets were average H+ fluxes during 1-h exposure to 100 mM NaCl stress. Salt-induced H2O2 production was higher in nudt7 than npr1-5 In vivo imaging of H2O2 production in root tissue was done 4 h and 24 h after 100 mM NaCl addition (Fig. 6). The salt- induced H2O2 production was several folds higher at 4 h than 24 h in all the genotypes tested, necessitating specific settings (described in the Materials and methods section) to acquire images for each time point to avoid oversaturation and pho- tobleaching. Among the genotypes, mutant npr1-5 with SA nudt7 and npr1-5 differ in the magnitude of NaCl- induced depolarization of the plasma membrane The resting membrane potential in the mature zones of Arabidopsis roots was not significantly different among the three genotypes under control conditions (Fig. 5B). Adding 100 mM NaCl to the bathing medium resulted in highly significant (P≤0.01) membrane depolarization in all three Arabidopsis genotypes tested. The time-course of membrane potential changes (Fig. 5B) mirrored both Na+ (Fig. 5a) and K+ flux (Fig. 3) data, with the maximum membrane 1870 \| Jayakannan et al. Net ion fluxes influenced by oxidative stress differ between nudt7 and npr1-5 mutants Net ion fluxes influenced by oxidative stress differ between nudt7 and npr1-5 mutants treating 4- to 5-day-old seedlings in a hydroxyl-radical- producing medium (1 mM copper-ascorbate or 10 mM H2O2) for 1 h (Fig. 8). Fluorescence microscopy showed that 1-h exposure to hydroxyl radicals caused severe dam- age to the roots of npr1-5 and less so to the wild type Col-0 (Fig. 8). No damage was found in nudt7 mutant (Fig. 8). Furthermore, in npr1-5 treated with copper-ascorbate the damage was detected in the root tips as well as in the mature root part, whereas in Col-0 plants only the mature zone showed damage symptoms (Fig. 8). With respect to H2O2, the damage was smaller in Col-0 and nudt7 in comparison to npr1-5 (Fig. 8). The damage was detected only in the cortex of the mature roots of Col-0 and nudt7 (Fig. 8), whereas the whole roots were severely affected by H2O2 stress in npr1-5. treating 4- to 5-day-old seedlings in a hydroxyl-radical- producing medium (1 mM copper-ascorbate or 10 mM H2O2) for 1 h (Fig. 8). Fluorescence microscopy showed that 1-h exposure to hydroxyl radicals caused severe dam- age to the roots of npr1-5 and less so to the wild type Col-0 (Fig. 8). No damage was found in nudt7 mutant (Fig. 8). Furthermore, in npr1-5 treated with copper-ascorbate the damage was detected in the root tips as well as in the mature root part, whereas in Col-0 plants only the mature zone showed damage symptoms (Fig. 8). With respect to H2O2, the damage was smaller in Col-0 and nudt7 in comparison to npr1-5 (Fig. 8). The damage was detected only in the cortex of the mature roots of Col-0 and nudt7 (Fig. 8), whereas the whole roots were severely affected by H2O2 stress in npr1-5. Application of 1 mM of hydroxyl-radical-generating cop- per-ascorbate mix caused a large K+ efflux from the mature root zone of all three Arabidopsis genotypes (Fig. 9A). This hydroxyl-radical-induced K+ efflux was not instantaneous, but increased gradually over time, reaching a peak value 5 min after the commencement of the oxidative stress treatment in Col-0 and nudt7 and 10 min for npr1-5 (Fig. 9A). The magni- tude of K+ efflux was the lowest in nudt7 and the highest in npr1-5 (Fig. 9A; 2-fold difference; significant at P≤0.05). Salicylic acid signalling during salinity stress \| 1871 Because the hydrogen peroxide fluorescence was much higher at 4 h than at 24 h, different settings were used to acquire images to show difference between genotypes at each time point. (C, D) Quantification of fluorescence in the roots of the different genotypes after exposure to salt stress for the indicated times. Each bar represents mean±SEM of 8–12 seedlings. Different letters above the bars in the bar graphs indicate significant differences. (This figure is available in colour at JXB online.) drogen peroxide production in the root tissue of Arabidopsis thaliana seedlings after NaCl treatment. (A, B) Fig. 6. In vivo detection of hydrogen peroxide production in the root tissue of Arabidopsis thaliana seedlings after NaCl treatment. (A, B) Images of Arabidopsis thaliana seedling roots, after being exposed to the indicated salt concentrations for 4 or 24 h. Samples were stained with 2′,7′-dichlorofluorescein diacetate for imaging under a fluorescence microscope. Roots for treatments were taken from 4- to 5-day-old seedlings grown in basal salt medium (BSM) containing 0.4% (w/v) agar. Because the hydrogen peroxide fluorescence was much higher at 4 h than at 24 h, different settings were used to acquire images to show difference between genotypes at each time point. (C, D) Quantification of fluorescence in the roots of the different genotypes after exposure to salt stress for the indicated times. Each bar represents mean±SEM of 8–12 seedlings. Different letters above the bars in the bar graphs indicate significant differences. (This figure is available in colour at JXB online.) Salicylic acid signalling during salinity stress \| 1871 1871 Salicylic acid signalling during salinity stress \| 1871 Salicylic acid signalling during salinity stress \| 1871 5-day-old seedlings in a hydroxyl-radical- dium (1 mM copper-ascorbate or 10 mM (Fig. 8). Fluorescence microscopy showed ure to hydroxyl radicals caused severe dam Net ion fluxes influenced by oxidative stress differ between nudt7 and npr1-5 mutants Application of 1 mM of hydroxyl-radical-generating cop- d b d a d c 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 control 100 mM NaCl Col-0 nudt7 npr1-5 b b b a b b 0 1 2 3 4 5 6 Control 100 mM NaCl Col-0 nudt7 npr1-5 Average fluorescence (arb. units) 4 h 24 h A B C D ction of hydrogen peroxide production in the root tissue of Arabidopsis thaliana seedlings after NaCl treatment. (A, B) psis thaliana seedling roots, after being exposed to the indicated salt concentrations for 4 or 24 h. Samples were stained with scein diacetate for imaging under a fluorescence microscope. Roots for treatments were taken from 4- to 5-day-old seedlings grown in (BSM) containing 0.4% (w/v) agar. Because the hydrogen peroxide fluorescence was much higher at 4 h than at 24 h, different settings re images to show difference between genotypes at each time point. (C, D) Quantification of fluorescence in the roots of the different posure to salt stress for the indicated times. Each bar represents mean±SEM of 8–12 seedlings. Different letters above the bars in the e significant differences. (This figure is available in colour at JXB online.) at The University of Western Australia on April 6, 2016 http://jxb.oxfordjournals.org/ Downloaded from A B B A d b d a d c 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 control 100 mM NaCl Col-0 nudt7 npr1-5 b b b a b b 0 1 2 3 4 5 6 Control 100 mM NaCl Col-0 nudt7 npr1-5 Average fluorescence (arb. units) 4 h 24 h C D 6 D D b Fig. 6. In vivo detection of hydrogen peroxide production in the root tissue of Arabidopsis thaliana seedlings after NaCl treatment. (A, B) Images of Arabidopsis thaliana seedling roots, after being exposed to the indicated salt concentrations for 4 or 24 h. Samples were stained with 2′,7′-dichlorofluorescein diacetate for imaging under a fluorescence microscope. Roots for treatments were taken from 4- to 5-day-old seedlings grown in basal salt medium (BSM) containing 0.4% (w/v) agar. nudt7 and npr1-5 mutants vary in their oxidative stress tolerance The viability staining was used to evaluate the responses of Arabidopsis genotypes during oxidative stress by Net ion fluxes influenced by oxidative stress differ between nudt7 and npr1-5 mutants a c f a b e a d g 0 3 6 9 12 Control 50 mM NaCl 100 mM NaCl Col-0 nudt7 npr1-5 g e b g f c g d a 0 2 4 6 8 Shoot K (g kg-1 D W) Shoot Na (g kg-1 D W) A B Fig. 7. Effect of different NaCl treatment on concentrations of Na+ (A) and K+ (B) in Arabidopsis shoots after 25 d of growth in the full-strength MS medium with 2% w/v phytogel. Each bar represents mean±SEM. Different letters above the bars indicate significant differences. a c f a b e a d g 0 3 6 9 12 Control 50 mM NaCl 100 mM NaCl Col-0 nudt7 npr1-5 g e b g f c g d a 0 2 4 6 8 Shoot K (g kg-1 D W) Shoot Na (g kg-1 D W) A B Fig. 7. Effect of different NaCl treatment on concentrations of Na+ (A) and K+ (B) in Arabidopsis shoots after 25 d of growth in the full-strength MS medium with 2% w/v phytogel. Each bar represents mean±SEM. Different letters above the bars indicate significant differences. -1500 -1200 -900 -600 -300 0 0 5 10 15 20 25 30 35 Col-0 nudt7 npr1-5 1 mM Cu/Asc mix b a c -700 -500 -300 -100 Col-0 nudt7 npr1-5 Net K+ flux, nmol m-2 s-1 Time, min A B C b a c b a d -60 -50 -40 -30 -20 -10 0 Col-0 nudt7 npr1-5 1 mM H2O2 10 mM H2O2 nudt7 npr1-5 H2O2 H2O2 nudt7 npr1-5 Fig. 9. K+ fluxes in response to 1 mM Cu-ascorbate. (A) Transient K+ fluxes in response to 1 mM Cu-ascorbate applied after 5 min. (B) Average K+ fluxes during 1-h exposure to 1 mM Cu-ascorbate or (C) 1 or 10 mM H2O2 stress. K+ fluxes measured at the mature root zone of 4- to 5-day-old Arabidopsis thaliana seedlings. Each point or bar represents mean±SEM of 8–12 seedlings. Different letters below the bars indicate significant differences. Net ion fluxes influenced by oxidative stress differ between nudt7 and npr1-5 mutants a c f a b e a d g 0 3 6 9 12 Control 50 mM NaCl 100 mM NaCl Col-0 nudt7 npr1-5 g e b g f c g d a 0 2 4 6 8 Shoot K (g kg-1 D W) Shoot Na (g kg-1 D W) A B -1500 -1200 -900 -600 -300 0 0 5 10 15 20 25 30 35 Col-0 nudt7 npr1-5 1 mM Cu/Asc mix Time, min A A b a c -700 -500 -300 -100 Col-0 nudt7 npr1-5 Net K+ flux, nmol m-2 s-1 , B C b a c b a d -60 -50 -40 -30 -20 -10 0 Col-0 nudt7 npr1-5 1 mM H2O2 10 mM H2O2 nudt7 npr1-5 H2O2 H2O2 nudt7 npr1-5 Shoot K (g kg-1 D W) Fig. 7. Effect of different NaCl treatment on concentrations of Na+ (A) and K+ (B) in Arabidopsis shoots after 25 d of growth in the full-strength MS medium with 2% w/v phytogel. Each bar represents mean±SEM. Different letters above the bars indicate significant differences. Col-0 npr1-5 nudt7 Fig. 8. Viability staining of 4- to 5-day-old Arabidopsis thaliana roots exposed to 1 mM Cu-ascorbate or 10 mM H2O2 for 1 h. The seedlings were grown in basal salt medium (BSM) containing 0.4 % w/v agar for 4–5 d, were pre-treated with either 1 mM CuCl2+1 mM ascorbate or 10 mM H2O2 in the BSM background for 1 h and then stained with fluorescein diacetate–propidium iodide for observations under a fluorescence microscope. (This figure is available in colour at JXB online.) Col-0 npr1-5 npr1-5 Fig. 9. K+ fluxes in response to 1 mM Cu-ascorbate. (A) Transient K+ fluxes in response to 1 mM Cu-ascorbate applied after 5 min. (B) Average K+ fluxes during 1-h exposure to 1 mM Cu-ascorbate or (C) 1 or 10 mM H2O2 stress. K+ fluxes measured at the mature root zone of 4- to 5-day-old Arabidopsis thaliana seedlings. Each point or bar represents mean±SEM of 8–12 seedlings. Different letters below the bars indicate significant differences. measured over a 60-min Cu-ascorbate treatment period was 2-fold higher in npr1-5 than nudt7 (Fig. 9B).l The average K+ fluxes during 1-h exposure to either 1 or 10 mM H2O2 treatment revealed no significant dose-depend- ency in Arabidopsis genotypes nudt7 and Col-0 (Fig. 9C). However, the npr1-5 mutant had 2-fold greater K+ efflux at 10 than at 1 mM H2O2 (Fig. 9C). Net ion fluxes influenced by oxidative stress differ between nudt7 and npr1-5 mutants This mutant had greater K+ efflux than nudt7 and Col-0 regardless of the H2O2 concentra- tion used (Fig. 9C). nudt7 Though the initial H+ flux from the mature root zone of Arabidopsis was higher at 10 mM H2O2 than 1 mM H2O2, the steady state H+ flux (from 10 min onwards) is similar for differ- ent genotypes exposed to either concentration of H2O2 (Fig. 10). In general, the salt-sensitive npr1-5 mutant showed significantly higher (4- to 5-fold) H+ influx compared with the other two gen- otypes (nudt7 and Col-0) in either 1 or 10 mM H2O2 (Fig. 10). npr1-5 Net ion fluxes influenced by oxidative stress differ between nudt7 and npr1-5 mutants The K+ flux gradually recovered after reaching the peak, although it remained negative for the treatment duration in all three Arabidopsis genotypes (Fig. 9A). The average K+ efflux 1872 \| Jayakannan et al. 1872 \| Jayakannan et al. measured over a 60-min Cu-ascorbate treatment period was 2-fold higher in npr1-5 than nudt7 (Fig. 9B). The average K+ fluxes during 1-h exposure to either 1 or 10 mM H2O2 treatment revealed no significant dose-depend- ency in Arabidopsis genotypes nudt7 and Col-0 (Fig. 9C). However, the npr1-5 mutant had 2-fold greater K+ efflux at 10 than at 1 mM H2O2 (Fig. 9C). This mutant had greater K+ efflux than nudt7 and Col-0 regardless of the H2O2 concentra- tion used (Fig. 9C). Though the initial H+ flux from the mature root zone of Arabidopsis was higher at 10 mM H2O2 than 1 mM H2O2, the steady state H+ flux (from 10 min onwards) is similar for differ- ent genotypes exposed to either concentration of H2O2 (Fig. 10). In general, the salt-sensitive npr1-5 mutant showed significantly higher (4- to 5-fold) H+ influx compared with the other two gen- otypes (nudt7 and Col-0) in either 1 or 10 mM H2O2 (Fig. 10). Discussion Th NPR1 d d SA i lli i i l f N + Col-0 npr1-5 nudt7 Fig. 8. Viability staining of 4- to 5-day-old Arabidopsis thaliana roots exposed to 1mM Cu-ascorbate or 10mM H2O2 for 1h. The seedlings -1500 -1200 -900 -600 -300 0 0 5 10 15 20 25 30 35 Col-0 nudt7 npr1-5 1 mM Cu/Asc mix b a c -700 -500 -300 -100 Col-0 nudt7 npr1-5 Net K+ flux, nmol m-2 s-1 Time, min A B C b a c b a d -60 -50 -40 -30 -20 -10 0 Col-0 nudt7 npr1-5 1 mM H2O2 10 mM H2O2 nudt7 npr1-5 H2O2 H2O2 nudt7 npr1-5 Fig. 9. K+ fluxes in response to 1 mM Cu-ascorbate. (A) Transient K+ fluxes in response to 1 mM Cu-ascorbate applied after 5 min. (B) Average K+ fluxes during 1-h exposure to 1 mM Cu-ascorbate or (C) 1 or 10 mM H2O2 stress. K+ fluxes measured at the mature root zone of 4- to 5-day-old Arabidopsis thaliana seedlings. Each point or bar represents mean±SEM of 8–12 seedlings. Different letters below the bars indicate significant differences. Discussion Fig. 8. Viability staining of 4- to 5-day-old Arabidopsis thaliana roots exposed to 1 mM Cu-ascorbate or 10 mM H2O2 for 1 h. The seedlings were grown in basal salt medium (BSM) containing 0.4 % w/v agar for 4–5 d, were pre-treated with either 1 mM CuCl2+1 mM ascorbate or 10 mM H2O2 in the BSM background for 1 h and then stained with fluorescein diacetate–propidium iodide for observations under a fluorescence microscope. (This figure is available in colour at JXB online.) The NPR1-dependent SA signalling is pivotal for Na+ exclusion from roots and shoots The NPR1-dependent SA signalling is pivotal for Na+ exclusion from roots and shoots Maintaining relatively low Na+ concentration in shoots is an important trait for salt tolerance in glycophytes (Colmer et al., Salicylic acid signalling during salinity stress 1873 -1 2 5 8 11 14 17 0 5 10 15 20 25 30 35 Col-0 nudt7 npr1-5 -1 2 5 8 11 14 17 0 5 10 15 20 25 30 35 Col-0 nudt7 npr1-5 et H+ flux, nmol m-2 s-1 1 mM H2O2 10 mM H2O2 A B C -1 2 5 8 11 14 17 0 5 10 15 20 25 30 35 Col-0 nudt7 npr1-5 -1 2 5 8 11 14 17 0 5 10 15 20 25 30 35 Col-0 nudt7 npr1-5 Time, min Net H+ flux, nmol m-2 s-1 1 mM H2O2 10 mM H2O2 c c b c c a 0 1 2 3 4 5 6 7 Col-0 nudt7 npr1-5 1 mM H2O2 10 mM H2O2 nudt7 npr1-5 H2O2 H2O2 A B C Fig. 10. H+ fluxes in response to treatment with H2O2. (A, B) Transient H+ fluxes measured at the mature root zone of 4- to 5-day-old Arabidopsis thaliana seedlings in response to 1 or 10 mM H2O2. (C) Average H+ fluxes during 1-h exposure to 1 or 10 mM H2O2. Each point or bar represents mean±SEM of 8–12 seedlings. Different letters above the bars in bar graph indicate significant differences. -1 2 5 8 11 14 17 0 5 10 15 20 25 30 35 Col-0 nudt7 npr1-5 1 mM H2O2 A preventing the entry of Na+ into root tissue and its subsequent transport into the shoots. In contrast to npr1-5, the nudt7 mutant had the lowest Na+ influx into root tissue (Fig. 5a). This may be attributable to either decreased Na+ entry or enhanced Na+ extrusion via H+-ATPase- energized SOS1 (a Na+/H+ exchanger) activity in the plasma membrane (Cuin et al., 2011). Four lines of evidence favour the latter explanation for the nudt7 mutant. First, the initial Na+ entry into the epidermis of root tissue during acute salt stress is ther- modynamically passive and is poorly controlled in glycophytes (Tester and Davenport, 2003). The NPR1-dependent SA signalling is pivotal for Na+ exclusion from roots and shoots Second, the inherent stability of SOS1 mRNA is poor (with a half-life of only 10 min), and it was shown that exogenous H2O2 application increased the stability of SOS1 in a rapid (within 30 min) concentration-dependent man- ner (Chung et al., 2008). If this is the case, sustained elevation of H2O2 production in the root tissue of nudt7 mutant (Fig. 6) dur- ing salt stress is expected to result in improved SOS1 mRNA sta- bility. Thirdly, SOS1 transcripts were found to be higher in roots of the salt-tolerant mutant over-expressing haem oxygenase (EC 1.14.99.3) (Bose et al., 2013). Indeed, a 3-fold higher induc- tion of putative haem oxygenase (At1g69720) was found in the nudt7 mutant when grown under nutrient stress (Jambunathan et al., 2010). Finally, the nudt7 mutant showed either H+ efflux or reduced net H+ influx during acute salt stress (Fig. 4) in compari- son with the wild type and npr1-5 mutant, which is usually the result of enhanced H+-ATPase activity fuelling SOS1 operation (Bose et al., 2013; Jayakannan et al., 2013). Overall, the above results suggest that the nudt7 mutant has enhanced capacity to decrease both the loading of Na+ into the root tissue and the transport of Na+ into the shoot (Fig. 7A). 0 5 10 15 20 25 30 35 -1 2 5 8 11 14 17 0 5 10 15 20 25 30 35 Col-0 nudt7 npr1-5 et H+ flux, nmol m-2 s-1 10 mM H2O2 B C B Net H+ flux, nmol m-2 s-1 B at The University of Western Australia on A http://jxb.oxfordjournals.org/ Downloaded from Time, min Ne c c b c c a 0 1 2 3 4 5 6 7 Col-0 nudt7 npr1-5 1 mM H2O2 10 mM H2O2 nudt7 npr1-5 H2O2 H2O2 C a npr1-5 npr1-5 The NPR1-dependent SA signalling assists plants in retaining K+ during salt stress by controlling both depolarization-activated KOR and ROS-activated NSCC channels Fig. 10. H+ fluxes in response to treatment with H2O2. (A, B) Transient H+ fluxes measured at the mature root zone of 4- to 5-day-old Arabidopsis thaliana seedlings in response to 1 or 10 mM H2O2. (C) Average H+ fluxes during 1-h exposure to 1 or 10 mM H2O2. Each point or bar represents mean±SEM of 8–12 seedlings. Different letters above the bars in bar graph indicate significant differences. The NPR1-dependent SA signalling is pivotal for Na+ exclusion from roots and shoots Salinity stress has ionic, hyperosmotic, and oxidative stress components that severely hamper plant growth and produc- tivity. Apart from hyperosmotic stress, both the ionic stress through depolarization-activated KOR and the oxidative stress through ROS-activated non-selective cation channels (NSCC) exacerbate K+ loss, thereby depleting the cytosolic K+ pool available for metabolic functions, which eventually leads to cell death (Shabala and Cuin, 2008; Shabala, 2009). Hence, the magnitude of salt-induced K+ loss can be used as a measure of salt tolerance of diverse plant species, including Arabidopsis (Bose et al., 2013; Jayakannan et al., 2013). Acute salt stress in the study presented here resulted (as expected) in a K+ loss from both the elongation and mature root zones of all three genotypes tested (Fig. 3). However, the salt-induced K+ loss was lowest in the nudt7 mutant and highest in the npr1-5 mutant (Fig. 3), suggesting NPR1-dependent SA sig- nalling is critical for decreasing the K+ loss during salt stress. 2005; Munns and Tester, 2008). The main mechanisms employed by the glycophytes to minimize Na+ accumulation in shoots are linked to the enhanced capacity of plants to (i) restrict the ini- tial entry of Na+ ions into the root tissue, (ii) excrete Na+ from root tissue back into the rhizosphere, (iii) sequester Na+ inside the root vacuoles, and (iv) reduce the long-distance transport of Na+ into the shoots (Cuin et al., 2011). Given that Arabidopsis is a glycophyte, shoot Na+ concentration analysis and root Na+ flux measurements were employed to ascertain the operation of the above mechanisms in two SA-signalling mutants. The npr1- 5 mutant lacking NPR1-dependent SA-signalling recorded the highest Na+ influx into root tissue in comparison with the wild type and nudt7 mutant (Fig. 5A). If npr1-5 was efficient in sequestering Na+ in root vacuoles or excluding Na+ out of root cells, there would have been significant improvement in growth accompanied by reduction in the shoot Na+ concentration. However, poor growth (Fig. 1 and Supplementary Fig. S1) and viability of root cells (Fig. 2) along with the highest shoot Na+ concentration (Fig. Acknowledgements g An increase in the production of superoxide (Borsani et al., 2001), hydrogen peroxide (Xie et al., 2011), and hydroxyl rad- icals (Demidchik et al., 2010) was noted in Arabidopsis roots exposed to salt stress. These ROS species can promote K+ loss through NSCC channels (Demidchik et al., 2003; Zepeda- Jazo et al., 2011) and/or through KOR channels (Demidchik et al., 2010; Tran et al., 2013). The results here (Fig. 9) showed that hydroxyl radicals caused a severe K+ loss (about 15- to 20-fold higher) compared with up to 10 mM H2O2. Among the genotypes, the npr1-5 mutant showed a higher K+ loss than the wild type and nudt7 mutant under hydroxyl radical and hydrogen peroxide treatments (Fig. 9), suggesting npr1-5 was more sensitive to these ROS species in comparison with the wild type and nudt7 mutant. The viability staining results confirmed this, whereby a 1-h treatment with either hydroxyl radicals or 10 mM hydrogen peroxide affected root cells more severely in npr1-5 than in the nudt7 mutant (Fig. 8). The nudt7 mutant was able to increase the salt-induced H2O2 produc- tion in root tissue over a 24 h period, but the npr1-5 mutant was not (Fig. 6) suggesting NPR1 is a key regulator of salt- induced H2O2 production in plants. Because the nudt7 mutant produced more ROS than wild type and npr1-5 during salt stress, it is reasonable to assume that H2O2-induced K+ efflux would be greater in nudt7. However, in the exogenous H2O2 treatment (1 and 10 mM), the K+ efflux of nudt7 mutant did not differ from the wild type, and was lower than in the npr1-5 mutant (Fig. 9C). This suggests that the presence of an NPR1-mediated SA signalling component in the nudt7 mutant makes K+-efflux transporters insensitive to elevated H2O2 concentration during salt stress. Overall, these results provide evidence that (i) NPR1-mediated SA signalling is pivotal for H2O2 production during salt stress, and also for decreasing K+ loss through the NSCC and KOR channels Maheswari Jayakannan is a recipient of the Australian Postgraduate Award (APA) and University of Western Australia Postgraduate Award (UPA). This work was supported by the Australian Research Council grants to Z. Rengel (DP0988193 and DP130104825) and S. Shabala (DP0987402 and DP1094663), and Spanish MICINN grant (BFU2010-14873) to C. Poschenrieder. Supplementary data Supplementary data are available at JXB online Figure S1. Effect of salt stress on dry weight and water content of Arabidopsis thaliana seedlings grown in the full- strength MS medium with 2% w/v phytogel for two weeks. 1874 \| Jayakannan et al. activated by hydrogen peroxide and hydroxyl radicals, and (ii) the nudt7 mutant shows no response to hydrogen peroxide and is tolerant to hydroxyl radicals. 1/4 through H2O2-activated channels (Jayakannan et al., 2013). Superoxide (Tran et al., 2013) and hydroxyl radicals (Demidchik et al., 2010) can also induce K+ loss through the GORK channel. Thus, the contrasting capacity of nudt7 and npr1-5 mutants to retain K+ in roots (Fig. 3) and shoots (Fig. 7B) during salt stress may be underpinned by their dif- ferential K+ loss through KOR and/or ROS-activated NSCC channels. In summary, an npr1-5 mutant lacking the NPR1-dependent SA signalling was unable to control both the entry of Na+ into roots and its long-distance transport into the shoot, and to pre- vent K+ loss via depolarization-activated KOR and the ROS- activated NSCC channels during salt stress. As a result, the npr1-5 mutant was sensitive to salt stress. On the other hand, the constitutive expression of NPR1-dependent SA signalling enhanced the salt tolerance of a nudt7 mutant by controlling Na+ entry into the root tissue and subsequent transport to the shoot, as well as minimizing K+ loss during salt stress. In con- clusion, NPR1-dependent SA signalling is a crucial compo- nent of salt and oxidative stress tolerance in Arabidopsis. The entry of positively charged Na+ (Fig. 5A) and H+ (Fig. 4) ions into root tissue during acute 100 mM NaCl stress resulted in net depolarization of the plasma membrane in all three genotypes tested (Fig. 5b), implying that the bulk of the NaCl-induced K+ loss (Fig. 3) might have been through depolarization-activated KOR channels. Among the genotypes, H+ and Na+ uptake (Figs 4, 5A) as well as NaCl- induced membrane depolarization were highest in the npr1-5 mutant followed by the wild type, and were lowest in the nudt7 mutant. Moreover, approximately a 15–25 mV difference was observed between npr1-5 and nudt7 mutants (the latter being less depolarized) throughout the measurement period (Fig. 5B). Such a difference in depolarization voltage may be associated with a lower NaCl-induced K+ loss in nudt7 com- pared with npr1-5. It is evident that NPR1-mediated SA sig- nalling plays a key role in regulating the membrane potential during salt stress. The NPR1-dependent SA signalling is pivotal for Na+ exclusion from roots and shoots 7A) in comparison with the wild type and nudt7 mutant implied that the npr1-5 mutant was defective in In Arabidopsis, comparison of the depolarization-acti- vated KOR knock-out mutant gork1-1 with rbhoD (a mutant lacking ROS production via NADPH oxidase) during acute 100 mM NaCl stress revealed that 3/4 of K+ loss were medi- ated by depolarization-activated KOR and the remaining 1874 \| Jayakannan et al. Salicylic acid signalling during salinity stress \| 1875 Tran D, El-Maarouf-Bouteau H, Rossi M, Biligui B, Briand J, Kawano T, Mancuso S, Bouteau F. 2013. Post-transcriptional regulation of GORK channels by superoxide anion contributes to increases in outward-rectifying K+ currents. New Phytologist 198, 1039–1048. Tester M, Davenport R. 2003. Na+ tolerance and Na+ transport in higher plants. Annals of Botany 91, 503–527. Jambunathan N, Mahalingam R. 2006. Analysis of Arabidopsis growth factor gene 1 (GFG1) encoding a nudix hydrolase during oxidative signaling. Planta 224, 1–11. Vlot AC, Dempsey DMA, Klessig DF. 2009. Salicylic acid, a multifaceted hormone to combat disease. Annual Review of Phytopathology 47, 177–206. Jambunathan N, Penaganti A, Tang Y, Mahalingam R. 2010. Modulation of redox homeostasis under suboptimal conditions by Arabidopsis nudix hydrolase 7. BMC Plant Biology 10, 173. Jayakannan M, Babourina O, Rengel Z. 2011. Improved measurements of Na+ fluxes in plants using calixarene-based microelectrodes. Journal of Plant Physiology 168, 1045–1051. Wang D, Amornsiripanitch N, Dong X. 2006. A genomic approach to identify regulatory nodes in the transcriptional network of systemic acquired resistance in plants. PLoS Pathol 2, e123. Arabidopsis nudix hydrolase 7. BMC Plant Biology 10, 173. Jayakannan M, Babourina O, Rengel Z. 2011. Improved measurements of Na+ fluxes in plants using calixarene-based microelectrodes. Journal of Plant Physiology 168, 1045–1051. Wang H, Lu Y, Liu P, Wen W, Zhang J, Ge X, Xia Y. 2012. The ammonium/nitrate ratio is an input signal in the temperature-modulated, SNC1-mediated and EDS1-dependent autoimmunity of nudt6-2 nudt7. The Plant Journal , doi: 10.1111/tpj.12032. Jayakannan M, Bose J, Babourina O, Rengel Z, Shabala S. 2013. Salicylic acid improves salinity tolerance in Arabidopsis by restoring membrane potential and preventing salt-induced K+ loss via a GORK channel. Journal of Experimental Botany 64, 2255–2268. Wu Y, Zhang D, Chu JY, Boyle P, Wang Y, Brindle ID, De Luca V, Després C. 2012. The Arabidopsis NPR1 protein is a receptor for the plant defense hormone salicylic acid. Cell Reports 1, 639–647. Kraszewska E. 2008. The plant Nudix hydrolase family. Acta Biochimica Polonica 55, 663–671. Lee S, Kim SG, Park CM. 2010. Salicylic acid promotes seed germination under high salinity by modulating antioxidant activity in Arabidopsis. New Phytologist 188, 626–637. Xie Y-J, Xu S, Han B, Wu M-Z, Yuan X-X, Han Y, Gu Q, Xu D-K, Yang Q, Shen W-B. 2011. Evidence of Arabidopsis salt acclimation induced by up-regulation of HY1 and the regulatory role of RbohD-derived reactive oxygen species synthesis. Salicylic acid signalling during salinity stress \| 1875 Physiologia Plantarum 133, 651–669. Demidchik V, Shabala SN, Coutts KB, Tester MA, Davies JM. 2003. Free oxygen radicals regulate plasma membrane Ca2+- and K+-permeable channels in plant root cells. Journal of Cell Science 116, 81–88. Shabala S, Cuin TA, Prismall L, Nemchinov LG. 2007. Expression of animal CED-9 anti-apoptotic gene in tobacco modifies plasma membrane ion fluxes in response to salinity and oxidative stress. Planta 227, 189–197. Dempsey DMA, Vlot AC, Wildermuth CM, Klessig FD. 2011. Salicylic acid biosynthesis and metabolism. The Arabidopsis Book 9, e0156. Shabala S, Hariadi Y. 2005. Effects of magnesium availability on the activity of plasma membrane ion transporters and light-induced responses from broad bean leaf mesophyll. Planta 221, 56–65. Fu ZQ, Yan S, Saleh A, Wang W, Ruble J, Oka N, Mohan R, Spoel SH, Tada Y, Zheng N. 2012. NPR3 and NPR4 are receptors for the immune signal salicylic acid in plants. Nature 486, 228–232. Shah J, Kachroo P, Klessig DF. 1999. The Arabidopsis ssi1 mutation restores pathogenesis-related gene expression in npr1 plants and renders defensin gene expression salicylic acid dependent. The Plant Cell Online 11, 191–206. Ge X, Li GJ, Wang SB, Zhu H, Zhu T, Wang X, Xia Y. 2007. AtNUDT7, a negative regulator of basal immunity in Arabidopsis, modulates two distinct defense response pathways and is involved in maintaining redox homeostasis. Plant Physiology 145, 204–215. Shah J, Tsui F, Klessig DF. 1997. Characterization of salicylic acid- insensitive mutant (sai1) of Arabidopsis thaliana, identified in a selective screen utilizing the SA-inducible expression of the tms2 gene. Molecular Plant–Microbe Interactions 10, 69–78. Hao L, Zhao Y, Jin D, Zhang L, Bi X, Chen H, Xu Q, Ma C, Li G. 2012. Salicylic acid-altering Arabidopsis mutants response to salt stress. Plant and Soil 354, 81–95. Straus MR, Rietz S, Ver Loren van Themaat E, Bartsch M, Parker JE. 2010. Salicylic acid antagonism of EDS1-driven cell death is important for immune and oxidative stress responses in Arabidopsis. The Plant Journal 62, 628–640. Horváth E, Szalai G, Janda T. 2007. Induction of abiotic stress tolerance by salicylic acid signaling. Journal of Plant Growth Regulation 26, 290–300. Ishikawa K, Ogawa T, Hirosue E, Nakayama Y, Harada K, Fukusaki E, Yoshimura K, Shigeoka S. 2009. Modulation of the poly (ADP-ribosyl) ation reaction via the Arabidopsis ADP-ribose/NADH pyrophosphohydrolase, AtNUDX7, is involved in the response to oxidative stress. Plant Physiology 151, 741–754. Salicylic acid signalling during salinity stress \| 1875 Munns R, Tester M. 2008. Mechanisms of salinity tolerance. Annual Review of Plant Biology 59, 651–681. Bose J, Xie Y, Shen W, Shabala S. 2013. Haem oxygenase modifies salinity tolerance in Arabidopsis by controlling K+ retention via regulation of the plasma membrane H+-ATPase and by altering SOS1 transcript levels in roots. Journal of Experimental Botany 64, 471–481. Newman IA. 2001. Ion transport in roots: measurement of fluxes using ion-selective microelectrodes to characterize transporter function. Plant, Cell and Environment 24, 1–14. Cao Y, Zhang ZW, Xue LW, Du JB, Shang J, Xu F, Yuan S, Lin HH. 2009. Lack of salicylic acid in Arabidopsis protects plants against moderate salt stress. Zeitschrift fur Naturforschung C, Journal of Biosciences 64, 231–238. Ondrasek G, Rengel Z, Veres S. 2011. Soil salinisation and salt stress in crop production. In: Shanker A, Venkateswarlu B, eds. Abiotic stress in plants—mechanisms and adaptations. Rijeka: InTech, 171–190. Chung JS, Zhu JK, Bressan RA, Hasegawa PM, Shi H. 2008. Reactive oxygen species mediate Na+-induced SOS1 mRNA stability in Arabidopsis. The Plant Journal 53, 554–565. Parida AK, Das AB. 2005. Salt tolerance and salinity effects on plants: a review. Ecotoxicology and Environmental Safety 60, 324–349. Poór P, Szopkó D, Tari I. 2012. Ionic homeostasis disturbance is involved in tomato cell death induced by NaCl and salicylic acid. In Vitro Cellular and Developmental Biology—Plant 48, 377–382. Colmer TD, Munns R, Flowers TJ. 2005. Improving salt tolerance of wheat and barley: future prospects. Australian Journal of Experimental Agriculture 45, 1425–1443. Rengasamy P. 2006. World salinization with emphasis on Australia. Journal of Experimental Botany 57, 1017–1023. Cuin TA, Bose J, Stefano G, Jha D, Tester M, Mancuso S, Shabala S. 2011. Assessing the role of root plasma membrane and tonoplast Na+/H+ exchangers in salinity tolerance in wheat: in planta quantification methods. Plant, Cell and Environment 34, 947–961. Rivas-San Vicente M, Plasencia J. 2011. Salicylic acid beyond defence: its role in plant growth and development. Journal of Experimental Botany 62, 3321–3338. Demidchik V, Cuin TA, Svistunenko D, Smith SJ, Miller AJ, Shabala S, Sokolik A, Yurin V. 2010. Arabidopsis root K+-efflux conductance activated by hydroxyl radicals: single-channel properties, genetic basis and involvement in stress-induced cell death. Journal of Cell Science 123, 1468–1479. Shabala S. 2009. Salinity and programmed cell death: unravelling mechanisms for ion specific signalling. Journal of Experimental Botany 60, 709–711. Shabala S, Cuin TA. 2008. Potassium transport and plant salt tolerance. References Asensi-Fabado M, Munné-Bosch S. 2011. The aba3-1 mutant of Arabidopsis thaliana withstands moderate doses of salt stress by modulating leaf growth and salicylic acid levels. Journal of Plant Growth Regulation 30, 456–466. Attaran E, He SY. 2012. The long-sought-after salicylic acid receptors. Molecular Plant 5, 971–973. Bartsch M, Gobbato E, Bednarek P, Debey S, Schultze JL, Bautor J, Parker JE. 2006. Salicylic acid–independent ENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenase FMO1 and the nudix hydrolase NUDT7. The Plant Cell 18, 1038–1051. Blanco F, Salinas P, Cecchini N, Jordana X, Hummelen P, Alvarez M, Holuigue L. 2009. Early genomic responses to salicylic acid in Arabidopsis. Plant Molecular Biology 70, 79–102. Borsani O, Valpuesta V, Botella MA. 2001. Evidence for a role of salicylic acid in the oxidative damage generated by NaCl and osmotic stress in Arabidopsis seedlings. Plant Physiology 126, 1024–1030. Bose J, Babourina O, Shabala S, Rengel Z. 2010a. Aluminium-induced ion transport in Arabidopsis: the relationship between Al tolerance and root ion flux. Journal of Experimental Botany 61, 3163–3175. Bose J, Babourina O, Shabala S, Rengel Z. 2010b. Aluminum- dependent dynamics of ion transport in Arabidopsis: specificity of low pH and aluminum responses. Physiologia Plantarum 139, 401–412. Bose J, Shabala L, Pottosin I, Zeng F, Velarde-Buendía A-M, Massart A, Poschenrieder C, Hariadi Y, Shabala S. 2014. Kinetics of xylem loading, membrane potential maintenance, and sensitivity of K+-permeable channels to reactive oxygen species: physiological traits that differentiate salinity tolerance between pea and barley. Plant, Cell and Environment 37, 589–600. Salicylic acid signalling during salinity stress \| 1875 The Plant Journal 66, 280–292. Martinez-Beltran J, Manzur CL. 2005. Overview of salinity problems in the world and FAO strategies to address the problem. Proceedings of the International Salinity Forum. California: Riverside. Zepeda-Jazo I, Velarde-Buendía AM, Enríquez-Figueroa R, Bose J, Shabala S, Muñiz-Murguía J, Pottosin I. 2011. Polyamines interact with hydroxyl radicals in activating Ca2+ and K+ transport across the root epidermal plasma membranes. Plant Physiology 157, 2167–2180. Miura K, Sato A, Ohta M, Furukawa J. 2011. Increased tolerance to salt stress in the phosphate-accumulating Arabidopsis mutants siz1 and pho2. Planta 234, 1191–1199.
https://openalex.org/W4317514682	https://www.researchsquare.com/article/rs-2481423/latest.pdf	English	null	Negative feedback regulation of MAPK signaling is an important driver of CLL progression	Research Square (Research Square)	2,023	cc-by	8,951	Negative feedback regulation of MAPK signaling is an important driver of CLL progression Veronika Ecker Technical University of Munich Lisa Brandmeier Technical University of Munich Martina Stumpf University Hospital of Erlangen Piero Giansanti Technical University of Munich Aida Varela Moreira Utrecht University Lisa Pfeuffer Technical University of Munich M l F Lisa Pfeuffer Technical University of Munich Lisa Pfeuffer Technical University of Munich Marcel Fens Utrecht University Marcel Fens Utrecht University Junyan Lu European Molecular Biology Laboratory https://orcid.org/0000-0002-9211-0746 Bernhard Küster Technical University of Munich Thomas Engleitner Technical University of Munich Simon Heidegger Technical University of Munich Page 1/24 Maike Buchner (  maike.buchner@tum.de ) Technical University of Munich https://orcid.org/0000-0002-4196-096X DOI: https://doi.org/10.21203/rs.3.rs-2481423/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 2/24 Abstract Despite several potent targeted treatments for chronic lymphocytic leukemia (CLL), the clinical challenge of treating drug-resistant disease is emerging. In this study, we discovered that the dual-specific phosphatases DUSP1 and DUSP6 are required to negatively regulate Mitogen-activated protein kinases (MAPK) and thus counterbalance excessive MAPK activity to prevent apoptosis in CLL. We show that DUSP1 and DUSP6 are widely expressed in CLL and high expression of DUSP6 in CLL correlates with a poor clinical prognosis, which may reflect high levels of MAPK activity. Importantly, genetic deletion of the inhibitory phosphatase DUSP1 or DUSP6 and blocking DUSP1/6 function using a small molecule are toxic for CLL cells in vitro and in vivo. Analyzing downstream effects using global phospho-proteome approaches, we observed that acute activation of MAPK signaling by DUSP1/6 inhibition induces DNA damage response and thereby apoptotic cell death in CLL cells. This cell death is mediated by CHK kinases and can function independent of p53 and ATM, both effectors of DNA damage response, which are frequently deleted in CLL. Finally, we observed that DUSP1/6 inhibition is particularly effective against treatment-resistant CLL and therefore suggest transient DUSP1/6 inhibition as a promising novel treatment concept to eliminate drug-resistant CLL cells. Introduction Chronic lymphocytic leukemia (CLL) is among the most frequent B-cell malignancies in the Western world and is characterized by clonal accumulation of mature CD5-positive B-cells. Signaling from the B-cell receptor (BCR) plays a significant role in promoting CLL pathogenesis.1–4 Inhibiting BCR downstream effectors with the BTK inhibitor ibrutinib, among others, is highly effective in lowering the disease burden and has transformed CLL therapy in recent years. Despite these targeted therapies, CLL remains an incurable disease for most patients, and the emergence of drug-resistant CLL demands the exploration of alternative treatments.5 Activation of the mitogen-activated protein kinase (MAPK) pathway promotes CLL cell proliferation and survival and occurs downstream of surface receptors, including the BCR. In addition, the MAPK-signaling pathway is frequently activated by mutations in CLL, with 8.7% of patients carrying at least one mutation in MAPK-pathway genes such as NRAS, KRAS, and BRAF.6 Importantly, patients with MAPK-activating mutations in CLL comprise a distinct subgroup with poor clinical characteristics such as shorter treatment-free survival7, 8 and higher drug resistance 9–11. However, clinical studies of FDA-approved MEK-or BRAF-inhibitors for CLL have yielded poor outcomes.12 As a result, despite its importance in CLL etiology, elevated MAPK signaling is yet to be addressed therapeutically. P 3/24 The physiological outcome of MAPK signalling in B-cells varies greatly depending on the maturation stage and activation status.13 ERK1/2 activation, for example, is predominantly known for its pro-survival effects. However, ERK1/2 activation can trigger apoptotic processes in developing B-cells, which are important for eliminating autoreactive B-cells.14, 15 Thus, the actual role of MAPK activation is highly cell- and context-dependent. MAPK is negatively regulated by the expression of dual-specificity protein Page 3/24 Page 3/24 phosphatases (DUSPs), which specifically dephosphorylate phosphothreonine and -tyrosine residues that activate MAPKs. Two distinct members of the DUSP family, DUSP1 and DUSP6, inactivate ERK1/2 by dephosphorylation. Although DUSP6 is specific for ERK1/2, DUSP1 also dephosphorylates JNK and p38. Both DUSPs are dysregulated in various diseases, including cancer. They can either function as tumor suppressors by inhibiting ERK activation16, 17 or as oncogenes by assisting tumor cells in adapting to high levels of MAPK signaling18, 19. Although adaption to signaling levels is critical for a number of B-cell malignancies,19 the role of MAPK negative feedback via the DUSP family in CLL remains largely unknown. Introduction In the present study, we explored the importance of DUSP1- and DUSP6-mediated negative regulation of MAPK in CLL and identified DUSP6 overexpression as a marker of enhanced MAPK activity, and hence, poor clinical prognosis in CLL. We showed that DUSP1 and DUSP6 are necessary in CLL to avoid apoptosis via the DNA damage response pathway by counterbalancing excessive oncogenic MAPK activation. Therefore, the inhibition of DUSP1 and DUSP6 significantly reduced the CLL burden in preclinical models and was highly effective in the treatment of drug-resistant CLL. In the present study, we explored the importance of DUSP1- and DUSP6-mediated negative regulation of MAPK in CLL and identified DUSP6 overexpression as a marker of enhanced MAPK activity, and hence, poor clinical prognosis in CLL. We showed that DUSP1 and DUSP6 are necessary in CLL to avoid apoptosis via the DNA damage response pathway by counterbalancing excessive oncogenic MAPK activation. Therefore, the inhibition of DUSP1 and DUSP6 significantly reduced the CLL burden in preclinical models and was highly effective in the treatment of drug-resistant CLL. Expression of the MAPK negative regulator DUSP6 but not DUSP1 is associated with poor clinical prognosis in CLL and may reflect ERK activity. To investigate the potential role of negative feedback regulation of MAPK signaling in CLL, we first analyzed the mRNA expression levels of DUSP1 and DUSP6 in a total number of 210 CLL samples.20 We observed that both DUSP1 and DUSP6 were readily expressed in all tested CLL samples, with higher levels of DUSP6 and lower levels of DUSP1 in CLL cases carrying unmutated BCRs, which is one of the strongest predictive factors for poor disease outcome (Fig. 1a, b). We also evaluated whether DUSP1 and DUSP6 expression is associated with CLL prognosis. We correlated the mRNA expression levels of DUSP1 and DUSP6 with time to treatment (TTT) and overall survival (OS).20, 21 DUSP1 expression was not associated with clinical parameters (Suppl. Figure 1a, b), we found that high expression of DUSP6 in CLL samples was associated with adverse clinical outcomes, indicated by decreased TTT (Fig. 1c) and OS (Fig. 1d). Analysis of DUSP6 expression in the mutated or unmutated CLL subsets indicated that high DUSP6 levels are only significantly associated with shorter TTT in the mutated CLL subset, which could be due to the limited number of patients analyzed (Suppl. Figure 1c). However, we noticed that CLL samples with mutations in the KRAS and BRAF genes (causing high levels of MAPK signaling) expressed very high levels of DUSP6 (Fig. 1e). However, this was not the case when we compared DUSP1 levels in CLL samples with KRAS and BRAF mutations (Suppl. Figure 1d). As negative regulators are commonly induced in response to stimulation,22, 23 we hypothesized that the levels of DUSP6 in CLL cells are directly related to the degree of MAPK signaling 24. To test whether MAPK signaling affects DUSP6 expression levels, we analyzed the effects of inhibition of MAPK signaling by the small molecule inhibitor PD0325901 (PD901) on DUSP6 levels and observed a significant reduction in DUSP6 in conjunction with Page 4/24 Page 4/24 reduced ERK phosphorylation (Fig. 1f, g). Stimulation of CLL cells by microenvironmental factors engages the BCR and MAPK signaling pathways in CLL cells, so we also analyzed DUSP6 expression in matched samples collected from the peripheral blood and lymph nodes using publicly available data.25 Indeed, DUSP6 and DUSP1 expression was higher in lymph node-derived CLL cells than in the respective peripheral blood CLL samples (Fig. 1h-i). Expression of the MAPK negative regulator DUSP6 but not DUSP1 is associated with poor clinical prognosis in CLL and may reflect ERK activity. Taken together, we conclude that the MAPK-negative regulators DUSP1 and DUSP6 are commonly expressed in CLL and are dynamically upregulated in response to microenvironmental stimulation. High levels of DUSP6 expression, but not DUSP1 expression, define a subset of patients with poor prognosis, particularly those with KRAS and BRAF mutations. DUSP1/6 inhibition is toxic specifically for CLL cells To investigate the functional relevance of DUSP1- and DUSP6-mediated negative MAPK regulation in CLL, we tested the effects of the DUSP1/6- specific small molecule inhibitor BCI26 on CLL cells. We treated 21 primary CLL samples harvested from the peripheral blood of patients for 48h with increasing doses of BCI and found a dose-dependent induction of specific cell death in vitro (Fig. 2a). We calculated the specific cell death in vehicle-treated control CLL cells undergoing spontaneous apoptosis in cell culture.27 There was no significant difference in the response of CLL to BCI based on the mutational status of their BCR (Suppl. Figure 1e). In addition, cell death was specifically induced in CLL cells, whereas B-cells derived from healthy donors remained largely unaffected by DUSP1/6 inhibition at the tested concentrations (Fig. 2b). Furthermore, we confirmed the selective toxicity of the DUSP1/6 inhibitor to CLL using the CLL-derived cell line MEC-1 compared to most other tested B-cell and T-cell lymphoma cell lines (Fig. 2c). To test whether the pharmacological inactivation of DUSP1/6 is a potential therapeutic option for CLL, we also determined its effect on CLL progression in vivo, using a TCL1-driven mouse model.28 For in vivo treatments, we used a derivate of BCI (BCI-215) with reduced in vivo toxicity.29,30 We injected CLL-bearing splenocytes from aged TCL1-tg mice into WT recipients and confirmed CLL engraftment prior to treatment initiation (Fig. 2d). Then the mice were randomized to treatment or control groups and were treated with either BCI-215 (10 mg/kg) or vehicle daily for 10 consecutive days. This resulted in significantly reduced CLL content in the spleen and the peritoneal cavity, the major target organs for TCL1-derived murine CLL,28 in the BCI215 treated mice as compared to the control mice (Fig. 2e, f). Taken together, we showed that CLL cells are highly sensitive to DUSP1/6 inhibition, suggesting that negative regulation of the MAPK signaling pathway is important for their survival and disease progression in vitro and in vivo. Genetic disruption of DUSP1 or DUSP6 impairs CLL cell expansion To exclude the possibility that the cytotoxic effects of BCI observed in CLL are primarily due to off-target effects by the small-molecule inhibitor, we next performed genetic knockout experiments to determine the role of DUSP1 and DUSP6 in CLL cell survival. To this end, we generated CRISPR/Cas9-mediated Page 5/24 Page 5/24 knockout lines of CLL-derived MEC-1 cells. Upon successful gene knockout of DUSP1 or DUSP6 in expanded single clones, as verified by western blotting (Fig. 2g, h), we performed in vitro competitor growth assays to compare growth behaviors. For this, we mixed the expanded GFP-expressing clones (carrying either the DUSP1 or DUSP6 knockout or the CAS9 control vector) with untransduced MEC-1 cells to visualize changes in their growth behavior under similar growth conditions. While the respective Cas9/GFP + control cells were not affected (Fig. 2i, j), both DUSP1 and DUSP6 knockout clones were outcompeted by WT MEC-1 cells, confirming the selective disadvantage of CLL cells lacking functional DUSP1 or DUSP6. Our attempts to generate DUSP1/DUSP6 double-knockout MEC-1 lines failed, suggesting that these are lethal (not shown). Notably, DUSP1 and DUSP6 knockout cells showed a markedly diminished response to the DUSP1/6 inhibitor, indicating that the observed specific apoptosis was largely attributed to on-target effects (Fig. 2k). It should be noted that DUSP1 and DUSP6 knockout and CAS9 + control MEC-1 cells were expanded from single cells prior to these experiments, which may limit the observed effects and the reproducibility of the effects of acute DUSP1/6 inhibition. Nevertheless, these experiments confirm that the expression and activity of DUSP1 and DUSP6 are required for optimal CLL cell growth and survival. DUSP1/6 inhibition induces BCR/MAPK signaling in CLL cells followed by DNA damage response and apoptosis To assess DUSP1/6-mediated signaling events in CLL, we used an unbiased screening approach and performed global phospho-proteome analysis of BCI- or vehicle-treated CLL cells. We first analyzed the early time points of BCI treatment for total phospho-proteomic alterations to validate the on-target effects of DUSP1/6 inhibition and to study the initial events using patient-derived CLL cells (workflow depicted in Fig. 3a). After 10 min of DUSP1/6 inhibitor treatment, we observed significant changes in the phosphorylation profile (Fig. 3b; Suppl. Figure 2a, b). Phospho-proteomic pathway analysis revealed that the phospho-sites of the BCR and MAPK networks were significantly deregulated (Fig. 3c). As expected, we observed an increase in the phosphorylation of both ERK1 and ERK2 in DUSP1/6 inhibitor-treated CLL cells compared to control cells, confirming the on-target specificity of the inhibitor. Using western blotting, we validated the early phosphorylation of ERK1/2 upon treatment with the DUSP1/6 inhibitor in three additional primary CLL samples (Suppl. Figure 2c). To account for the heterogeneity among individual CLL patients in the signaling responses induced by DUSP1/6 inhibition, we used the MEC-1 cell line as a model to study the downstream signaling events of DUSP1/6 inhibition in CLL. Here, we performed a time-course experiment with subsequent phospho- proteome analysis using MEC-1 CLL cells, using later time points (0, 15, and 45 min) to gain insight into the molecular events associated with cell death. A heatmap with differentially phosphorylated proteins over all analyzed time points is shown, with four different clusters identified (Fig. 4a, b). We then performed kinase prediction analysis to identify the kinases that mediate the observed downstream effects (Fig. 4c). We identified the target sites of several kinases, including CDK1, PKCβ, and CK2α. As expected, ERK1/2 target sites were significantly phosphorylated, confirming ERK1/2 activation upon DUSP1/6 inhibition. However, when we analyzed which kinases were most active upon DUSP1/6 Page 6/24 Page 6/24 inhibition, we observed that HIPK2-regulated target sites were highly phosphorylated upon DUSP1/6 inhibitor treatment at both time points (Fig. 4d; analysis for the remaining kinases depicted in Suppl. Figure 2d, e). DUSP1/6 inhibition induces BCR/MAPK signaling in CLL cells followed by DNA damage response and apoptosis HIPK2 is a serine/threonine-protein kinase involved in p53/TP53-mediated cellular apoptosis and the DNA damage response (DDR) pathway31 and is a downstream target of the MAPK pathway.32 Consistent with this, subsequent pathway analysis revealed that the DDR pathway is one of the most strongly regulated pathways upon DUSP1/6 inhibitor treatment in MEC-1 cells, with activating phosphorylation events on ATF2, c-JUN, and CHK1/2 kinases (Fig. 4e).33,34 We validated the activation of these pathways following DUSP1/6 inhibition in primary CLL samples (Suppl. Figure 2f, g). In addition, we observed differential phosphorylation of molecules associated with the apoptotic pathway upon DUSP1/6 inhibition in our phospho-proteome screen, which is in line with the observed cell death upon DUSP1/6 inhibition in CLL (Fig. 4f). Taken together, our phospho-proteome analysis suggests that DUSP1/6 inhibition induces MAPK signaling, followed by activation of the DDR and apoptotic pathways in CLL Functional relevance of downstream signaling mediated by DUSP1/6 inhibition in CLL Our phospho-proteome and western blot analyses revealed enhanced activation of the MAPK signaling pathway upon DUSP1/6 inhibition. To determine whether MAPK activation is associated with cell death induced by DUSP1/6 inhibition in CLL, we evaluated whether preventing ERK1/2 activation would mitigate the apoptotic effects of DUSP1/6 inhibition. Although MEK inhibition itself is toxic to CLL cells,35 we observed that co-treatment with the MEK inhibitor PD901 partially reversed the induction of specific cell death by the DUSP1/6 inhibitor (Fig. 5a), indicating that ERK activation contributes to DUSP1/6- mediated cell death. Based on the activation of the DDR pathway observed in our phospho-proteome screen, we investigated whether DUSP1/6 inhibition promotes DNA damage in CLL cells. To this end, we analyzed the phosphorylation of γH2AX, one of the earliest cellular responses to DNA double-strand breaks, using flow cytometry. Indeed, phospho-γH2AX levels increased upon DUSP1/6 inhibitor treatment as compared to the control patient-derived CLL cells in a dose-dependent manner (Fig. 5b), while isotype control-stained cells did not show increased fluorescence (Suppl. Figure 3a). It should be noted that γH2AX phosphorylation occurs only after 1–4 hours of DUSP1/6 inhibitor treatment and was therefore not detected in our global phospho-proteome analysis, where we used early time points to avoid secondary effects on high levels of cell death. To determine whether the DNA damage response was a consequence of increased MAPK signaling induced by DUSP1/6 inhibition, we analyzed the phosphorylation of γH2AX with and without pretreatment with the MEK-inhibitor PD901 in MEC1, OSU- CLL, and EHEB CLL cell lines. We observed that PD901 treatment significantly reduced the DUSP1/6- mediated upregulation of phospho-γH2AX in multiple independent experiments (Fig. 5c). In addition, we analyzed whether DUSP1/6 inhibition induces classical apoptosis in CLL, as indicated by phospho-proteome analysis. Annexin V staining of primary human CLL cells treated with DUSP1/6 inhibitor revealed strong exposure of phosphatidylserine on the outer membrane (Fig. 5d). Consistent with this, co-treatment with the pan-caspase inhibitor QVD (or Emericasan) significantly reduced the toxic Page 7/24 Page 7/24 Page 7/24 effect of DUSP1/6 inhibition alone (Fig. 5e, Suppl. Figure 3b). Absolute CLL cell viability increased upon caspase inhibition by reducing spontaneous apoptosis in cultured CLL cells (Suppl. Figure 3c). Next, we investigated whether the observed activation of the DNA damage response pathway promoted the cytotoxic effects of DUSP1/6 inhibition in CLL cells. Functional relevance of downstream signaling mediated by DUSP1/6 inhibition in CLL In addition, p53, a major effector in the induction of apoptosis as a consequence of DNA damage, seems to be dispensable for mediating DUSP1/6 inhibition-promoted cell death, as the MEC-1 CLL cell line, which carries both 17p deletion (Del17p) and TP53 mutations, is sensitive to DUSP1/6 inhibition (shown in Fig. 2c). Taken together, our analysis confirmed the functional relevance of the DDR pathway in apoptosis induction in CLL cells upon DUSP1/6 inhibition, which is at least partially mediated by the activation of CHK1/2 and functions in the absence of ATM or p53 activity. concomitant treatment of CLL with DUSP1/6 and CHK1/2 inhibitors ameliorated the toxic effect of DUSP1/6 inhibition alone (Fig. 5f). The CHK1 inhibitor similarly reduced cell death induced by DUSP1/6 inhibition (Suppl. Figure 3e). Nevertheless, ATM inhibition did not significantly alter the effects of DUSP1/6 inhibition on CLL cell survival, indicating that this pathway is less important in the downstream response (Suppl. Figure 3f). In addition, p53, a major effector in the induction of apoptosis as a consequence of DNA damage, seems to be dispensable for mediating DUSP1/6 inhibition-promoted cell death, as the MEC-1 CLL cell line, which carries both 17p deletion (Del17p) and TP53 mutations, is sensitive to DUSP1/6 inhibition (shown in Fig. 2c). Taken together, our analysis confirmed the functional relevance of the DDR pathway in apoptosis induction in CLL cells upon DUSP1/6 inhibition, which is at least partially mediated by the activation of CHK1/2 and functions in the absence of ATM or p53 activity. Functional relevance of downstream signaling mediated by DUSP1/6 inhibition in CLL Therefore, we investigated whether inhibition of CHK1/2 kinases, critical effectors of the DDR pathway (which were also differentially phosphorylated in our MEC-1 phospho-proteome screen) reduced the cytotoxic effects of DUSP1/6 inhibition. CHK1/2 inhibition alone was not toxic to the CLL cells at the tested concentrations (Suppl. Figure 3d), concomitant treatment of CLL with DUSP1/6 and CHK1/2 inhibitors ameliorated the toxic effect of DUSP1/6 inhibition alone (Fig. 5f). The CHK1 inhibitor similarly reduced cell death induced by DUSP1/6 inhibition (Suppl. Figure 3e). Nevertheless, ATM inhibition did not significantly alter the effects of DUSP1/6 inhibition on CLL cell survival, indicating that this pathway is less important in the downstream response (Suppl. Figure 3f). In addition, p53, a major effector in the induction of apoptosis as a consequence of DNA damage, seems to be dispensable for mediating DUSP1/6 inhibition-promoted cell death, as the MEC-1 CLL cell line, which carries both 17p deletion (Del17p) and TP53 mutations, is sensitive to DUSP1/6 inhibition (shown in Fig. 2c). Taken together, our analysis confirmed the functional relevance of the DDR pathway in apoptosis induction in CLL cells upon DUSP1/6 inhibition, which is at least partially mediated by the activation of CHK1/2 and functions in the absence of ATM or p53 activity. effect of DUSP1/6 inhibition alone (Fig. 5e, Suppl. Figure 3b). Absolute CLL cell viability increased upon caspase inhibition by reducing spontaneous apoptosis in cultured CLL cells (Suppl. Figure 3c). Next, we investigated whether the observed activation of the DNA damage response pathway promoted the cytotoxic effects of DUSP1/6 inhibition in CLL cells. Therefore, we investigated whether inhibition of CHK1/2 kinases, critical effectors of the DDR pathway (which were also differentially phosphorylated in our MEC-1 phospho-proteome screen) reduced the cytotoxic effects of DUSP1/6 inhibition. CHK1/2 inhibition alone was not toxic to the CLL cells at the tested concentrations (Suppl. Figure 3d), ( pp g ) concomitant treatment of CLL with DUSP1/6 and CHK1/2 inhibitors ameliorated the toxic effect of DUSP1/6 inhibition alone (Fig. 5f). The CHK1 inhibitor similarly reduced cell death induced by DUSP1/6 inhibition (Suppl. Figure 3e). Nevertheless, ATM inhibition did not significantly alter the effects of DUSP1/6 inhibition on CLL cell survival, indicating that this pathway is less important in the downstream response (Suppl. Figure 3f). Discussion In this study, we discovered a previously unknown role for DUSP1 and DUSP6 in limiting MAPK signaling in CLL, thereby preventing apoptosis. We discovered that high mRNA expression of the negative regulator DUSP6 is linked to a poor clinical outcome in CLL. Both DUSP1 and DUSP6 expression are necessary for maintaining optimal CLL cell proliferation. Inhibition of DUSP1 and DUSP6 phosphatase activities limits CLL cell survival in vitro and disease development in vivo. Mechanistically, we discovered that inhibiting DUSP1/6 triggers activation of the BCR/MAPK signaling pathway, causing DNA damage-mediated apoptosis. This pro-apoptotic effect of DUSP1/6 inhibition was strong in drug-resistant CLL. Therefore, we identified the MAPK negative regulators DUSP1 and DUSP6 as novel treatment targets, particularly for drug-resistant CLL. By acting as negative regulators of the MAPK signaling pathway, dual-specific phosphatases can restrict tumor growth and promote carcinogenesis and drug resistance.39 Understanding the downstream processes of DUSP1/6 inhibition should aid in determining malignancies that are responsive to the drug. We used an unbiased phospho-proteome screen to determine the cause of cell death in response to DUSP1/6 inhibition in CLL. ERK1/2 activation was found to be important for inducing CLL apoptosis upon DUSP1/6 inhibition, which may appear surprising as ERK1/2 activity is most commonly associated with cell proliferation and survival. However, active ERK1/2 has also been linked to cell death during negative selection of B-cells to prevent autoimmunity.14 We hypothesized that CLL-specific checkpoints that limit B-cell growth stem from the inherent sensitivity of B-cells to strong BCR/MAPK signaling that mimics autoimmunity, resulting in the indolent nature of the disease. Consequently, CLL cells die rather than proliferate rapidly when MAPK signaling is induced to physiological levels. In contrast to DUSP6, DUSP1 mRNA levels do not correlate with disease progression, which could be due to the regulation of DUSP1 at the post-transcriptional level.40 Interestingly, Richter’s transformation is associated with an increased mutation frequency in the MAPK signaling pathway and co-occurring alterations in genes involved in the DNA damage response pathway.41 For example, loss of CHK1 and ATR was frequently observed in Richter transformed cases, but not in indolent CLL.41 We found that both molecules were strongly activated by DUSP1/6 inhibition and potential promoters of DUSP1/6 inhibition-mediated cell death in CLL. This suggests that growth-limiting checkpoints are lost, for example, by deletion of important DNA damage response mediators, as CLL transforms into aggressive lymphoma, where robust and sustained signaling is well tolerated. DUSP1/6 inhibition is effective in drug resistant CLL Similar to the MEC-1 cell line, primary CLL cells frequently harbor genetic alterations in the DDR pathway. Up to 8% of chemotherapy-naïve patients carry Del17p, and up to 80% of these carry mutations in TP53 on the second allele.36, 37 This loss of functional p53 is associated with resistance to chemotherapeutic agents. In addition, ATM is frequently inactivated in CLL and is associated with reduced apoptosis induction in response to chemotherapeutic agents.38 To determine whether genetic alterations in the DDR pathway affect the response rate to DUSP1/6 inhibition in CLL, we compared the cytotoxic response towards DUSP1/6 inhibition in CLL samples carrying p53 mutations or Del17p or Del11q, leading to a loss of functional p53 or ATM, respectively, as compared to WT p53 or ATM expressing cases. Although there was a minor reduction in the mean cell death induction, there was no significant difference in the cytotoxic response to DUSP1/6 inhibition between cases with and without functional p53/ATM (Fig. 5g). Although this analysis is based on a limited number of samples with the respective genetic alterations, it indicates that DUSP1/6 inhibition remains highly effective in killing CLL cells carrying mutations that disrupt a functional ATM/p53-mediated DDR. Based on the clinical need to identify novel treatment options for clinically ibrutinib-refractory CLL, we compared the effects of DUSP1/6 inhibition on primary CLL in treatment-naïve patients with ibrutinib- refractory cases. Here, we found that ibrutinib-resistant CLL cells remained sensitive to BCI treatment with similar induction of specific cell death as compared to treatment-naïve CLL cells analyzed in parallel (Fig. 5h). Taken together, we showed that DUSP1/6 inhibition is highly effective in inducing cytotoxicity in all tested CLL subsets and may be particularly useful for treating treatment-resistant and refractory CLL. Page 8/24 Page 8/24 Discussion Therefore, the vulnerability of CLL cells to DUSP1/6 inhibition is likely characterized by effective CHK- and/or ATR-mediated apoptosis in response to MAPK hyperactivation. Further understanding of the biology of Richter’s transformation will help clarify this. Drug resistance is a major obstacle to the treatment of CLL. Despite high initial response rates, a considerable percentage of patients receiving chemotherapy or targeted therapies experience relapse with progressive and refractory disease. Drug resistance has been linked to the mutation of specific drug- binding sites or the activation of alternative pro-survival pathways, including the MAPK signaling pathway.9–11 In addition, particular genetic abnormalities are associated with drug resistance to Page 9/24 Page 9/24 Page 9/24 chemotherapeutic treatments, such as the deletion of ATM or p53, which are important molecules in DNA damage-mediated apoptosis 36, 38. Although we observed that the DNA damage response was also involved in DUSP1/6 inhibitor-mediated cytotoxicity, DUSP1/6 suppression efficiently killed p53-mutated and ATM-deleted CLL, suggesting that alternative pathways that can induce DDR-mediated cell death, such as CHK1 and/or ATR activation, remain intact.42 Activation of ERK1/2, JNK, and p38 determines the sensitivity to therapy in a variety of cancers, including CLL43,44 and resistance emerging from patients treated with the PI3K inhibitor idelalisib results in elevated MAPK signaling to counteract PI3K pathway inhibition.45 Based on our findings, we hypothesize that cells with high levels of ERK signaling would be equally, if not more, vulnerable to DUSP1/6 inhibition, rendering DUSP1/6 inhibition an interesting approach for drug-resistant CLL. Negative regulation of MAPK via DUSP1 (via JNK suppression) also promotes drug resistance in cancer entities,46 including osteosarcoma,47 lung cancer cells,48 and ovarian cancer.49 This JNK regulation may contribute to the toxicity induced in CLL by DUSP1/6 inhibition. In clinical patient care, limited responses against the BTK inhibitor ibrutinib are highly relevant; therefore, we investigated whether DUSP1/6 inhibition is also effective against ibrutinib-refractory cases. Based on our findings, these refractory CLL cells are equally vulnerable to DUSP1/6 inhibition in treatment-naïve patients. Therefore, DUSP1/6 inhibition may be equally effective in treating drug-resistant CLL. Taken together, our findings reveal that in CLL, negative control of MAPK signaling is required to avoid DNA damage response-mediated cell death. We show that high DUSP6 expression is associated with poor clinical outcomes in CLL and may indicate high MAPK signaling, for example, induced by activating mutations of NRAS or BRAF. Discussion Furthermore, we demonstrated that inhibiting the activity of DUSP1/6 phosphatases causes DNA damage and cell death in CLL, and that treatment-resistant CLL cases remain sensitive to DUSP1/6 inhibition. As a result, we propose intermittent DUSP1/6 inhibition as a novel approach for CLL therapy, either in combination with kinase inhibition, to potentially boost the efficacy of both solo treatments or to treat kinase inhibitor-resistant disease. ACKNOWLEGMENTS: We would like to thank Tanja Neumeier and Nicole Beck for excellent technical support. This work was funded by the German Cancer Aid (Deutsche Krebshilfe, Max Eder Grant to MB, Project ID:70114720), German Research Foundation (Deutsche Forschungsgemeinschaft, DFG) – Project-ID 360372040 – SFB 1335/P02 awarded to MB, P01 to JR, and Wilhelm Sander Foundation (Project ID 2018.111.1 to MB). AUTHOR CONTRIBUTIONS: Veronika Ecker and Lisa Brandmeier: designed and performed the research; Martina Stumpf: designed and performed the research; Piero Giansanti: designed and performed the research; Aida Varela Moreira: designed and performed the research; Lisa Pfeuffer: designed and performed the research; Junyan Lu: analyzed the data; Marcel H. A. M. Fens: designed and performed the research; Bernhard Küster: designed the research; Thomas Engleitner: analyzed the data. Simon Heidegger: contributed CLL samples; Ingo Ringshausen: designed the research; Thorsten Zenz: contributed CLL samples; Clemens-Martin Wendtner: contributed CLL samples; Markus Müschen: designed the research; Jürgen Ruland: designed the research; Maike Buchner: designed the research, performed the research, and wrote the paper. DATA AVAILABILITY STATEMENT: The datasets generated during and analyzed during the current study are available from the corresponding author on reasonable request. Phospho-proteome data supporting this publication are deposited in the ProteomeXchange Consortium with the dataset identifier PXD032039. Methods Human subjects. Primary CLL samples were obtained from the peripheral blood of patients treated at the Klinikum rechts der Isar or München Klinik Schwabing, Munich, Germany, or the National Center for Tumor Diseases, Heidelberg, Germany (Suppl. Table 1). Patient sampling was approved by the local ethics committee, and informed consent was obtained from all patients. Healthy donor-derived blood samples were obtained from Bavarian Red Cross (Munich, Germany). Inhibitor treatment and subsequent analysis. Primary patient-derived CLL cells, MEC-1 cells, B- and T-cell lymphoma, or healthy peripheral blood MACS-isolated B-cells were seeded at 200.000 cells/well in 96- well-plates or 500.000 cells in 24-well plates and treated with BCI (Axon Medchem) alone or in combination with the indicated inhibitors. Viability was analyzed by flow cytometry. All antibodies used are listed in Suppl. Table 2. Please refer to the Supplemental Data for further details. Page 10/24 Page 10/24 splanted 2x10e7 splenocytes from aged TCL1-tg mice 28 into C57BL/6 wt Mice. We transplanted 2x10e7 splenocytes from aged TCL1-tg mice 28 into C57BL/6 wt immunocompetent mice and waited for the detection of murine CLL cells in the peripheral blood. BCI-215 treatment was initiated daily for a total of 10 doses, and CLL content in the spleen and peritoneal cavity was determined thereafter. Please refer to the Supplemental Data for details. Phospho-proteome screen. Freshly isolated primary CLL cells were treated with BCI for 3 and 10 min; MEC-1 cells were incubated for 24 h at a cell density of 1x10e6/ml and then treated with BCI for 15 and 45 min before isolating protein. A comprehensive description of the sample processing and subsequent analysis is provided in the Supplemental Data. All other methods are described in detail in the Supplemental Data. COMPETING INERESTS: The authors declare no competing financial interests References Page 11/24 1. Duhren-von Minden M, Ubelhart R, Schneider D, Wossning T, Bach MP, Buchner M, et al. Chronic lymphocytic leukaemia is driven by antigen-independent cell-autonomous signalling. Nature 2012 Sep 13; 489(7415): 309-312. 2. Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK. Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 1999 Sep 15; 94(6): 1848-1854. 2. Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK. Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 1999 Sep 15; 94(6): 1848-1854. 3. Mauerer K, Zahrieh D, Gorgun G, Li A, Zhou J, Ansen S, et al. Immunoglobulin gene segment usage, location and immunogenicity in mutated and unmutated chronic lymphocytic leukaemia. British journal of haematology 2005 May; 129(4): 499-510. 3. Mauerer K, Zahrieh D, Gorgun G, Li A, Zhou J, Ansen S, et al. Immunoglobulin gene segment usage, location and immunogenicity in mutated and unmutated chronic lymphocytic leukaemia. British journal of haematology 2005 May; 129(4): 499-510. 4. Messmer BT, Albesiano E, Efremov DG, Ghiotto F, Allen SL, Kolitz J, et al. Multiple distinct sets of stereotyped antigen receptors indicate a role for antigen in promoting chronic lymphocytic leukemia. The Journal of experimental medicine 2004 Aug 16; 200(4): 519-525. 4. Messmer BT, Albesiano E, Efremov DG, Ghiotto F, Allen SL, Kolitz J, et al. Multiple distinct sets of stereotyped antigen receptors indicate a role for antigen in promoting chronic lymphocytic leukemia. The Journal of experimental medicine 2004 Aug 16; 200(4): 519-525. 5. Kittai AS, Woyach JA. Resistance Mechanisms to Targeted Agents in Chronic Lymphocytic Leukemia. Cancer J 2019 Nov/Dec; 25(6): 428-435. 5. Kittai AS, Woyach JA. Resistance Mechanisms to Targeted Agents in Chronic Lymphocytic Leukemia. Cancer J 2019 Nov/Dec; 25(6): 428-435. 6. Landau DA, Tausch E, Taylor-Weiner AN, Stewart C, Reiter JG, Bahlo J, et al. Mutations driving CLL and their evolution in progression and relapse. Nature 2015 Oct 22; 526(7574): 525-530. 6. Landau DA, Tausch E, Taylor-Weiner AN, Stewart C, Reiter JG, Bahlo J, et al. Mutations driving CLL and their evolution in progression and relapse. Nature 2015 Oct 22; 526(7574): 525-530. 7. Gimenez N, Martinez-Trillos A, Montraveta A, Lopez-Guerra M, Rosich L, Nadeu F, et al. Mutations in the RAS-BRAF-MAPK-ERK pathway define a specific subgroup of patients with adverse clinical features and provide new therapeutic options in chronic lymphocytic leukemia. immunology 2011 May; 12(5): 425-433. 15. Seger R, Krebs EG. The MAPK signaling cascade. FASEB J 1995 Jun; 9(9): 726-735. 16. Zhang Z, Kobayashi S, Borczuk AC, Leidner RS, Laframboise T, Levine AD, et al. Dual specificity phosphatase 6 (DUSP6) is an ETS-regulated negative feedback mediator of oncogenic ERK signaling in lung cancer cells. Carcinogenesis 2010 Apr; 31(4): 577-586. 17. Furukawa T, Sunamura M, Motoi F, Matsuno S, Horii A. Potential tumor suppressive pathway involving DUSP6/MKP-3 in pancreatic cancer. Am J Pathol 2003 Jun; 162(6): 1807-1815. 18. Ramkissoon A, Chaney KE, Milewski D, Williams KB, Williams RL, Choi K, et al. Targeted Inhibition of the Dual Specificity Phosphatases DUSP1 and DUSP6 Suppress MPNST Growth via JNK. Clin Cancer Res 2019 Jul 1; 25(13): 4117-4127. 19. Shojaee S, Caeser R, Buchner M, Park E, Swaminathan S, Hurtz C, et al. Erk Negative Feedback Control Enables Pre-B Cell Transformation and Represents a Therapeutic Target in Acute Lymphoblastic Leukemia. Cancer cell 2015 Jul 13; 28(1): 114-128. 20. Lu J, Cannizzaro E, Meier-Abt F, Scheinost S, Bruch PM, Giles HA, et al. Multi-omics reveals clinically relevant proliferative drive associated with mTOR-MYC-OXPHOS activity in chronic lymphocytic leukemia. Nat Cancer 2021 Aug; 2(8): 853-864. 21. Dietrich S, Oles M, Lu J, Sellner L, Anders S, Velten B, et al. Drug-perturbation-based stratification of blood cancer. J Clin Invest 2018 Jan 2; 128(1): 427-445. 21. Dietrich S, Oles M, Lu J, Sellner L, Anders S, Velten B, et al. Drug-perturbation-based stratification of blood cancer. J Clin Invest 2018 Jan 2; 128(1): 427-445. 22. Amit I, Citri A, Shay T, Lu Y, Katz M, Zhang F, et al. A module of negative feedback regulators defines growth factor signaling. Nat Genet 2007 Apr; 39(4): 503-512. 22. Amit I, Citri A, Shay T, Lu Y, Katz M, Zhang F, et al. A module of negative feedback regulators defines growth factor signaling. Nat Genet 2007 Apr; 39(4): 503-512. 23. Saito T, Yamasaki S. Negative feedback of T cell activation through inhibitory adapters and costimulatory receptors. Immunol Rev 2003 Apr; 192: 143-160. 23. Saito T, Yamasaki S. Negative feedback of T cell activation through inhibitory adapters and costimulatory receptors. Immunol Rev 2003 Apr; 192: 143-160. 24. Bhalla US, Ram PT, Iyengar R. MAP kinase phosphatase as a locus of flexibility in a mitogen- activated protein kinase signaling network. Science 2002 Aug 9; 297(5583): 1018-1023. 24. Bhalla US, Ram PT, Iyengar R. References Haematologica 2019 Mar; 104(3): 576-586. 8. Vendramini E, Bomben R, Pozzo F, Benedetti D, Bittolo T, Rossi FM, et al. KRAS, NRAS, and BRAF mutations are highly enriched in trisomy 12 chronic lymphocytic leukemia and are associated with shorter treatment-free survival. Leukemia 2019 Aug; 33(8): 2111-2115. 9. Herling CD, Abedpour N, Weiss J, Schmitt A, Jachimowicz RD, Merkel O, et al. Clonal dynamics towards the development of venetoclax resistance in chronic lymphocytic leukemia. Nat Commun 2018 Feb 20; 9(1): 727. 10. Pandzic T, Larsson J, He L, Kundu S, Ban K, Akhtar-Ali M, et al. Transposon Mutagenesis Reveals Fludarabine Resistance Mechanisms in Chronic Lymphocytic Leukemia. Clin Cancer Res 2016 Dec 15; 22(24): 6217-6227. 11. Takahashi K, Hu B, Wang F, Yan Y, Kim E, Vitale C, et al. Clinical implications of cancer gene mutations in patients with chronic lymphocytic leukemia treated with lenalidomide. Blood 2018 Apr 19; 131(16): 1820-1832. 12. Chen Y, Germano S, Shelmani G, Kluczna D, Jayne S, Dyer MJS, et al. Paradoxical activation of alternative pro-survival pathways determines resistance to MEK inhibitors in chronic lymphocytic leukaemia. British journal of haematology 2018 Sep; 182(6): 921-924. 13. Wagner EF, Nebreda AR. Signal integration by JNK and p38 MAPK pathways in cancer development. Nat Rev Cancer 2009 Aug; 9(8): 537-549. 13. Wagner EF, Nebreda AR. Signal integration by JNK and p38 MAPK pathways in cancer development. Nat Rev Cancer 2009 Aug; 9(8): 537-549. 14. Limnander A, Depeille P, Freedman TS, Liou J, Leitges M, Kurosaki T, et al. STIM1, PKC-delta and RasGRP set a threshold for proapoptotic Erk signaling during B cell development. Nature 14. Limnander A, Depeille P, Freedman TS, Liou J, Leitges M, Kurosaki T, et al. STIM1, PKC-delta and RasGRP set a threshold for proapoptotic Erk signaling during B cell development. Nature Page 12/24 Page 12/24 immunology 2011 May; 12(5): 425-433. MAP kinase phosphatase as a locus of flexibility in a mitogen- activated protein kinase signaling network. Science 2002 Aug 9; 297(5583): 1018-1023. 25. Herishanu Y, Perez-Galan P, Liu D, Biancotto A, Pittaluga S, Vire B, et al. The lymph node microenvironment promotes B-cell receptor signaling, NF-kappaB activation, and tumor proliferation in chronic lymphocytic leukemia. Blood 2011 Jan 13; 117(2): 563-574. 26. Molina G, Vogt A, Bakan A, Dai W, Queiroz de Oliveira P, Znosko W, et al. Zebrafish chemical screening reveals an inhibitor of Dusp6 that expands cardiac cell lineages. Nat Chem Biol 2009 Sep; 5(9): 680-687. 27. Mougiakakos D, Johansson CC, Jitschin R, Bottcher M, Kiessling R. Increased thioredoxin-1 production in human naturally occurring regulatory T cells confers enhanced tolerance to oxidative stress. Blood 2011 Jan 20; 117(3): 857-861. 28. Bichi R, Shinton SA, Martin ES, Koval A, Calin GA, Cesari R, et al. Human chronic lymphocytic leukemia modeled in mouse by targeted TCL1 expression. Proceedings of the National Academy of Sciences of the United States of America 2002 May 14; 99(10): 6955-6960. 28. Bichi R, Shinton SA, Martin ES, Koval A, Calin GA, Cesari R, et al. Human chronic lymphocytic leukemia modeled in mouse by targeted TCL1 expression. Proceedings of the National Academy of Sciences of the United States of America 2002 May 14; 99(10): 6955-6960. Page 13/24 Page 13/24 29. Chan LN, Murakami MA, Robinson ME, Caeser R, Sadras T, Lee J, et al. Signalling input from divergent pathways subverts B cell transformation. Nature 2020 Jul; 583(7818): 845-851. 30. Korotchenko VN, Saydmohammed M, Vollmer LL, Bakan A, Sheetz K, Debiec KT, et al. In vivo structure-activity relationship studies support allosteric targeting of a dual specificity phosphatase. Chembiochem 2014 Jul 7; 15(10): 1436-1445. 31. Sombroek D, Hofmann TG. How cells switch HIPK2 on and off. Cell Death Differ 2009 Feb; 16(2): 187-194. 32. Di Segni M, Virdia I, Verdina A, Amoreo CA, Baldari S, Toietta G, et al. HIPK2 Cooperates with KRAS Signaling and Associates with Colorectal Cancer Progression. Mol Cancer Res 2022 May 4; 20(5): 686-698. 33. Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 2002 Dec 6; 298(5600): 1911-1912. 34. Sehgal V, Ram PT. Network Motifs in JNK Signaling. Genes Cancer 2013 Sep; 4(9-10): 409-413. 34. Sehgal V, Ram PT. Network Motifs in JNK Signaling. Genes Cancer 2013 35. Crassini K, Stevenson WS, Mulligan SP, Best OG. immunology 2011 May; 12(5): 425-433. The MEK1/2 inhibitor, MEKi-1, induces cell death in chronic lymphocytic leukemia cells under conditions that mimic the tumor microenvironment and is synergistic with fludarabine. Leuk Lymphoma 2015; 56(12): 3407-3417. 36. Campo E, Cymbalista F, Ghia P, Jager U, Pospisilova S, Rosenquist R, et al. TP53 aberrations in chronic lymphocytic leukemia: an overview of the clinical implications of improved diagnostics. Haematologica 2018 Dec; 103(12): 1956-1968. 37. Dohner H, Stilgenbauer S, Benner A, Leupolt E, Krober A, Bullinger L, et al. Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med 2000 Dec 28; 343(26): 1910-1916. 38. Knittel G, Liedgens P, Reinhardt HC. Targeting ATM-deficient CLL through interference with DNA repair pathways. Front Genet 2015; 6: 207. 39. Prabhakar S, Asuthkar S, Lee W, Chigurupati S, Zakharian E, Tsung AJ, et al. Targeting DUSPs in glioblastomas - wielding a double-edged sword? Cell Biol Int 2014 Feb; 38(2): 145-153. 40. Brondello JM, Pouyssegur J, McKenzie FR. Reduced MAP kinase phosphatase-1 degradation after p42/p44MAPK-dependent phosphorylation. Science 1999 Dec 24; 286(5449): 2514-2517. 40. Brondello JM, Pouyssegur J, McKenzie FR. Reduced MAP kinase phosphatase-1 degradation after p42/p44MAPK-dependent phosphorylation. Science 1999 Dec 24; 286(5449): 2514-2517. 41. Klintman J, Appleby N, Stamatopoulos B, Ridout K, Eyre TA, Robbe P, et al. Genomic and transcriptomic correlates of Richter transformation in chronic lymphocytic leukemia. Blood 2021 May 20; 137(20): 2800-2816. 42. Pabla N, Ma Z, McIlhatton MA, Fishel R, Dong Z. hMSH2 recruits ATR to DNA damage sites for activation during DNA damage-induced apoptosis. J Biol Chem 2011 Mar 25; 286(12): 10411-10418. 42. Pabla N, Ma Z, McIlhatton MA, Fishel R, Dong Z. hMSH2 recruits ATR to DNA damage sites for activation during DNA damage-induced apoptosis. J Biol Chem 2011 Mar 25; 286(12): 10411-10418. 43. Golding SE, Rosenberg E, Neill S, Dent P, Povirk LF, Valerie K. Extracellular signal-related kinase positively regulates ataxia telangiectasia mutated, homologous recombination repair, and the DNA damage response. Cancer Res 2007 Feb 1; 67(3): 1046-1053. 43. Golding SE, Rosenberg E, Neill S, Dent P, Povirk LF, Valerie K. Extracellular signal-related kinase positively regulates ataxia telangiectasia mutated, homologous recombination repair, and the DNA damage response. Cancer Res 2007 Feb 1; 67(3): 1046-1053. 44. Wu QN, Liao YF, Lu YX, Wang Y, Lu JH, Zeng ZL, et al. Pharmacological inhibition of DUSP6 suppresses gastric cancer growth and metastasis and overcomes cisplatin resistance. Cancer Lett 44. immunology 2011 May; 12(5): 425-433. Wu QN, Liao YF, Lu YX, Wang Y, Lu JH, Zeng ZL, et al. Pharmacological inhibition of DUSP6 suppresses gastric cancer growth and metastasis and overcomes cisplatin resistance. Cancer Lett Page 14/24 Page 14/24 2018 Jan 1; 412: 243-255. 2018 Jan 1; 412: 243-255. 45. Scheffold A, Jebaraj BMC, Tausch E, Bloehdorn J, Ghia P, Yahiaoui A, et al. IGF1R as druggable target mediating PI3K-delta inhibitor resistance in a murine model of chronic lymphocytic leukemia. Blood 2019 Aug 8; 134(6): 534-547. 46. Zandi Z, Kashani B, Alishahi Z, Pourbagheri-Sigaroodi A, Esmaeili F, Ghaffari SH, et al. Dual- specificity phosphatases: therapeutic targets in cancer therapy resistance. J Cancer Res Clin Oncol 2022 Jan; 148(1): 57-70. 47. Wang Z, Zhou JY, Kanakapalli D, Buck S, Wu GS, Ravindranath Y. High level of mitogen-activated protein kinase phosphatase-1 expression is associated with cisplatin resistance in osteosarcoma. Pediatr Blood Cancer 2008 Dec; 51(6): 754-759. 48. Vicent S, Garayoa M, Lopez-Picazo JM, Lozano MD, Toledo G, Thunnissen FB, et al. Mitogen- activated protein kinase phosphatase-1 is overexpressed in non-small cell lung cancer and is an independent predictor of outcome in patients. Clin Cancer Res 2004 Jun 1; 10(11): 3639-3649. 48. Vicent S, Garayoa M, Lopez-Picazo JM, Lozano MD, Toledo G, Thunnissen FB, et al. Mitogen- activated protein kinase phosphatase-1 is overexpressed in non-small cell lung cancer and is an independent predictor of outcome in patients. Clin Cancer Res 2004 Jun 1; 10(11): 3639-3649. 49. Gao Y, Li H, Han Q, Li Y, Wang T, Huang C, et al. Overexpression of DUSP6 enhances chemotherapy- resistance of ovarian epithelial cancer by regulating the ERK signaling pathway. J Cancer 2020; 11(11): 3151-3164. 49. Gao Y, Li H, Han Q, Li Y, Wang T, Huang C, et al. Overexpression of DUSP6 enhances chemotherapy- resistance of ovarian epithelial cancer by regulating the ERK signaling pathway. J Cancer 2020; 11(11): 3151-3164. Figures Page 15/24 Figure 2 DUSP1/6 inhibition is toxic specifically for CLL cells a) RNASeq counts of DUSP1 mRNA and b) DUSP6 mRNA in IGVH-mutated (M-CLL; n=94) compared to IGVH-unmutated (UM-CLL; n=95) CLL patient-derived samples. CLL cells were obtained from the a) RNASeq counts of DUSP1 mRNA and b) DUSP6 mRNA in IGVH-mutated (M-CLL; n=94) compared to IGVH-unmutated (UM-CLL; n=95) CLL patient-derived samples. CLL cells were obtained from the Page 16/24 Page 16/24 peripheral blood. Data are presented as log2 x 104 values ± SD. Statistical significance was assessed by a two-tailed unpaired Student’s t-test (DUSP1 p=0.0161; DUSP6 p=0.0008) 21. c) Correlation of mRNA expression levels of DUSP6 in CLL samples (n=210). DUSP6 high and DUSP6 low samples (defined by above or below the median expression level) were displayed as time to treatment (TTT) curve (p=0.0109) and d) overall survival (OS) curve (p=0.0075) 21. Statistical analysis was performed using the Log Rank (Mantle Cox) test. e) RNASeq counts of DUSP6 mRNA in CLL samples with mutated BRAF and KRAS genes. Comparison of KRAS/BRAF wildtype (wt; n=189) compared to KRAS/BRAF mutation (n=21). Data are presented as log2 x 104 values ± SD. Statistical significance was assessed by a two-tailed unpaired Student’s t-test (DUSP6 p<0.0001). f) Analysis of the DUSP6 and g) corresponding phospho-ERK mean fluorescence intensity (MFI) of CLL-derived cell lines treated with 5 µM MEK inhibitor (PD901) for 24 hours (n=8, pooled data from 2 independent experiments). Statistical significance was assessed by a two-tailed paired Student’s t-test (DUSP6 p<0.0155) (pERK p<0.0033). h) Comparison of reanalyzed gene expression publicly available data 25 of DUSP1 expression (n=14; p<0.0001) and i) DUSP6 expression (n=14; p<0.0001) specified as log2 fold-change (logFC) in matched peripheral blood samples and lymph node samples. Page 17/24 Figure 2 DUSP1/6 inhibition is toxic specifically for CLL cells a) Cytotoxic dose-response to increasing concentrations of the DUSP1/DUSP6 inhibitor BCI (0-5 µM) in primary CLL samples (n=21). Viability was determined after 48 hours treatment by flow cytometry via DAPI staining. The percentage of specific cell death was calculated as follows: 100 × (% dead cells − % baseline dead cells)/(100% − % baseline dead cells). Data are presented as mean values ± SD. b) Specific cell death was calculated after viability measurement upon 48 hours treatment with 1.25 µM BCI in vitro in CD19+ healthy donor-derived B-cells (n=5) compared to primary CLL cells (n=21). Data are presented Page 18/24 Page 18/24 Page 18/24 as mean values ± SD. Statistical significance was assessed by a two-tailed unpaired Student’s t-test (p<0.0001). c) Time course of in vitro treatment of CLL-derived cell line MEC-1 compared to human diffuse large B-cell lymphoma cell line (HBL-1) and T-cell lymphoma cell lines (Jurkat and HUT78). Specific cell death was determined after 24-72 hours treatment with 5 µM BCI (n=2; independent experiments). Data are presented as mean values ± SD. d-f) Analysis of in vivo treatment with the BCI derivate BCI-215. Splenocytes from TCL-1 tg mice were transplanted in wt mice and treated with 10 mg/kg BCI-215 (n=5) or vehicle control (5 % DMSO in PBS) (n=5) for 10 days. Content of CLL cells in peripheral blood (PB), spleen (SP) or peritoneal cavity (PC) in % was determined by flow cytometry in vehicle control group and BCI-215 treatment group. Representative result for 2 independent experiments is shown. Data are presented as mean values ± SD. d) Evidence of CLL engraftment in wt mice prior treatment. e) Content of total CLL counts (x108) in spleen. Statistical significance was assessed by a two tailed unpaired Student’s t-test (p=0.0026). f) Analysis of total CLL counts (x107) in the PC. Statistical significance was assessed by a two-tailed unpaired Student’s t-test (p=0.0029). g-k) Evaluation of genetic knockout experiments by CRISPR/Cas9 system in MEC-1 cell line. g) Representative immunoblot of DUSP1 protein expression in MEC-1 wt cells compared to KO clone. Beta actin served as a loading control. h) Immunoblot of DUSP6 expression in MEC-1 wt cells compared to KO clones. Loading: wt MEC1, M = marker, ko1, ko2 clone. Beta Actin served as a control. i-j) In vitro competitor growth assays of successfully generated gene knockouts in MEC-1 cell line: i) DUSP1 knockout clones (n=2) and GFP+ control clones (n=2). DUSP1/6 inhibition is toxic specifically for CLL cells j) DUSP6 knockout clones (n=2) and GFP+ control clones (n=2). Fold-change of % GFP was determined after mixing GFP+ control cells and knockout cells. Data are presented as mean values (with ± SD for knockout clones) of the fold change of GFP expression over time. k) Specific cell death of wt control cell line versus DUSP1 and DUSP6 knockout clones was calculated after viability measurement by flow cytometry via DAPI staining after 48h treatment with 1.25 µM BCI or vehicle control. Pooled data from 4 independent experiments; statistical significance was assessed by a two- tailed unpaired Student’s t-test (DUSP1 p=0.0939; DUSP6 p=0.0159). Page 19/24 Page 19/24 Page 19/24 DUSP1/6 inhibition induces MAPK signaling in CLL cells DUSP1/6 inhibition induces MAPK signaling in CLL cells a) Experimental setup of global phospho-proteome screen in primary CLL cells after DUSP1/DUSP6 inhibition (BCI; 2 µM). b) Heatmap of the phosphorylation profile of primary CLL cells treated for 3 or 10 minutes with the DUSP1/6 inhibitor BCI compared to untreated control (in 3 technical replicates) with a Page 20/24 Page 20/24 list of differentially phosphorylated proteins within the indicated clusters. c) Phospho-proteome analysis of primary CLL cells treated for 10 minutes with 2 µM BCI compared to untreated CLL cells. Map of regulated phospho-sites of BCR and MAPK network based on the KEGG pathway database. Figure 4 Prolonged DUSP1/6 inhibition induces the activation of DNA damage response and apoptosis in CLL Page 21/24 Page 21/24 a) Heatmap of the phosphorylation profile of MEC-1 cells treated for 15 or 45 minutes with the DUSP1/6 inhibitor (BCI; 5 µM) compared to untreated control MEC-1 cells (in 3 technical replicates) with a list of differentially phosphorylated proteins within the indicated region. b) Unsupervised hierarchical cluster analysis revealed 4 clusters of differentially regulated phospho‑sites; cluster 1: up at 15 and 45 min treatment; cluster 2: up only at 45 min treatment; cluster 3: up only at 15 min treatment; cluster 4: down at 15 and 45 min treatment; gene ontology analysis was performed for enrichment of target proteins in specific pathways and listed below the individual clusters. c) Prediction of upstream kinases responsible for the observed differentially regulated phosphorylation sites after 15 min DUSP1/6 inhibition using the NetworKIN algorithm. d) Pie chart of up and downregulated target sites of the HIPK2 kinase indicate that this kinase is highly active upon DUSP1/6 inhibition. e, f) Phospho-proteome analysis of MEC-1 treated for 15 and 45 min with DUSP1/6 inhibitor (BCI; 5 µM) compared to untreated MEC-1 cells using ANOVA analysis. DNA damage network (e) and apoptosis network (f) were identified as differentially regulated using Cytoscape and the PhosphoPath plugin. Page 22/24 a) Specific cell death was calculated after flow cytometric viability analysis by DAPI staining. CLL-derived cell lines were pre-treated for 1 hour with 1 µM of the MEK inhibitor (PD901) followed by DUSP1/6 a) Specific cell death was calculated after flow cytometric viability analysis by DAPI staining. CLL-derived cell lines were pre-treated for 1 hour with 1 µM of the MEK inhibitor (PD901) followed by DUSP1/6 Page 23/24 Page 23/24 inhibitor treatment (BCI; 1.25 µM) or control treatment for 24 hours (n=4, pooled data from 2 independent experiments). Statistical significance was assessed by a two-tailed paired Student’s t-test (p=0.01740174). b) Analysis of the phosphorylation of γH2AX mean fluorescence intensity (MFI) of primary CLL cells treated with BCI (2.5 µM and 5 µM). MFI values ± SD. Statistical significance was assessed by a two-tailed paired Student’s t-test (2.5 µM p=0.0425; 5 µM p=0.0074). c) γH2AX mean fluorescence intensity (MFI) of CLL cell lines (MEC-1, OSU-CLL,and EHEB) (n=6, pooled data from 2 independent experiments) were pre-treated for 1 hour with 5 µM of MEK inhibitor (PD901) or control followed by DUSP1/6 inhibition (BCI; 5 µM). Statistical significance was assessed using ANOVA analysis (p=0.0030). d) Flow cytometric analysis of Annexin V staining of primary CLL cells treated with DUSP1/6 inhibitor (BCI; 0-5 µM) for 24 hours. Classification of the cells in necrotic, late apoptotic, apoptotic and viable cell stages specified as % of all cells; representative example for n=4 primary CLL cases. e) Comparison of the effects of DUSP1/6 inhibition (1 µM) and the combination of DUSP1/6 with the pan- caspase inhibitor QVD (5 µM; n=7; p=0.0030) or f) DUSP1/6 inhibitor (BCI; 1 µM) with CHK1/2 kinase inhibition (AZD6672; 50 nM; n=6; p=0.0237). Cells were pre-treated for 1 hour with inhibitor before DUSP1/6i was added, viability was analyzed by flow cytometry after 48 hours. Specific cell death was calculated after viability measurement by DAPI staining. Statistical significance was assessed by a two- tailed paired Student’s t-test. g) Response of primary CLL cells with Del11q or Del17p/p53 mutation towards DUSP1/6 inhibitor (BCI; 1.25 µM) as compared to patients without these alterations. Data represented as specific cell death (%) after DAPI measurement by flow cytometry. Data are presented as mean values ± SD. Statistical significance was assessed by a two-tailed unpaired Student’s t-test (n.s. p=0.2803) h) Specific cell death of treatment naïve (n=6) and ibrutinib resistant (n=6) primary CLL samples after 48 hours BCI treatment (1 µM). Statistical significance was assessed by a two-tailed unpaired Student’s t-test (n.s. p=0.8099). Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplFiguresLeukemia.pdf SupplMaterialMethodsLeukemia.pdf Page 24/24
https://openalex.org/W2016649910	https://link.springer.com/content/pdf/10.1007/s12274-014-0681-y.pdf	English	null	Tuning the translational freedom of DNA for high speed AFM	Nano research	2,015	cc-by	7,174	ABSTRACT Direct observation is arguably the preferred way to investigate the interactions between two molecular complexes. With the development of high speed atomic force microscopy (AFM), it is becoming possible to observe directly DNA-protein interactions with relevant spatial and temporal resolutions. These interactions are of central importance to biology, bionanotechnology, and functional biologically inspired materials. As in all microscopy studies, sample preparation plays a central role in AFM observation and minimal perturbation of the sample is desired. Here, we demonstrate the ability to tune the interactions between DNA molecules and the surface to create an association strong enough to enable high-resolution AFM imaging while also providing sufficient translational freedom to allow the relevant protein-DNA interactions to take place. Furthermore, we describe a quantitative method for measuring DNA mobility, while also determining the individual forces contributing to DNA movement. We found that for a weak surface association, a significant contribution to the movement arises from the interaction of the AFM tip with the DNA. In combination, these methods enable the tuning of the surface translational freedom of DNA molecules to allow the direct study of a wide range of nucleo-protein interactions by high speed atomic force microscopy. © The author(s) 2014. This article is published with open access at Springerlink.com Tuning the translational freedom of DNA for high speed AFM 1 Bioelectronics, School of Electronic and Electrical Engineering, University of Leeds, Leeds, LS2 9JT, UK 2 Department of Physics and Astronomy, University of Sheffield, Sheffield, S3 7RH, UK Received: 13 August 2014 Revised: 02 December 2014 Accepted: 03 December 2014 © The author(s) 2014. This article is published with open access at Springerlink.com KEYWORDS high speed atomic force microscopy (HS-AFM), DNA, protein, bionanotechnology Received: 13 August 2014 Revised: 02 December 2014 Accepted: 03 December 2014 Received: 13 August 2014 Revised: 02 December 2014 Accepted: 03 December 2014 Address correspondence to c.walti@leeds.ac.uk Tuning the translational freedom of DNA for high speed Tuning the translational freedom of DNA for high speed AFM Nano Research 2015, 8(6): 1811–1821 DOI 10.1007/s12274-014-0681-y Nano Research 2015, 8(6): 1811–1821 DOI 10.1007/s12274-014-0681-y Nano Research 2015, 8(6): 1811–1821 DOI 10.1007/s12274-014-0681-y 1 Introduction However, the dynamic nature of these processes poses significant challenges for AFM investigations and, until recently, time-course studies of such interactions were limited by the acquisition rate of the AFM. g g The 2D confinement of the molecules inherently perturbs any biological interaction of interest. It is therefore necessary to mediate a surface absorption that allows the retention of sufficient translational and rotational freedom while securely adhering the molecules against the instantaneous lateral forces imposed by the scanning probe. Additionally, it is necessary to maintain a suitable reduced physiological environment for biological activity to be retained. Previous studies employing methods to modify the mica surface chemically [23], using cyclic dielectric fields [24, 25] and cyclic buffer exchanges [10, 26, 27], have attempted to mitigate confinement effects by cycling the surface between a loose association— allowing reactions to proceed freely—and a tight association to enable high-resolution imaging. It has also been shown that modulation of surface voltage can be used to control adhesion forces [28] but this requires the use of metal electrodes as substrates and very low salt concentrations. Additionally, the use of DNA origami tiles as support structures to isolate the biological interactions being imaged from the surface has been demonstrated [29]. These alternative methods either put significant constraints on the experimental setup or do not provide the required functionality, and therefore pre-incubation of mica with divalent cations remains the common practice. The recent development of high speed atomic force microscopy (HS-AFM), and in particular HS-AFM in fluid, has allowed for the direct examination of discrete biological interactions and nano-scale self-assembly processes in physiological buffer at relevant spatial and temporal resolution [5]. This technology not only enables the confirmation of established hypotheses in biology, such as the hand-over-hand motion of myosin [6], but also has the potential to further the understanding of how protein complexes and nucleic acids interact, something that is critical to all forms of life [7–13]. HS-AFM can also be applied in the study of a wide range of biologically inspired materials. HS-AFM work builds upon two decades of biological studies with conventional AFM [14] which, to-date, has achieved significant success on model systems such as RNA polymerase [15, 16], bacteriophage Lambda Cro protein [17], DNA photolyase [18], nucleosomes [19], and restriction enzymes [20]. 1 Introduction it offers significant potential for the development of novel functional materials that are heterogeneously structured at the nano-scale. However, in order to exploit the potential of this technology, a solid understanding of the interactions that drive the bottom-up assembly of nano-scale structures has to be established. DNA nanotechnology is a promising approach for the bottom-up construction of sophisticated nano-scale complexes. In combination with methods such as hierarchical assembly using location-specific arrange- ments of proteins upon structural DNA scaffolds [1–3], 1812 Nano Res. 2015, 8(6): 1811–1821 The sample preparation procedure and its impact on the experiment is a key concern in all forms of microscopy. Here, AFM has distinct advantages over radiative-based alternatives, as it does not require the labeling or modification of delicate biological structures. However, AFM employs a physical probe and therefore requires the deposition of the sample on a solid support surface [14]. Muscovite mica surfaces are typically used as the substrate owing to their perfect cleavage along a <001> plane, yielding large atomically flat areas. Mica consists of layers of an aluminum phyllosilicate lattice ionically bonded through interstitial K+ ions. Upon cleavage, these K+ ions are highly mobile and are readily exchanged with divalent cation species at the solid–liquid interface [21]. This exchange results in a positive overcharging of the mica surface, which enables the deposition of molecules that hold a net negative charge, such as DNA [22]. In recent years, a significant understanding of the complex interactions between proteins and nucleic acids in vivo has been developed. When attempting to utilize these interactions outside their native biological context, however, additional factors have to be taken into account. For example, the presence of solid surfaces leads to additional interactions between DNA and proteins, and the presence of unusual topological features in the DNA molecules, for instance branch- points such as those present in DNA origami [4], can have an impact on the interactions. For the full potential of these approaches to be exploited, it is important to understand in detail the relevant interactions in the context that they are to be employed. Arguably, direct observation using atomic force microscopy (AFM) would be the preferred way to study these dynamic protein-DNA interactions in vitro and in the proximity of solid surfaces. \| www.editorialmanager.com/nare/default.asp Nano Res. 2015, 8(6): 1811–1821 Nano Res. 2015, 8(6): 1811–1821 1813 It is well established that upon cleavage of mica layers, the interstitial K+ ions are split between the two cleavage faces and tend to cluster. The resulting vacant sites are easily filled upon exposure to divalent cations. Transition metal cations such as Ni2+, Co2+, and Zn2+ bind irreversibly to mica and have been demonstrated to displace residual K+ ions. They interact with the hydroxyl groups of mica cooperatively [22, 30], leading to phase-separated domains of positive surface charge [31]. Where transition metal cations are utilized, a strong adhesion between DNA and mica is achieved due to directional bonding of the d-orbitals of the divalent cation. Such deposited DNA molecules adopt a kinetically trapped form [31], sufficient for high-resolution imaging in aqueous buffer. organisms, enabling its use as a strong surface binding agent. When employing the idealized polka dot represen- tation, it is possible that pre-incubation of the mica surface with different concentrations of Ni2+ allows for control over the localized surface-DNA association. If the DNA molecules are now deposited in Mg2+ buffer, Ni2+ should act to restrain the translational freedom through a few specific anchor points while Mg2+ mediates a more global and distributed but weak association. Thus, the mobility of a DNA molecule is governed by the number of Ni2+ interactions and is controlled by the Ni2+ concentration and exposure time during surface pre-incubation. By modulating the concentration of Ni2+ for mica pre-treatment, we demonstrate the ability to control the surface translational freedom of DNA molecules while maintaining a fixed Mg2+ concentration within the imaging buffer, which can be tailored to optimize the relevant nucleo-protein interactions. This allows us to maintain a transient surface interaction of nucleic acids strong enough for imaging with HS-AFM under fluid, while simultaneously providing enough mobility of the DNA to allow the relevant nucleo- protein interactions to take place. We employ a novel methodology for quantifying DNA mobility and use it to demonstrate our ability to tune the surface interaction from tightly surface-tethered to highly mobile DNA. In contrast, Mg2+ cannot form directional bonds because it is continually exchanging with residual K+ and H+ ions on the mica surface and in the imaging buffer. As a consequence, Mg2+ only mediates a relatively weak and diffuse adhesion between DNA and the mica surface, allowing DNA molecules to adopt an equilibrated form [22, 31–33]. Nano Res. 2015, 8(6): 1811–1821 This complex spatial distribution has been rationalized by others [31, 33], where the phase-separated domains of ionic species are represented as an idealized polka dot pattern: An ionic patchwork of evenly distributed positively charged Ni2+ clusters interspersed across a mica surface undergoing dynamic exchanges between K+, H+, and other cations such as Mg2+. Previous work has taken advantage of this difference in binding capacities by adjusting the ratio between different monovalent and monovalent-to-divalent ion concentrations in the deposition buffer to alter the surface adhesion of DNA in air [20, 34, 35]. www.theNanoResearch.com∣www.Springer.com/journal/12274 \| Nano Research 1 Introduction HS-AFM may therefore have a central role to play in bionano- technology and experimental biology, complementing current biochemical techniques and crystallography while elucidating fundamental biological processes. \| www.editorialmanager.com/nare/default.asp \| www.editorialmanager.com/nare/default.asp 2.1 Quantifying the mobility of DNA molecules In order to establish the methodology for quantifying the mobility of surface-immobilized DNA molecules, mica substrates were pre-incubated with 15 mM Ni2+. Subsequently, 3.5-kbp DNA molecules were deposited in Tris-Mg buffer and imaged by AFM. A Ni2+ con- centration of 15 mM was expected to provide strong surface adhesion, allowing contrasting small regions of chain mobility to be easily identified. Areas of 1 μm2 of sample were continuously imaged in solution to observe the motion of the DNA molecules. For each sequence of AFM images, several representative DNA molecules were selected and processed using a Interestingly, Mg2+ is the primary co-factor for the majority of enzymes that manipulate all biological polyphosphates including ATP, DNA, and RNA [36]. The transient nature of Mg2+ association allows it to be utilized, in equilibrium, as both a surface binding agent and enzymatic co-factor. Typically, cellular Mg2+ ranges from 5–20 mM, where 95% is chelated with proteins and nucleic acids, primarily ATP [36]. However, a concentration below 10 mM is sufficient for most nucleo-protein interactions. In contrast, Ni2+ exhibits little to no physiological role in higher www.theNanoResearch.com∣www.Springer.com/journal/12274 \| Nano Research 1814 Nano Res. 2015, 8(6): 1811–1821 chain-fitting algorithm. A chain of co-ordinates was fitted to the height peaks in the AFM image, generating a vector representation of the molecule’s conformation (Figs. 1(a) and 1(b)). This method eliminates the noise and image artifacts that arise during high-speed AFM imaging in liquid. Each selected DNA molecule was tracked sequentially through an image series, where fitting was repeated for sections of the chain that deviated from the previous image, allowing their motion to be quantified. Each chain was then divided into >100 equidistant points and a minimization algorithm was used to map corresponding points between successive frames in a time series, mitigating errors in imaging and fitting around the chain ends. The amount of motion for each pair of points could then be calculated, averaged, and normalized to yield movement velocities for individual DNA chains or sections of a chain (Figs. 1(c) and 1(d)). g To quantify the lateral motion of each DNA molecule over time, the movements of individual molecules were analyzed. Figure 2(a) shows the time-course of two representative DNA molecules over the course of an image sequence. The time evolution is represented by a color scale, with the darkest red line representing the final state and the lightest blue line the initial state. 2.1 Quantifying the mobility of DNA molecules The total distance travelled by the DNA chain, normalized by the length of the DNA chain, was determined by summing up all the distances travelled by the equidistant points on the chain. The results for the two representative DNA molecules are shown as a function of time in Fig. 2(b). The movements were found to be linear in time and hence, from the graphs in Fig. 2(b), a velocity per unit of DNA in nanometers per 100 base pairs length per second (nm·100 bp–1·s–1) was then derived as the characteristic quantity to Figure 1 A schematic diagram depicting the chain fitting and evolution processing from the raw AFM images. (a) Sequential AFM images following a single DNA molecule. (b) Derived chain vectors. (c) Mapping of chain vectors into equidistant points. (d) Point mapping is indicated across a short section of the molecule; the relevant section of the molecule is indicated by the boxes in (c). The scale bar is 100 nm. Figure 2 (a) Sequential HS-AFM images of two representative 3.5-kbp DNA molecules over long time scales at Ni2+ surface pre-incubation concentrations of 15 mM. The time evolution of the DNA chain is represented by a color scale, with the lightest blue line representing the initial state and the darkest red line the final state. Under these binding conditions, DNA molecules have little mobility, as indicated by the tight overlap through subsequent frames. Panel (b) shows the chain movement per 100 bp for both DNA molecules, from which an average chain mobility of 17 nm· 100 bp–1·s–1 can be calculated. Figure 2 (a) Sequential HS-AFM images of two representative 3.5-kbp DNA molecules over long time scales at Ni2+ surface pre-incubation concentrations of 15 mM. The time evolution of the DNA chain is represented by a color scale, with the lightest blue line representing the initial state and the darkest red line the final state. Under these binding conditions, DNA molecules have little mobility, as indicated by the tight overlap through subsequent frames. Panel (b) shows the chain movement per 100 bp for both DNA molecules, from which an average chain mobility of 17 nm· 100 bp–1·s–1 can be calculated. Figure 2 (a) Sequential HS-AFM images of two representative 3.5-kbp DNA molecules over long time scales at Ni2+ surface pre-incubation concentrations of 15 mM. 2.1 Quantifying the mobility of DNA molecules The time evolution of the DNA chain is represented by a color scale, with the lightest blue line representing the initial state and the darkest red line the final state. Under these binding conditions, DNA molecules have little mobility, as indicated by the tight overlap through subsequent frames. Panel (b) shows the chain movement per 100 bp for both DNA molecules, from which an average chain mobility of 17 nm· 100 bp–1·s–1 can be calculated. Figure 2 (a) Sequential HS-AFM images of two representative 3.5-kbp DNA molecules over long time scales at Ni2+ surface pre-incubation concentrations of 15 mM. The time evolution of the DNA chain is represented by a color scale, with the lightest blue line representing the initial state and the darkest red line the final state. Under these binding conditions, DNA molecules have little mobility, as indicated by the tight overlap through subsequent frames. Panel (b) shows the chain movement per 100 bp for both DNA molecules, from which an average chain mobility of 17 nm· 100 bp–1·s–1 can be calculated. Figure 1 A schematic diagram depicting the chain fitting and evolution processing from the raw AFM images. (a) Sequential AFM images following a single DNA molecule. (b) Derived chain vectors. (c) Mapping of chain vectors into equidistant points. (d) Point mapping is indicated across a short section of the molecule; the relevant section of the molecule is indicated by the boxes in (c). The scale bar is 100 nm. \| www.editorialmanager.com/nare/default.asp Nano Res. 2015, 8(6): 1811–1821 1815 freedom of DNA and enable relevant nucleo-protein interactions to take place. measure lateral mobility (see movie S1 in Electronic Supplementary Material (ESM) for details). This detailed analysis of the lateral motion of DNA molecules reveals a number of intricacies in the surface interaction and the influence of the microscope probe on the sample. 2.2 Tuning DNA surface mobility with Ni2+ pre- incubation 2.3 Influence of the HS-AFM probe AFM images are acquired through the monitoring of the interaction of a sharp solid tip with the surface- immobilized molecules. When imaging biological systems, the tip radius is generally of the same order of magnitude as the surface-immobilized molecules; hence, it can be expected that the interaction is signi- ficant. Furthermore, in the investigation reported here, the DNA molecules are deliberately only weakly associated with the surface, leading to increased impact from the tip-molecule interactions. Vectorization of the chain motion employed here enables behavioral analysis of individual segments of each DNA molecule and allows for the tip-DNA molecule interaction to be analyzed. When a particular nucleo-protein interaction is to be observed, it is necessary to be able to image specific molecules continuously over the time course of the reaction. If the surface association is too weak, the DNA molecule may be able to move out of the frame and key events will inevitably be missed. Therefore, optimal binding conditions need to present weak surface associations without large overall translational movements of the DNA across the mica surface, while at the same time allowing individual segments of the DNA molecule to move relatively freely. We concluded from the data presented in Fig. 3 that for the 3.5-kbp DNA molecules used in this study, optimal conditions are achieved at around 5 mM Ni2+, which results in optimal spatial resolution and chain flexi- bility, with a mobility of 38 ± 2 nm·100 bp–1·s–1. This is proposed to be sufficient for imaging enzymatic interactions with minimal hindrance from the mica support surface, while the DNA molecules remain relatively immobile within a 1 μm2 imaging area. As a proof-of-concept, we investigated the influence of different translational mobilities on the efficiency of restriction enzyme cleavage of surface-associated DNA. EcoRI was able to cleave DNA deposited on a 5 mM Ni2+ surface, allowing real-time imaging of individual cleavage events, while on 15 mM Ni2+ surfaces, the The direction of movement for each DNA chain segment is known and therefore a polar histogram of the magnitude of the movement versus the direction can be plotted for the whole dataset across all Ni2+ concentrations (Fig. 4(a)). A distinct preference for movements along a particular direction can be observed, and an anisotropy of 1.4 was obtained by fitting an ellipsoid to the data. Random thermal motion of the DNA molecule is not expected to lead to any directional preference. 2.2 Tuning DNA surface mobility with Ni2+ pre- incubation To investigate whether the DNA mobility can be controlled via tuning the interaction of the surface- immobilized Ni2+ ions by varying their density, the above approach was applied across a range of Ni2+ concentrations from 1 mM to 15 mM (Fig. 3). Varying the ion concentration was chosen over increasing the incubation time, which may lead to unwanted crystallization of Ni-salts. A lower Ni2+ concentration is expected to lead to a lower density of positively charged sites on the surface, providing fewer pinning points for a DNA molecule and allowing it more translational freedom. Analyzing the motion of DNA molecules is not a novel concept. A previous study has attempted the direct superposition of sequential images aligned to immobile parts of the molecule [37] or to a reference elsewhere in the frame [38]. Deriving additional details by analysis of vectorized molecules has also been undertaken through hand-tracing methods [39] in order to examine quantities such as elastic bending energies [40] and the influence of the AFM probe [11]. However, such studies lack detail due to the crude alignment and vector derivation methodologies, which yield mobility analyses based on single point mapping of center-of-mass or chain termini only. Consequently, these analyses lack the power to provide the understanding necessary to tune the translational Figure 3(a) shows the chain mobility as a function of Ni2+ concentration. Each point on the graph represents an average across a number of DNA molecules and the error bars represent the standard deviation. It can Figure 3 (a) DNA molecule translational mobility (nm·100 bp–1·s–1) as a function of Ni2+ surface pre-incubation concentration. A distinct decrease in mobility is associated with an increasing Ni2+ concentration. Error bars indicate the standard deviation for each data point. Example chain vector evolution profiles for (b) 2 mM, (c) 3.5 mM, (d) 5 mM, (e) 10 mM, and (f) 15 mM Ni2+ surface pre-incubation concentrations. Figure 3 (a) DNA molecule translational mobility (nm·100 bp–1·s–1) as a function of Ni2+ surface pre-incubation concentration. A distinct decrease in mobility is associated with an increasing Ni2+ concentration. Error bars indicate the standard deviation for each data point. Example chain vector evolution profiles for (b) 2 mM, (c) 3.5 mM, (d) 5 mM, (e) 10 mM, and (f) 15 mM Ni2+ surface pre-incubation concentrations. www.theNanoResearch.com∣www.Springer.com/journal/12274 \| Nano Research 1816 Nano Res. 2015, 8(6): 1811–1821 cleavage efficiency was minimal (see Figs. 2.2 Tuning DNA surface mobility with Ni2+ pre- incubation S1 and S2, Table S1 and Movie S3 in the ESM). be seen that the DNA translational mobility increases significantly with decreasing Ni2+ concentration. Where the surface concentration of Ni2+ is at the highest level investigated, large regions of the chain remain stationary through many consecutive images in a series, indicating a very tight surface association (see Fig. 3(f) for an example). In contrast, below 5 mM, the surface Ni2+ becomes sufficiently sparse such that large stretches of the DNA chain show considerable movement over the duration of the experiment (Fig. 3(d)). This is also reflected in the chain mobility, which was calculated to be 38 ± 2 nm·100 bp–1·s–1 at 5 mM Ni2+, with even larger degrees of freedom found for 3.5 and 2 mM Ni2+ (70 ± 8 nm·100 bp–1·s–1 and 171 ± 15 nm·100 bp–1·s–1, respectively). For the lowest Ni2+ concentration investigated (1 mM), a high velocity of motion was observed (see Movie S2 in ESM), which meant that a meaningful quantification of the mobility was not possible; hence, this concentration is not represented in the graph. We note that while the values quoted here have been normalized with respect to the sequence length, the absolute size of the DNA molecule is of importance. The 3.5-kbp DNA molecules studied above were compared to shorter, 890-bp DNA molecules, which undergo large “worm-like” movements across the surface under comparable conditions (data not shown). Therefore, while the surface preparation can be tuned to produce the desired degree of nucleic acid association, it may be necessary to adjust conditions to account for the length and charge of the particular molecules of study. \| www.editorialmanager.com/nare/default.asp 2.3 Influence of the HS-AFM probe In contrast, the AFM tip is scanned back and forth very fast along one axis (the fast scan axis) and relatively slowly along the other axis. Therefore, the AFM tip-molecule interactions are expected to be predominately in the direction of the fast scan axis, and hence would lead to an anisotropic histogram. \| www.editorialmanager.com/nare/default.asp \| www.editorialmanager.com/nare/default.asp 1817 Nano Res. 2015, 8(6): 1811–1821 Figure 4 Polar histograms indicating the magnitude and directionality of chain movements (a) including all Ni2+ concentrations, (b) including only high Ni2+ concentrations (10 mM and 15 mM), and (c) including only low Ni2+ concentration (2 mM and 3.5 mM). The movements of all segments are binned according to the direction and weighted by the magnitude of the movement. The grey scale delineates the contributions of different DNA chains. The degree of the anisotropy of each histogram is indicated by the elliptical fit to the data (indicated by the red ellipsoid). than in Fig. 4(a) (anisotropy of 1.5), demonstrating that the anisotropy increases with increasing lateral mobility. Notably, the skew seen in the magnitude data is found to be in line with the image acquisition direction, suggesting that the AFM tip-DNA interaction contributes significantly to the observed motion of loosely bound DNA molecules. Conversely, higher Ni2+ preparations are characterized by smaller magnitude motions and are largely isotropic in nature. This would be consistent with molecules undergoing small conformational changes as a result of thermal fluctua- tions, notably constrained against large translational AFM tip-induced motions. The lateral forces imparted by the AFM tip naturally perturb the sample of interest and may influence the nucleo-protein interaction. However, the ability to extract detailed information regarding the behavior of molecules during the imaging process may lead to a better understanding of the nature of the observed biological interactions. www.theNanoResearch.com∣www.Springer.com/journal/12274 \| Nano Research 3 Conclusion When attempting to observe biological processes in vitro with the HS-AFM, it is essential to ensure that the measurement only minimally perturbs the interactions of interest. For the investigation of nucleo-protein interactions, the DNA molecules have to be associated with the surface strongly enough to enable stable imaging over relevant timescales, while at the same time providing enough translational freedom to allow the proteins to interact with the DNA with minimal disturbances. A number of different approaches have been reported in the literature for controlling the surface association of DNA molecules. However, none of the existing methods offer the ability to mediate the surface interaction and thus the translational freedom of the DNA to enable large ranges of diverse investigations within the additional constraints of maintaining suitable reduced physiologi- cal conditions. Here, we demonstrated an approach to tune the surface interaction of DNA that fulfills these requirements. Both stable imaging and reaction conditions are orchestrated simultaneously without the need to perturb the system throughout the reaction time-course. Figure 5 Area plots depicting the cumulative movement of chain segments for representative DNA chains for different Ni2+ concentrations (left panels). Distinct regions of the DNA molecule are seen to be anchored at particular locations (indicated by arrows). The density of minima increases with Ni2+ concentrations, suggesting that Ni2+ ions provide specific anchor points on the surface to increase DNA surface association. The grey scale indicates successive frames in a time series. The panels on the right show the chain evolution with the minima location indicated by arrows. Figure 5 Area plots depicting the cumulative movement of chain segments for representative DNA chains for different Ni2+ concentrations (left panels). Distinct regions of the DNA molecule are seen to be anchored at particular locations (indicated by arrows). The density of minima increases with Ni2+ concentrations, suggesting that Ni2+ ions provide specific anchor points on the surface to increase DNA surface association. The grey scale indicates successive frames in a time series. The panels on the right show the chain evolution with the minima location indicated by arrows. The number of such minima, and hence the number of stationary points, decreases with decreasing Ni2+ concentration (Figs. 5(b)–(d)). We note that all of the molecules imaged on surfaces pre-incubated with ≥10 mM Ni2+ and in excess of half of the molecules imaged on 5 mM Ni2+ surfaces show distinct pinning at one or more locations. 2.4 Identification of discrete surface anchor points To consider the role of Ni2+ within our system, we adopted the idealized polka dot surface model discussed by others [30, 32]. It is reasonable to assume that the density of surface-associated Ni2+ ions increases with increasing Ni2+ concentration during incubation. It has previously been demonstrated that the distri- bution of Ni2+ on the surface is highly non-uniform [40] and lacks homogeneity [30]. However, it may be possible to gain further insight into the Ni2+/Mg2+- governed surface interactions by identifying regions of specific surface association by examining the motion of the DNA chains. When plotting the cumulative movement for each segment along individual DNA molecules (Fig. 5), a series of minima can be observed (indicated by arrows). Figure 5(a) shows the results for a representative DNA molecule immobilized on a 15 mM Ni2+ pre-incubated surface, and four distinct minima can be identified. The right hand panel shows the time evolution of the DNA molecule and in this representation, particular regions of the chain appear pinned to the surface, while other chain regions move large distances. The pinned, stationary positions coincide with the observed minima in movement and are again indicated by arrows. Figure 4 Polar histograms indicating the magnitude and directionality of chain movements (a) including all Ni2+ concentrations, (b) including only high Ni2+ concentrations (10 mM and 15 mM), and (c) including only low Ni2+ concentration (2 mM and 3.5 mM). The movements of all segments are binned according to the direction and weighted by the magnitude of the movement. The grey scale delineates the contributions of different DNA chains. The degree of the anisotropy of each histogram is indicated by the elliptical fit to the data (indicated by the red ellipsoid). When examined more closely, it can be seen that the anisotropy arises mainly from highly mobile DNA molecules that appear on low Ni2+ sample preparations. Figure 4(b) shows the polar histogram of the magnitude of the movement versus the direction but only with Ni2+ concentrations of 10 mM and 15 mM included. An anisotropy of only 1.2 is obtained from the fit of the ellipsoid to the data. In contrast, when considering only the very weakly bound DNA molecules, which show significant lateral mobility (2 mM and 3.5 mM Ni2+, Fig. 4(c)), the anisotropy is even more pronounced www.theNanoResearch.com∣www.Springer.com/journal/12274 \| Nano Research Nano Res. 2.4 Identification of discrete surface anchor points 2015, 8(6): 1811–1821 1818 Figure 5 Area plots depicting the cumulative movement of chain segments for representative DNA chains for different Ni2+ concentrations (left panels). Distinct regions of the DNA molecule are seen to be anchored at particular locations (indicated by arrows). The density of minima increases with Ni2+ concentrations, suggesting that Ni2+ ions provide specific anchor points on the surface to increase DNA surface association. The grey scale indicates successive frames in a time series. The panels on the right show the chain evolution with the minima location indicated by arrows. sequence, while others appear in subsequent frames, suggesting that specific surface interactions break and re-form continuously. This behavior is more prominent with molecules imaged with Ni2+ concentrations of 5 mM or lower. Although a direct correlation with surface distributions of Ni2+ would be purely speculative without further in depth studies, the results together with previous findings on the distribution of Ni2+ ions [30, 40] suggest that positively charged Ni2+ domains act as anchor points for the negatively charged DNA chains, with the interstitial regions governed by weak Mg2+ associations, allowing for the desired mobility. \| www.editorialmanager.com/nare/default.asp 3 Conclusion Interestingly, in some cases minima disappear over the course of the imaging We have developed an analytical method for quantifying the mobility of DNA under different surface association conditions, and we showed that for physiological conditions, the interaction of the DNA with the mica surface could be tuned from tightly surface-associated to highly mobile. This approach, therefore, provides a highly flexible method to establish \| www.editorialmanager.com/nare/default.asp Nano Res. 2015, 8(6): 1811–1821 1819 conditions which enable the investigation of a large range of nucleo-protein interactions. The presented analytical approach allowed us to investigate the impact of the AFM tip interactions with the surface- associated DNA. We showed that the overall transla- tional movement of the DNA is mostly isotropic for a stronger surface association, while increasing move- ment in the fast-scanning direction of the AFM tip is observed with decreasing strength of the surface association, indicating that the AFM tip-DNA molecule interaction contributes significantly to the overall movement. resolution of 128 × 128 pixels. The drive amplitude and set point used were typically around 3 V and 200 mV, respectively, but adjusted for optimum resolution as needed. Image analysis and chain fitting: AFM images were processed with a low-pass filter and a linear vector (chain) was fitted to the intensity peaks of the image using the ImageJ software (http://rsbweb.nih.gov/ij/) with the JFilament plugin [42]. Subsequently, the chains were split into >100 equidistant points and overlaid frame by frame (Fig. 1). Finally, the point- by-point differences between two sequential chains in a series were calculated and averaged to represent the chain movement in nm per 100 bp per second (nm·100 bp–1·s–1). This process is discussed in more detail in the main text. The cumulative movement of individual segments along DNA molecules was calculated by adding up the distances travelled by the segments between time frames. The distribution of segments along the chains of individual time frames was chosen so that local minima in movement were aligned. The tools presented here confer the ability to orchestrate and evaluate discrete biological interactions for novel applications in experimental biology and bionanotechnology. 4 Experimental section Reagents: Linear DNA (3.5 kbp) was amplified by PCR from a modified pGEM-T plasmid vector [41]. Linear DNA (890 bp) was amplified from lambda phage DNA. Magnesium-acetate, Tris-acetate, and nickel chloride, which were prepared as 100 mM stocks with a pH of 7.4, were all purchased from Sigma Aldrich, St Louis, USA. 4.1 HS-AFM sample preparation Mica (Agar Scientific, USA) was freshly cleaved and pre-incubated with NiCl2 (20 μl) at concentrations ranging from 1 mM to 15 mM for 1 minute. Excess NiCl2 solution was removed and DNA solution (20 μl) was immediately applied to the mica surface and incubated for 5 minutes. Subsequently, the surface was partially de-wetted. All DNA samples were prepared using 10 mM Tris-acetate (pH 7.4), 10 mM Magnesium- acetate, and 3.5 kbp DNA (10 ng). Mica samples were re-immersed in Tris-Mg buffer (200 μl) for imaging, with a small volume (50 μl) applied directly to the AFM cantilever. Electronic Supplementary Material: A short movie depicting the chain fitting process through a set of sequential AFM images is supplied as a visual aid to the description given in the results and discussion section (Movie S1). A short movie depicting the surface mobility of 3.5 kbp DNA fragments as a function of surface-immobilized Ni2+ concentration is supplied in support of Fig. 3 (Movie S2). Acknowledgements This work was funded in part by the EPSRC Centre for Doctoral Training in Molecular Scale Engineering, and WELMEC, a Centre of Excellence in Medical Engineering funded by the Wellcome Trust and EPSRC, under grant number WT 088908/Z/09/Z. The authors would like to acknowledge Alexander Dulebo, Hartmut Stadler and Bruker AXS, Karlsruhe for their time and contribution to this work, and the equipment funding from the BBSRC. References [1] Sharma, R.; Davies, A. G.; Wälti, C. Directed assembly of 3-nm-long RecA nucleoprotein filaments on double-stranded DNA with nanometer resolution. ACS Nano 2014, 8, 3322– 3330. [16] Guthold, M.; Bezanilla, M.; Erie, D. A.; Jenkins, B.; Hansma, H. G.; Bustamante, C. Following the assembly of RNA polymerase-DNA complexes in aqueous solutions with the scanning force microscope. Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 12927–12931. [2] Sharma, R.; Davies, A. G.; Wälti, C. Nanoscale programmable sequence-specific patterning of DNA scaffolds using RecA protein. Nanotechnology 2012, 23, 365301. [3] Sharma, R.; Davies, A. G.; Wälti, C. RecA protein mediated nano-scale patterning of DNA scaffolds. J. Nanosci. Nanotechnol. 2011, 11, 10629–10632. [17] Erie, D. A.; Yang, G. L.; Schultz, H. C.; Bustamante, C. DNA bending by Cro protein and nonspecific complexes: Implications for protein site recognition and specificity. Science. 1994, 266, 1562–1566. [4] Rothemund, P. W. K. Folding DNA to create nanoscale shapes and patterns. Nature 2006, 440, 297–302. [18] van Noort, J.; Orsini, F.; Eker, A.; Wyman, C.; de Grooth, B.; Greve, J. DNA bending by photolyase in specific and non-specific complexes studied by atomic force microscopy. Nucleic Acids Res. 1999, 27, 3875–3880. [5] Ando, T. High-speed atomic force microscopy coming of age. Nanotechnology 2012, 23, 062001. [6] Kodera, N.; Yamamoto, D.; Ishikawa, R.; Ando, T. Video imaging of walking myosin V by high-speed atomic force microscopy. Nature 2010, 468, 72–76. [19] Leuba, S. H.; Yang, G.; Robert, C.; Samori, B.; van Holde, K.; Zlatanova, J.; Bustamante, C. Three-dimensional structure of extended chromatin fibers as revealed by tapping-mode scanning force microscopy. Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 11621–11625. [7] Hansma, H. G.; Bezanilla, M.; Zenhausern, F.; Adrian, M.; Sinsheimer, R. L. Atomic force microscopy of DNA in aqueous solutions. Nucleic Acids Res. 1993, 21, 505–512. [8] Hansma, H. G.; Laney, D. E.; Bezanilla, M.; Sinsheimer, R. L.; Hansma, P. K. Applications for atomic force microscopy of DNA. Biophys. J. 1995, 68, 1672–1677. [20] Sorel, I.; Piétrement, O.; Hamon, L.; Baconnais, S.; Le Cam, E.; Pastré, D. The EcoRI-DNA complex as a model for investigating protein-DNA interactions by atomic force microscopy. Biochemistry 2006, 45, 14675–14682. [9] van Noort, S. J. T.; van der Werf, K. O.; Eker, A. P. M.; Wyman, C.; de Grooth, B. G.; van Hulst, N. F.; Greve, J. Direct visualization of dynamic protein-DNA interactions with a dedicated atomic force microscpe. Biophys. J. 4.2 HS-AFM imaging in aqueous buffer All samples were imaged with a Dimension FastScan using FastScan-DX probes (Bruker AXS, USA). All images were collected at a scan rate of 38 Hz and a Additionally, AFM images showing cleavage of DNA strands by the EcoRI enzyme (Figs. S1 and S2), statistical analysis of the cleavage efficiency (Table S1), www.theNanoResearch.com∣www.Springer.com/journal/12274 \| Nano Research 1820 Nano Res. 2015, 8(6): 1811–1821 2007, 107, 184–190. and a short movie showing a single cleavage event (Movie S3) are provided. [12] Gilmore, J. L.; Suzuki, Y.; Tamulaitis, G.; Siksnys, V.; Takeyasu, K.; Lyubchenko, Y. L. Single-molecule dynamics of the DNA-EcoRII protein complexes revealed with high- speed atomic force microscopy. Biochemistry 2009, 48, 10492–10498. This information is available in the online version of this article at http://dx.doi.org/10.1007/s12274-014- 0681-y. [13] Suzuki, Y.; Shin, M.; Yoshida, A.; Yoshimura, S. H.; Takeyasu, K. Fast microsopical dissection of action scenes played by Escherichia coli RNA polymerase. FEBS Lett. 2012, 586, 3187–3192. Open Access: This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. [14] Binnig, G.; Quate, C. F.; Gerber, C. Atomic force microscope. Phys. Rev. Lett. 1986, 56, 930–933. [15] Kasas, S.; Thomson, N. H.; Smith, B. L.; Hansma, H. G.; Zhu, X. S.; Guthold, M.; Bustamante, C.; Kool, E. T.; Kashlev, M.; Hansma, P. K. Escherichia coli RNA polymerase activity observed using atomic force microscopy. Biochemistry 1997, 36, 461–468. References 1998, 74, 2840–2849. [21] Balmer, T. E.; Christenson, H. K.; Spencer, N. D.; Heuberger, M. The effect of surface ions on water adsorption to mica. Langmuir 2008, 24, 1566–1569. [22] Hansma, H. G.; Laney, D. E. DNA binding to mica correlates with cationic radius: Assay by atomic force. Biophys. J. 1996, 70, 1933–1939. [10] Thomson, N. H.; Fritz, M.; Radmacher, M.; Cleveland, J. P.; Schmidt, C. F.; Hansma, P. K. Protein tracking and detection of protein motion using atomic force microscopy. Biophys. J. 1996, 70, 2421–2431. [23] Lyubchenko, Y. L.; Shlyakhtenko, L. S.; Gall, A. A. Atomic force microscopy imaging and probing of DNA, proteins, and protein DNA complexes: Silatrane surface chemistry. Methods Mol. Biol. 2009, 543, 337–351. [11] Kobayashi, M.; Sumitomo, K.; Torimitsu, K. Real-time imaging of DNA–streptavidin complex formation in solution using a high-speed atomic force microscope. Ultramicroscopy \| www.editorialmanager.com/nare/default.asp \| www.editorialmanager.com/nare/default.asp 1821 Nano Res. 2015, 8(6): 1811–1821 theoretical and experimental study. Biophys. J. 2003, 85, 2507–2518. [24] Josephs, E. A.; Ye, T. Electric-field dependent conformations of single DNA molecules on a model biosensr surface. Nano Lett. 2012, 12, 5255–5261. [34] Ellis, J. S.; Abdelhady, H. G.; Allen, S.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. Direct atomic force microscopy observations of monvalent ion induced binding of DNA to mica. J. Microsc. 2004, 215, 297–301. [25] Josephs, E. A.; Ye, T. A single-molecule view of confor- mational switching of DNA tethered to a gold electrode. J. Am. Chem. Soc. 2012, 134, 10021–10030. [35] Pastré, D.; Hamon, L.; Landousy, F.; Sorel, I.; David, M.-O.; Zozime, A.; Le Cam, E.; Piétrement, O. Anionic polyelectrolyte adsorption on mica mediated by multivalent cations: A solution to DNA imaging by atomic force microscopy under high ionic strengths. Langmuir 2006, 22, 6651–6660. [26] Piétrement, O.; Pastre, D.; Fusil, S.; Jeusset, J.; David, M.-O.; Landousy, F.; Hamon, L.; Zozime, A.; Le Cam, E. Reversible binding of DNA on NiCl2-treated mica by varying the ionic strength. Langmuir 2003, 19, 2536–2539. [27] Abel, G. R.; Josephs, E. A.; Luong, N.; Ye, T. A switchable surface enables visualization of single DNA hybridization events with atomic force microscopy. J. Am. Chem. Soc. 2013, 135, 6399–6402. [36] Jahnen-Dechent, W.; Ketteler, M. Magnesium basics. Clin. Kidney J. 2012, 5, i3–i14. [37] Lyubchenko, Y. L.; Shlyakhtenko, L. S.; Ando, T. Imaging of nucleic acids with atomic force microscopy. Methods 2011, 54, 274–283. References [28] Erdmann, M.; David, R.; Fornof, A.; Gaub, H. E. Electrically controlled DNA adhesion. Nat. Nanotechnol. 2010, 5, 154– 159. [38] Guthold, M.; Zhu, X. S.; Rivetti, C.; Yang, G. L.; Thomson, N. H.; Kasas, S.; Hansma, H. G.; Smith, B.; Hansma, P. K.; Bustamante, C. Direct obsercation of one-dimensional diffusion and transcription by Escherichia Coli RNA polymerase. Biophys. J. 1999, 77, 2284–2294. [29] Endo, M.; Sugiyama, H. Single-molecule imaging of dynamic motions of biomolecules in DNA origami nanostructures using high-speed atomic force microscopy. Acc. Chem. Res. 2014, 47, 1645–1653. [30] Hsueh, C.; Chen, H. J.; Gimzewski, J. K.; Reed, J.; Abdel- Fattah, T. M. Localized nanoscopic surface measurements of nickel-modified mica for single-molecule DNA sequence sampling. ACS Appl. Mater. Interfaces 2010, 2, 3249–3256. [39] Bennink, M. L.; Nikova, D. N.; van der Werf, K. O.; Greve, J. Dynamic imaging of single DNA–protein interactions using atomic force. Anal. Chim. Acta 2003, 479, 3–15. [40] Suzuki, Y.; Higuchi, Y.; Hizume, K.; Yokokawa, M.; Yoshimura, S. H.; Yoshikawa, K.; Takeyasu, K. Molecular dynamics of DNA and nucleosomes in solution studied by fast-scanning atomic force microscopy. Ultramicroscopy 2010, 110, 682–688. [31] Billingsley, D. J.; Lee, A. J.; Johansson, N. A. B.; Walton, A.; Stanger, L.; Crampton, N.; Bonass, W. A.; Thomson, N. H. Patchiness of ion-exchanged mica revealed by DNA binding dynamics at short length scales. Nanotechnology 2014, 25, 025704. [41] Tosch, P.; Wälti, C. Middelberg, A. P. J.; Davies, A. G. Generic technique to generate large branched DNA complexes. Biomacromolecules 2006, 7, 677–681. [32] Zheng, J. P.; Li, Z.; Wu, A. G.; Zhou, H. L. AFM studies of DNA structures on mica in the presence of alkaline earth metal ions. Biophys. Chem. 2003, 104, 37–43. [42] Smith, M. B.; Li, H. S.; Shen, T.; Huang, X. L.; Yusuf, E.; Vavylonis, D. Segmentation and tracking of cytoskeletal filaments using open active contours. Cytoskeleton 2010, 67, 693–705. [33] Pastré, D.; Piétrement, O.; Fusil, S.; Landousy, F.; Jeusset, J.; David, M.-O.; Hamon, L.; Le Cam, E.; Zozime, A. Adsorption of DNA to mica mediated by divalent counterions: A www.theNanoResearch.com∣www.Springer.com/journal/12274 \| Nano Research www.theNanoResearch.com∣www.Springer.com/journal/12274 \| Nano Research
https://openalex.org/W4392352084	https://prin.or.id/index.php/nusantara/article/download/2712/2454	Indonesian	null	Edukasi Pencegahan Stunting di Kelurahan Pedurungan Tengah, Kota Semarang	Nusantara	2,024	cc-by-sa	1,909	g y Abstrak Stunting pada balita merupakan masalah kesehatan global yang serius, terutama di Kota Semarang. Kegiatan pengabdian masyarakat dilakukan di Kelurahan Pedurungan Tengah. Tujuan penelitian ini adalah untuk meningkatkan pemahaman tentang stunting dan pencegahannya. Metode ceramah dan diskusi digunakan untuk penyampaian materi, serta pembagian leaflet sebagai sumber informasi tambahan. Hasil kegiatan menunjukkan partisipasi yang baik dari 32 peserta. Diskusi aktif menunjukkan minat peserta dalam memahami topik stunting. Pengabdian masyarakat ini juga menyoroti pentingnya pengetahuan ibu terkait stunting, mengingat risiko stunting meningkat pada ibu dengan pengetahuan rendah. Pelaksanaan kegiatan diakhiri dengan evaluasi yang menunjukkan hasil yang memuaskan. Pengabdian masyarakat ini diharapkan dapat membantu meningkatkan pengetahuan dan kesadaran masyarakat tentang stunting, serta memberikan kontribusi dalam upaya pencegahan stunting di Kota Semarang. Kata Kunci: Stunting, Kesehatan Masyarakat, Pengetahuan Ibu * Silvia Nurvita, silviaunkartur@gmail.com NUSANTARA: Jurnal Pengabdian Kepada Masyarakat Vol. 4, No. 2 Mei 2024 e-ISSN: 2962-4800; p-ISSN: 2962-360X, Hal 42-47 DOI: https://doi.org/10.55606/nusantara.v4i2.2712 Edukasi Pencegahan Stunting di Kelurahan Pedurungan Tengah, Kota Semarang Education to Prevent Stunting in Pedurungan Tengah Village, Semarang City Silvia Nurvita 1, Siti Noor Chotimah2, Dody Indra Sumantiawan3, Viny Natalia Dewi4, Aerrosa Murenda Mayadilanuari5 1,2,3,4 Universitas Nasional Karangturi, Kota Semarang, Indonesia 5 Kaohsiung Medical University, Kaohsiung, Taiwan korespondensi Penulis : silviaunkartur@gmail.com Silvia Nurvita 1, Siti Noor Chotimah2, Dody Indra Sumantiawan3 Viny Natalia Dewi4, Aerrosa Murenda Mayadilanuari5 1,2,3,4 Universitas Nasional Karangturi, Kota Semarang, Indonesia 5 Kaohsiung Medical University, Kaohsiung, Taiwan korespondensi Penulis : silviaunkartur@gmail.com Abstract: Stunting in children under five years old is a serious global health problem, especially in Semarang City. Community service activities were carried out in Pedurungan Tengah Village. The aim of this study was to increase the understanding about stunting and its prevention. Discussion method was used to deliver the topics, as well as distributing leaflets as a source of additional information. The results of the activity showed good participation from 32 participants. Active discussions showed participants' interest in understanding the topic of stunting. This community service also highlights the importance of maternal knowledge regarding stunting, considering that the risk of stunting increases in mothers with low knowledge. The implementation of the activity ended with an evaluation that showed satisfactory results. It is hoped that this community service can help increase public knowledge and awareness about stunting, as well as contribute to efforts to prevent stunting in Semarang City. Article History: Received: Januari 31, 2024 Accepted: February 29, 2024 Published: Mei 31, 2024 Article History: Received: Januari 31, 2024 Accepted: February 29, 2024 Published: Mei 31, 2024 Keywords: Stunting, Public Health, Mother's Level Of Knowledge Keywords: Stunting, Public Health, Mother's Level Of Knowledge PENDAHULUAN Balita merupakan salah satu kelompok yang rentan terhadap masalah kesehatan, terutama masalah gizi kurang atau buruk. Usia balita sering disebut periode emas karena dalam periode ini terjadi perkembangan saraf otak khususnya mielinisasi sekaligus periode kritis. Periode emas dapat tercapai apabila kebutuhan gizi balita terpenuhi secara optimal. Status gizi balita adalah keadaan kesehatan balita yang ditentukan oleh derajat kebutuhan fisik akan energi dan zat-zat gizi yang diperoleh dari zat pangan atau makanan yang dampak fisiknya dapat diukur dengan antropometri. Edukasi Pencegahan Stunting di Kelurahan Pedurungan Tengah, Kota Semarang Menurut data dari WHO, di seluruh dunia, 178 juta anak di bawah usia lima tahun diperkirakan mengalami pertumbuhan terhambat karena stunting. Stunting adalah permasalahan gizi kronis yang disebabkan oleh kurangnya asupan gizi dalam rentang yang cukup waktu lama, umumnya hal ini karena asupan makan yang tidak sesuai dengan kebutuhan gizi. Permasalahan stunting terjadi mulai dari dalam kandungan dan baru akan terlihat ketika anak sudah menginjak usia dua tahun. Stunting atau pendek adalah status gizi yang ditandai dengan gangguan pertumbuhan (pendek) berdasarkan parameter atropetri tinggi badan yaitu Panjang Badan menurut Umur (PB/U) atau Tinggi Badan menurut Umur (TB/U). Hasil pengukuran berada pada ambang batas (Z-Score) <-2 SD sampai dengan -3 SD (pendek/stunted) dan <-3 SD (sangat pendek/severely stunted). Stunting adalah masalah kurang gizi kronis yang disebabkan oleh asupan zat gizi yang kurang dalam waktu cukup lama akibat pemberian makanan yang tidak sesuai dengan kebutuhan gizi. Stunting merupakan dampak dari berbagai faktor seperti berat lahir yang rendah, stimulasi dan pengasuhan anak kurang tepat, asupan nutrisi kurang, dan infeksi berulang serta berbagai faktor lingkungan lainnya. Stunting terjadi dimulai dari janin dalam kandungan serta akan nampak saat anak berusia dua tahun. Kekurangan zat gizi pada anak usia dini dapat meningkatkan angka kematian bayi dan anak, menyebabkan penderitanya mudah terserang penyakit, dan akan memiliki postur tubuh tidak maksimal saat dewasa. Stunting di Kota Semarang pada bulan Januari 2023 sampai dengan November 2023 terdapat kasus stunting sebanyak 912 kasus dengan Kecamatan Pedurungan menduduki peringkat 4 di Semarang. Berdasarkan latar belakang tersebut, maka perlu adanya kegiatan pengabdian masyarakat tentang edukasi cegah stunting sebelum genting agar masyarakat lebih sadar terhadap pengendalian kasus stunting di lingkungan keluarga. METODE Kegiatan pengabdian masyarakat ini dilakukan bagi penduduk di Kelurahan Pedurungan Tengah, Kota Semarang. Pengabdian masyarakat ini berlangsung pada 19 Januari 2024. Sampel kegiatan pengabdian masyarakat ini adalah warga yang berdomisili di Kelurahan Pedurungan Tengah sebanyak 32 orang. Tahapan pengabdian masyarakat ini diawali dengan survei pendahuluan, penentuan topik dan tujuan pengabdian masyarakat, pembuatan materi pengabdian masyarakat, pelaksanaan pengabdian masyarakat, dan evaluasi kegiatan pengabdian masyarakat. 43 NUSANTARA- Vol. 4, No. 2 Mei 2024 Kegiatan pengabdian masyarakat ini bertujuan untuk meningkatkan edukasi e-ISSN: 2962-4800; p-ISSN: 2962-360X, Hal 42-47 masyarakat di Kelurahan Pedurungan Tengah tentang bahanya stunting beserta pencegahannya. Mekanisme pelaksanaan pengabdian masyarakat yaitu metode ceramah dan diskusi tanya jawab. Pemateri memberikan materi dalam bentuk ceramah dengan bantuan Power Point yang berisi materi. Selanjutnya dilakukan diskusi dengan peserta terkait materi yang diberikan dan pembagian leaflet ke peserta. Tahap akhir kegiatan pengabdian masyarakat ini yaitu evaluasi. Evaluasi untuk mengetahui terjadinya peningkatan pengetahuan atau tidak dilakukan dengan cara menilai hasil diskusi dengan warga. HASIL Kegiatan pengabdian masyarakat telah dilaksanakan pada 18 Januari 2024 di Kelurahan Pedurungan Tengah dengan jumlah peserta sebanyak 32 orang. Peserta yang hadir dalam acara ini sudah melebihi target yang awalnya ditargetkan akan dihadiri minimal 15 peserta namun yang hadir sebanyak 32 peserta. Dalam pengabdian masyarakat ini juga memasang poster cegah stunting di depan pintu masuk supaya para peserta dapat membacanya (Gambar 1). Sebelum memulai sesi penyampaian materi, asisten narasumber membantu membagikan lembar materi yang berisi tentang pengertian stunting hingga cara pencegahan stunting. Lembar materi yang dibagikan ini bertujuan untuk memberikan informasi kepada peserta yang menghadiri kegiatan pengabdian kepada masyarakat ini dan serta dapat sekaligus menyebarkan informasi terkait stunting tersebut pada keluarga, kerabat maupun masyarakat lainnya yang berhalangan hadir saat kegiatan ini berlangsung. Materi ceramah yang disosialisasikan berisi tentang definisi stunting hingga cara untuk mencegah stunting sebelum semakin terlambat (Gambar 2). Diskusi ceramah berlangsung dengan baik dan beberapa peserta aktif bertanya terkait materi stunting yang telah diberikan (Gambar 3 dan Gambar 4) Gambar 1. Poster Cegah Stunting Sebelum Genting Gambar 1. Poster Cegah Stunting Sebelum Genting Edukasi Pencegahan Stunting di Kelurahan Pedurungan Tengah, Kota Semarang Edukasi Pencegahan Stunting di Kelurahan Pedurungan Tengah, Kota Semarang Edukasi Pencegahan Stunting di Kelurahan Pedurungan Tengah, Kota Semarang Gambar 2. Pemberian Materi Terkait Stunting Gambar 3. Sesi Tanya Jawab Gambar 4. Penutupan Kegiatan Pengabdian Masyarakat USI Prevalensi kasus stunting pada baduta (sebutan yang ditujukan untuk anak usia bawah Gambar 2 Pemberian Materi Terkait Stunting ARA- Vol. 4, No. 2 Mei 2024 Gambar 2. Pemberian Materi Terkait Stunting Gambar 3. Sesi Tanya Jawab Gambar 4. Penutupan Kegiatan Pengabdian Masyarakat nsi kasus stunting pada baduta (sebutan yang ditujukan untuk anak sekitar 0-24 bulan) sebanyak 22,6% di wilayah kerja Puskesmas Gambar 2. Pemberian Materi Terkait Stunting Gambar 2. Pemberian Materi Terkait Stunting g Gambar 3 Sesi Tanya Jawab Gambar 3. Sesi Tanya Jawab Gambar 4. Penutupan Kegiatan Pengabdian Masyarakat Gambar 4. Penutupan Kegiatan Pengabdian Masyarakat NUSANTARA- Vol. 4, No. 2 Mei 2024 DISKUSI Prevalensi kasus stunting pada baduta (sebutan yang ditujukan untuk anak usia bawah dua tahun atau sekitar 0-24 bulan) sebanyak 22,6% di wilayah kerja Puskesmas Bandarharjo Prevalensi kasus stunting pada baduta (sebutan yang ditujukan untuk anak usia bawah dua tahun atau sekitar 0-24 bulan) sebanyak 22,6% di wilayah kerja Puskesmas Bandarharjo Prevalensi kasus stunting pada baduta (sebutan yang ditujukan untuk anak usia bawah dua tahun atau sekitar 0-24 bulan) sebanyak 22,6% di wilayah kerja Puskesmas Bandarharjo NUSANTARA- Vol. 4, No. 2 Mei 2024 45 45 e-ISSN: 2962-4800; p-ISSN: 2962-360X, Hal 42-47 Semarang. Bahkan, Ibu dengan pengetahuan Responsive Feeding (RF) rendah berisiko 10,2 kali lebih besar memiliki anak stunting dibandingkan dengan ibu berpengetahuan cukup. Sehingga penting untuk meningkatkan pengetahuan ibu terkait stunting di Kota Semarang. Karena menurut penelitian pada tahun 2022, tingkat pengetahuan memiliki pengaruh pada kejadian stunting di Prambanan. Menurut Notoatmodjo (2007), dengan metode ceramah dan diskusi dapat terjadi proses perubahan perilaku kearah yang diharapkan melalui peran aktif sasaran dan saling tukar pengalaman sesama sasaran dalam penyuluhan kesehatan. Sehingga metode ceramah dan diskusi dipilih pada pengabdian masyarakat ini. Pembagian leaflet bagi para peserta diharapkan dapat meningkatkan pengetahuan bagi para peserta serta dapat dibawa pulang kerumah. Manfaat leaflet dalam promosi kesehatan yaitu leaflet memiliki manfaat untuk menjelaskan materi secara lebih rinci dan komperhensif. Pelaksanaan kegiatan masyarakat ini diakhiri dengan sesi tanya jawab, lima peserta aktif bertanya terkait hipertensi. Pemateri memberikan 3 pertanyaan terkait hipertensi ke peserta sebagai bahan evaluasi pemahaman peserta selama diberikan materi sosialisasi. Tiga peserta dapat menjawab dengan benar semua pertanyaan terkait pengertian hipertensi, faktor- faktor hipertensi, dan pencegahan hipertensi. Kemudian peserta yang menjawab pertanyaan dengan benar semua diberikan souvenir. KESIMPULAN Pengabdian masyarakat di Kelurahan Pedurungan Tengah telah berjalan dengan baik dan dapat menambah pengetahuan peserta tentang pencegahan stunting di keluarga masing- masing peserta. PENGAKUAN Terima kasih atas partisipasi warga di Kelurahan Pedurungan Tengah. Serta terima kasih atas dukungan dan pendanaan dari Universitas Nasional Karangturi Semarang. Apriluana, Gladys, and Sandra Fikawati. "Analisis Faktor-Faktor Risiko Terhadap Kejadian Stunting Pada Balita (0-59 Bulan) Di Negara Berkembang Dan Asia Tenggara." Media Penelitian dan Pengembangan Kesehatan 28, no. 4 (2018): 247-56. De Onis, Mercedes, and Francesco Branca. "Childhood Stunting: A Global Perspective." Maternal & child nutrition 12 (2016): 12-26. DAFTAR REFERENSI Apriluana, Gladys, and Sandra Fikawati. "Analisis Faktor-Faktor Risiko Terhadap Kejadian Stunting Pada Balita (0-59 Bulan) Di Negara Berkembang Dan Asia Tenggara." Media Penelitian dan Pengembangan Kesehatan 28, no. 4 (2018): 247-56. De Onis, Mercedes, and Francesco Branca. "Childhood Stunting: A Global Perspective." Maternal & child nutrition 12 (2016): 12-26. Edukasi Pencegahan Stunting di Kelurahan Pedurungan Tengah, Kota Semarang Devianto, Aan, Eltanina Ulfameytalia Dewi, and Dita Yustiningsih. "Hubungan Tingkat Pengetahuan Ibu Tentang Stunting Dengan Angka Kejadian Stunting Di Desa Sanggrahan Prambanan Klaten: The Correlation of Mother's Knowledge Levels About Stunting with Stunting Event Rate in Sanggrahan Prambanan Village Klaten." Journal Nursing Research Publication Media (NURSEPEDIA) 1, no. 2 (2022): 81-88. Dinkes Semarang. Profil Kesehatan Kota Semarang. Semarang: DKK.( 2023). Ifroh, Riza Hayati, Rahmi Susanti, Lies Permana, and Reny Noviasty. "Peran Petugas Promosi Kesehatan Dalam Penggunaan Audiovisual Sebagai Media Komunikasi Informasi Dan Edukasi." Jurnal Ilmu Kesehatan Vol 7, no. 2 (2019). Notoatmodjo, Soekidjo. "Pendidikan Dan Promosi Kesehatan." Jakarta: Rineka Cipta Probohastuti, Nadia Feryka, and Aloysius Rengga. "Implementation of Nutrition-Sensitive Interventions Policy for Stunting Decrease in Blora Regency." Journal of Public Policy and Management Review 8, no. 4 (2019): 251-66. Septamarini, Risna Galuh, Nurmasari Widyastuti, and Rachma Purwanti. "Hubungan Pengetahuan Dan Sikap Responsive Feeding Dengan Kejadian Stunting Pada Baduta Usia 6-24 Bulan Di Wilayah Kerja Puskesmas Bandarharjo, Semarang." Journal of Nutrition College 8, no. 1 (2019): 9-20. Widyastuti, Ririn, and Martinus V Ndona. "Reducing and Preventing Stunting through Integrated Posyandu with Fathers and or Child Caregivers (Posyandu Bersayap) at Watukapu Public Health Center in Ngada Regency, East Nusa Tenggara Province, Indonesia." GHMJ (Global Health Management Journal) 5, no. 1 (2022): 70-74. NUSANTARA- Vol. 4, No. 2 Mei 2024 47 47
https://openalex.org/W4213262988	https://www.frontiersin.org/articles/10.3389/feart.2022.824453/pdf	English	null	Distribution and Controlling Growth Factors of Ooids in Qinghai Lake, Northern Tibet Plateau, China	Frontiers in earth science	2,022	cc-by	7,990	ORIGINAL RESEARCH published: 21 February 2022 doi: 10.3389/feart.2022.824453 Distribution and Controlling Growth Factors of Ooids in Qinghai Lake, Northern Tibet Plateau, China Lewei Hao 1, Huifei Tao 1, Shutong Li 1, Xiaofeng Ma 1, Hongjie Ji 2 and Junli Qiu 1 1Key Laboratory of Petroleum Resources, Northwest Institute of Eco-Environment and Resources, Chinese Academy of Sciences, Lanzhou, China, 2State Key Laboratory for Nuclear Resources and Environment, East China University of Technology, Nanchang, China Ooids are coated carbonate grains, which exist in shallow water marine and lacustrine environments. There is an ongoing debate about whether the origin of ooids is inorganic or organic. Qinghai Lake is the largest inland lake in China, and ooids are seen on the lake shore. This paper focuses on whether environmental energy has an impact on the growth and size of ooids. Through hydrochemical analysis, thin section observation, and scanning electron microscope, the carbonate coats of beach sands from Qinghai Lake were studied. The research shows that the carbonate-coated grain content from the different shores of the lake present variations. The hydrodynamics and particularly the waves seem to control the distribution of carbonate coats in the lake shore, not the hydrochemical condition. In addition, the integrity and thickness of carbonate coats from the shores with a strong hydrodynamic force are high and thick, respectively. The carbonate coats are often observed on medium-grained sands, and the maximum carbonate-coated grain occurred under the strongest waves, indicating that ooids can be produced only when hydrodynamic force and particle size are well matched. Bacteria or extracellular polymeric substances are not observed within the ooid cortices by scanning electron microscopy. So, bacteria may not be a major factor in the formation and growth of ooids, but hydrodynamic forces appear to play a great role in carbonate grain coat distribution, integrity, thickness, and ooid grain size. Edited by: Zhang Chengjun, Lanzhou University, China Reviewed by: Yanzhong Wang, China University of Petroleum, (East China) China Meiyan Fu, Chengdu University of Technology, China Correspondence: Lewei Hao lwhao@lzb.ac.cn Huifei Tao tophic3@yeah.net Edited by: Zhang Chengjun, Lanzhou University, China Reviewed by: Yanzhong Wang, China University of Petroleum, (East China) China Meiyan Fu, Chengdu University of Technology, China Reviewed by: Yanzhong Wang, China University of Petroleum, (East China) China Meiyan Fu, Chengdu University of Technology, China Correspondence: Lewei Hao lwhao@lzb.ac.cn Huifei Tao tophic3@yeah.net Keywords: Qinghai Lake, hydrodynamics, grain size, hydrochemical condition, carbonate coats Specialty section: This article was submitted to Sedimentology, Stratigraphy and Diagenesis, a section of the journal Frontiers in Earth Science Received: 29 November 2021 Accepted: 18 January 2022 Published: 21 February 2022 INTRODUCTION Ooids are poorly to well-developed cortices accreted around a nucleus, which are found in both ancient and modern sedimentary environments (Ball, 1967; Harris et al., 1979; Beukes, 1983; Simonson and Jarvis, 1993; Li et al., 2013; Liu et al., 2021). Ooids have been used as paleoclimatic and paleoceanographic proxies for sea level, redox state, water depth, temperature, salinity, and hydrodynamic environment (Kump and Hine, 1986; Opdyke and Wilkinson, 1990; Lu et al., 2020). It has been debated for over a hundred years whether the formation of ooids is inorganic or organic. The most widely accepted ooid genesis is that ooids are inorganic carbonates formed by chemical precipitation from shallow, warm, carbonate-saturated, and agitated water (Davies et al., 1978; Tucker and Wright, 1990; Sumner and Grotzinger, 1993; Duguid et al., 2010; Trower et al., 2017). Field and laboratory studies suggest that tangential ooids are inorganic precipitated during agitated conditions, whereas radial ooids are formed in quiet water with the participation of organic Keywords: Qinghai Lake, hydrodynamics, grain size, hydrochemical condition, carbonate coats Citation: Hao L, Tao H, Li S, Ma X, Ji H and Qiu J (2022) Distribution and Controlling Growth Factors of Ooids in Qinghai Lake, Northern Tibet Plateau, China. Front. Earth Sci. 10:824453. doi: 10.3389/feart.2022.824453 Hao L, Tao H, Li S, Ma X, Ji H and Qiu J (2022) Distribution and Controlling Growth Factors of Ooids in Qinghai Lake, Northern Tibet Plateau, China. Front. Earth Sci. 10:824453. doi: 10.3389/feart.2022.824453 February 2022 \| Volume 10 \| Article 824453 Frontiers in Earth Science \| www.frontiersin.org 1 Hao et al. Controlling Growth Factors of Ooids FIGURE 1 \| Location of the study area (satellite data taken from Google Earth). FIGURE 1 \| Location of the study area (satellite data taken from Google Earth). matter (Kahle, 1974; Davies and Martin, 1976; Davies et al., 1978; Diaz and Eberli, 2019). Microbes may be a key role in the formation of ooids. Metabolic activities of microbes induce pH changes resulting in precipitation and extracellular polymeric substances (EPS) providing nucleation sites for carbonate mineral growth (Dupraz and Visscher, 2005; Diaz and Eberli, 2019). Photosynthetic microbes not only dominate carbonate precipitation in the early stages of ooids but also control the formation of the entire cortex in ooids from Lake Geneva (Plée et al., 2008; Pacton et al., 2012). Microorganisms are found to be important in the formation of ooids. However, Trower et al. (2017) demonstrated that the physical environment is more important generally in controlling ooid growth in laboratory experiments. The size of ooids is determined by a balance between growth by chemical precipitation and erosion by abrasion (Trower et al., 2017; Sipos et al., 2018). This oratory result lacks validation with ﬁeld data (Diaz and Eberli, 2019). Ariztegui et al. (2012) also showed a little inﬂuence of environmental energy on the size and form of the ooids in western Lake Geneva, Switzerland. So, whether the environmental energy has an impact on the growth of ooids needs more ﬁeld data. ocean and some particles with discontinuous or patchy micritic coatings (Davaud and Girardclos, 2001). In the Qinghai Lake located in the northeastern Tibetan Plateau (Figure 1), aragonitic ooids are found in the eastern lake shore (LZIG, 1979). However, further works about the origin, distribution, and morphology of ooids in Qinghai Lake have not been studied. Citation: The aims of this paper are to describe the distribution of the coated grains in Qinghai Lake and analyze the factors controlling their distribution and growth, especially environmental energy. Study area Qinghai Lake is the largest inland saltwater lake in China, which is located in the northeastern Tibetan Plateau (36°32′–37°15′N, 99°36′–100°47′E) (Figure 1). It is a closed lake with an area of 4,400 km2 and a catchment area of ca 2.96 × 104 km2 (Fan et al., 1994). The salinity is from 12.25 to 13.2 g/L, and the south of the lake is higher than the northern lake (LZIG, 1979). The lake transformed from open to close due to the tectonic uplift of the Riyue Mountains during the Middle to Late Pleistocene (Li and Fang, 1999; Yuan et al., 1990). Qinghai Lake is situated in a semiarid area inﬂuenced by the East Asian monsoon, Indian monsoon, and westerly winds (An et al., 2012). The average annual temperature is −0.7°C, and the average water depth is 21 m in the lake (Fan et al., 1994). The main rivers ﬂowing into Qinghai Lake are Buha River, Quanji River, Shaliu River, Haergai River, and Heima River, and the other rivers are small and seasonal (Figure 1) (LZIG, 1979; Li et al., 2007). The two meteorological stations Eri and Yl are located in the north and south of the lake, respectively (Figure 1). Eri Station located in the northern lake displays a bidirectional wind regime, and the wind predominantly blows from ESE and NE. The mean wind speed is 4.6 m/s (Hu et al., 2021). The prevailing winds at Yl Station located in the Most of the studies on modern ooids are concentrated in the ocean especially the Bahamian archipelago, which have been extensively studied (Ball, 1967; Harris et al., 1979; Wanless and Tedesco, 1993; Rankey et al., 2006; Harris et al., 2011). Besides the ocean, ooids are also found in lakes, such as the Great Salt Lake, Utah (Kahle, 1974; Halley, 1977), Pyramid Lake, Nevada (Popp and Wilkinson, 1983), Higgins Lake, Michigan (Wilkinson et al., 1980), and Lake Geneva, Switzerland (Davaud and Girardclos, 2001; Plée et al., 2008; Pacton et al., 2012). Most of these lacustrine ooids show a smaller size compared with the February 2022 \| Volume 10 \| Article 824453 Frontiers in Earth Science \| www.frontiersin.org 2 Controlling Growth Factors of Ooids Hao et al. TABLE 1 \| Hydrochemical properties and major chemical constituents of water samples from the study area (ionic concentrations in mg/L). Study area Sample type Sample Ca2+ K+ Mg2+ Na+ Cl− SO4 2- CO3 2- HCO3 − pH S River BHH 38.75 1.98 15.66 30.00 32.39 53.62 2.89 176.10 8.21 - DTH 66.96 7.17 36.96 99.33 132.48 72.30 17.32 346.34 8.43 - HEG 56.43 1.88 22.69 31.35 14.58 43.29 17.32 193.71 8.37 - HMH 29.58 3.25 13.38 52.17 30.02 22.77 5.77 205.45 8.44 - JHH1 74.55 2.58 9.73 32.58 20.01 29.94 5.77 217.19 8.38 - JHH2 55.62 3.66 7.75 30.51 22.77 22.86 5.77 146.75 8.30 - LNH1 78.00 2.02 21.21 42.63 48.69 28.29 5.77 281.77 8.32 - LNH2 72.39 3.45 18.00 39.66 43.41 45.06 8.66 211.32 8.34 - QJH 62.88 2.24 13.35 32.04 23.73 26.64 5.77 199.58 8.36 - SLH 27.00 1.89 14.99 75.33 35.94 96.24 5.77 187.84 8.37 - Lake DS1 8.23 160.56 688.20 2,480.54 4,602.00 1,922.40 288.70 851.17 8.87 11.84 DS2 7.23 160.62 706.20 2,490.48 4,765.80 1,960.20 288.70 851.17 8.86 10.18 HX1 6.38 156.72 679.80 2,830.22 4,486.80 1,860.60 317.57 836.49 8.94 10.02 HX2 7.87 162.00 721.20 2,903.49 5,092.20 2,148.60 317.57 821.82 8.95 11.34 HX3 8.26 142.20 607.20 2,568.00 4,066.20 1,710.00 317.57 821.82 8.91 14.20 JH1 6.59 118.32 472.20 2,247.76 3,290.40 1,396.20 288.70 777.79 8.90 12.43 JH2 6.58 119.40 452.80 2,264.74 2,079.00 931.20 303.13 719.09 8.98 15.58 LD1 9.54 104.04 430.50 2,118.60 3,911.40 1,639.20 274.26 704.41 9.04 16.35 LD2 9.35 127.44 505.74 2,192.40 2,698.20 1,171.20 274.26 645.71 9.00 18.04 LD3 10.15 121.50 530.58 2,176.20 3,381.60 1,430.40 274.26 645.71 9.02 17.71 LN1 7.08 192.72 765.60 2,725.00 4,813.80 1,971.00 346.44 763.12 8.99 11.40 LN2 5.44 164.52 738.00 2,390.94 4,686.60 1,899.00 360.87 733.77 9.03 9.55 LN3 7.48 113.28 486.54 1,936.80 3,057.00 1,261.20 245.39 851.17 8.84 12.71 QU1 9.14 107.58 459.30 1,792.20 2,878.80 1,182.60 332.00 821.82 8.96 21.76 QU2 5.67 132.96 577.74 2,364.00 3,531.00 1,447.80 332.00 821.82 8.94 11.20 QU3 9.31 125.82 520.38 2,183.40 3,816.00 1,609.20 317.57 821.82 8.97 17.58 QU4 5.99 117.00 507.12 2,067.60 3,292.20 1,380.60 346.44 821.82 8.95 13.35 Lagoon EH1 24.19 13.32 76.14 273.06 120.84 57.78 34.64 363.95 8.63 25.86 EH2 35.20 11.95 60.20 226.66 186.12 112.14 28.87 363.95 8.43 30.97 GH1 13.89 405.33 985.86 6,780.89 10,160.40 4,645.80 433.04 807.14 8.89 14.01 GH2 13.21 442.87 1,154.17 6,986.84 8,295.60 3,856.80 447.48 792.47 8.90 14.72 Note. S, calcium carbonate supersaturation value. bottles were rinsed twice with deionized water and local water before sampling. The concentrations of major cations and anions were detected by ICP–OES (OPTIMA 8000, PerkinElmer) and ion chromatography (ECO IC, Metrohm), respectively. Study area Carbonate and bicarbonate ions were measured by titration method according to the geological and mineral industry standard of the People’s Republic of China (DZ/T 0064.49-93: Groundwater quality analysis methods Part 49: Determination of carbonate, bicarbonate and hydroxide ions—Titration method). pH values were measured with handheld meters (SX-620; Sanxin, China). Sixteen sediment samples were selected from eight rivers, six beaches of lake and two lagoons for thin sections. The samples were obtained under polarization microscope with the photo- collecting system. The grains with carbonate coats are mostly smaller than 500 μm. On the basis of the analysis of thin sections, eight sediment samples with carbonate coats were selected and sieved less than 500 μm for scanning electronic microscopy (SEM) analysis. The samples were impregnated with epoxy resin for thin sections with a diameter of 1 cm and a thickness of 3 mm. The thin sections were mounted on an aluminum stub and coated with carbon to render them conductive. Samples were imaged using a Zeiss MERLIN Compact FESEM. The grain size and carbonate-coated thickness in every photomicrograph by SEM was obtained using the ImageJ software. The roundness of southern lake are from NNE and NE, and the mean wind speed is 3.7 m/s (Li et al., 2016, Li et al., 2018; Li, 2020). Wave height and length in coastal systems correspond to wind speed and fetch in lakes. Annual average wave height of Qinghai Lake is from 0.2 to 0.4 m according to the wind speed from 2.8 to 4.1 m/s (Zhang et al., 2018). Based on the wind data, it shows that the hydrodynamics of the northern lake are stronger than the southern lake. Two lagoons are distributed on the east coast of the lake, named Erhai and Gahai, respectively (Figure1). There are rivers ﬂowing into Erhai, while Gahai is a closed lagoon. The area of Erhai and Gahai are 4 and 12 km2, respectively. The maximum depths of the lagoon are from 4 to 5 m (LZIG, 1979). Due to the small area and shallow water depth, the waves formed in the lagoon are small. Beach deposits occur mostly along the coast of the lake and lagoons. Characteristics of carbonate coats Through thin section observation, carbonate coats are found in lake and lagoon sediments, while there are no carbonate coats on grains in river sediments (Figure 3A). Carbonate coats cover diverse framework grains (e.g., quartz, feldspars, heavy minerals, lithic fragments, and carbonates) in different samples, reﬂecting the nature of the source areas. Some particles exhibit patchy or discontinuous coatings, and other grains present continuous cortices with poorly developed concentric laminae (Figures 3B–D), similar to lacustrine ooids described from Lake Geneva (Davaud and Girardclos, 2001). Scanning electron microscopy analyses reveal needle-columnar and anhedral crystals within the cortices (Figures 3E,F). The number of particles with carbonate coats, the degree of carbonate coat coverage, and thickness of coats are highly variable in the sediments. The carbonate coat thickness, coverage, particle size, and roundness of nine sediment samples from lakes and lagoons were statistically analyzed (Table 2). In total, 117 to 242 grains were counted to identify whether carbonate coats are developed in the samples. The sediment samples show signiﬁcant differences in the percentage of particles with carbonate coats. The lagoon sediments have low values of 15.3% and 15.8% (Table 2). Compared with lagoons, the percentage of particles with carbonate coats in the lake METHODS A total of 31 water and sediment samples are collected from the lake and rivers. Among them, 10 samples are distributed in 10 rivers, 17 samples from 6 beaches of a lake (DS, HX, JH, LD, LN, QU), and 4 in the beaches of two lagoons (Figure 1 and Table 1). All water samples were ﬁltered by 0.45-μm membranes. The February 2022 \| Volume 10 \| Article 824453 Frontiers in Earth Science \| www.frontiersin.org 3 Hao et al. Hao et al. Controlling Growth Factors of Ooids FIGURE 2 \| Piper diagram plot for the study area (Piper, 1944). FIGURE 2 \| Piper diagram plot for the study area (Piper, 1944). 9.04 (Table 1). For the lagoons, one with a river inﬂow shows lower pH values than the other. the grains with carbonate coats was classiﬁed as 1 (angular), 2 (sub-angular), 3 (sub-rounded), 4 (rounded), and 5 (very rounded) using the scale from the petroleum and natural gas industry standards of the People’s Republic of China (SY/T 5368- 2016: Identiﬁcation for thin section of rocks). The proportion of carbonate coat covering the grain surface was described by a semiquantitative approach, from 1 (10% grain surface) to 10 (100% of grain surface). Hydrochemical characteristics of water Hydrochemical characteristics of water Hydrochemical data of water samples are presented in Table 1. It shows that the concentrations of most ions in lakes are generally higher than those in rivers, except calcium ion. The concentrations of ions in a lagoon with river inﬂow (EH1 and EH2) are between those of lakes and rivers (Table 1). Water samples in the closed lagoon (Gahai) present the highest the concentrations of ions in this study area. Figure 2 shows the Piper diagram plot for the study area. Water samples are classiﬁed as two different water types. The hydrochemical types of most waters in the rivers are HCO3–Ca·Na type. Water samples from the lake and closed lagoon present the Cl–Na type. Water samples of a lagoon with a river inﬂow show the HCO3–Na type. The pH values in the rivers range 8.21–8.44. The lake water samples present higher pH values, from 8.84 to February 2022 \| Volume 10 \| Article 824453 Frontiers in Earth Science \| www.frontiersin.org 4 Hao et al. Controlling Growth Factors of Ooids FIGURE 3 \| Representative microphotographs under optical microscope and scanning electronic microscopy (SEM) showing the textural characteristics of carbonate coats within the study area. (A) Photomicrograph of the sand sediments from Buha River. (B) Photomicrograph of the sample QU4, CC-carbonate coat. (C) SEM image of the sample HX4. (D) SEM image of the sample LD1. (E) SEM image reveals needle-columnar crystals within the cortices. NCC, needle-columnar crystals. (F) SEM image reveals anhedral crystals within the cortices. AC, anhedral crystals. FIGURE 3 \| Representative microphotographs under optical microscope and scanning electronic microscopy (SEM) showing the textural characteristics of carbonate coats within the study area. (A) Photomicrograph of the sand sediments from Buha River. (B) Photomicrograph of the sample QU4, CC-carbonate coat. (C) SEM image of the sample HX4. (D) SEM image of the sample LD1. (E) SEM image reveals needle-columnar crystals within the cortices. NCC, needle-columnar crystals. (F) SEM image reveals anhedral crystals within the cortices. AC, anhedral crystals. sediments have higher values, but there are also differences between the northern and southern parts of the lake. In the northern lake, the percentage of particles with carbonate coats ranges from 26.1% to 43.1%. The particles with carbonate coat percentages of two samples in the south of the lake are 20.7% and 29.0%, respectively (Table 2). samples include ﬁne sand and medium sand (Figure 4A). Hydrochemical characteristics of water To the particles with carbonate coats, they are mainly distributed in the coarser part of the samples (Figure 4B). The grain size of the particles with carbonate coats from the samples presents mean values ranging from 178.8 to 372.3 μm (Table 2). The particles with carbonate coats are mainly composed of medium sand except samples DS2 and HX4 (Figure 4B). The grain sizes of all samples display mean values ranging from 173.7 to 303.5 μm (Table 2). The sediments of samples belong to ﬁne sand (125–250 μm) and medium sand (250–500 μm). DS2 and HX4 consist of ﬁne sand (Figure 4A). LN3 and EH1 are mainly medium sand (Figure 4A). The other The roundness of the particles with carbonate coats shows mean values ranging from 2.9 to 3.9 (Table 2). The particle roundness from QU4 and EH1 present sub-angular to rounded. The other samples are mainly sub-rounded to even very rounded February 2022 \| Volume 10 \| Article 824453 Frontiers in Earth Science \| www.frontiersin.org 5 Controlling Growth Factors of Ooids Hao et al. TABLE 2 \| Data table for the samples from the lake and lagoon. Sampling location Sample N1 N2 P D1 D2 R I T South of the lake QU4 184 38 20.7 233.9 290.0 3.0 5.8 15.4 DS2 214 62 29.0 173.7 198.8 3.6 6.8 11.6 North of the lake HX4 230 60 26.1 185.9 210.7 3.8 7.0 11.4 JH2 242 88 36.4 222.5 276.2 3.6 7.7 12.8 LD1 117 44 37.6 281.2 372.3 3.5 7.7 18.8 LN3 209 90 43.1 277.1 336.1 3.6 7.5 13.3 Lagoon EH1 124 19 15.3 303.5 321.5 2.9 4.9 12.5 GH1 152 24 15.8 277.9 328.5 3.9 6.5 16.7 Note. N1, number of all grains; N2, number of coated grains; P, proportion of coated grains (%); D1, average size of all grains (μm); D2, average size of coated grains (μm); R, coated grain rounded (μm); I, carbonate coat integrity; T, carbonate coat thickness (μm). TABLE 2 \| Data table for the samples from the lake and lagoon. Note. N1, number of all grains; N2, number of coated grains; P, proportion of coated grains (%); D1, average size of all grains (μm); D2, average size of coated grains (μm); R, coated grain rounded (μm); I, carbonate coat integrity; T, carbonate coat thickness (μm). Carbonate coat formation conditions Carbonate coat formation conditions Carbonate supersaturation is the main factor controlling the distribution of ooid sands (Rankey and Reeder, 2009). When the content of a certain mineral in water reaches saturation, precipitation can occur. By calculating the supersaturation value of calcium carbonate, Qinghai Lake and its lagoons are in a supersaturated state (LZIG, 1979). Chemically formed micritic carbonate minerals, like high-Mg calcite and aragonite, are about 1–15 μm and are found in the upper 10 m water of Qinghai Lake and at the bottom of the lake, while they are not found in the lake shore (LZIG, 1979; Jin et al., 2013). Due to the strong hydrodynamic force at the lake shore (maximum wave height ranging from1.8 to 3.3 m), the microcrystalline carbonate was transported from the lake shore to the deep lake (LZIG, 1979; Zhang et al., 2018). Previous studies mainly collected the water in the deeper part of the lake to calculate the supersaturation value of calcium carbonate, and whether it has reached supersaturation for the lake shore (LZIG, 1979). According to the same method from LZIG (1979), we calculate the supersaturation value of calcium carbonate from the lake shore. Both Qinghai Lake and lagoon shores are greater than 1 (Table 1), although the values presented are different, indicating that calcium carbonate minerals can also be precipitated in the water of the lake shore. Moreover, it can be observed from the thin section that the sediments of the river are not carbonate coats, so the carbonate coats of the sediments in the lake and lagoons should be self-generated rather than imported from rivers. Therefore, part of the microcrystalline carbonate is transported to the deep lake by backwash from waves, and the other part covered with particles formed the carbonate coats. Grain size effect on carbonate coat growth The carbonate coats have often been observed around medium- grained sand, which are the coarser sand fraction in the sediments (Figure 4BandTable 1). Carbonate-coat growth requires three stages: suspension growth, resting, and sleeping stages (Davies et al., 1978). In the same sedimentary hydrodynamic condition, the different-sized grains experience different transport types, such as grain ﬂow, saltation, and suspension (Hao et al., 2019). At the lake shore, where the large wave occurs, the strong hydrodynamics can stir up the coarse and ﬁne sand leading to transient suspension. Hydrochemical characteristics of water Though strong hydrodynamics can take microcrystalline carbonate away from the lake shore to the deep lake, it is the essential condition to form the carbonate coats (Davies et al., 1978). Field studies and laboratory experiments suggest that agitation and supersaturation are two important conditions in the formation of ooids (Broecker and Takahashi, 1966; Davies et al., 1978). Sediments in this study are collected from the lake shores with strong hydrodynamics due to wind waves. Sands are introduced into turbulent supersaturated lake water by the strong hydrodynamics, and inorganic extremely small calcium carbonate crystal aggregates precipitate on their surfaces (Davies et al., 1978). Sedimentary particles with the same lake shore environment should have a similar behavior. However, not each particle has carbonate coats in the study area, and the highest percentage of particles with carbonate coats is only 43.1% at the lake shore (Table 2), which shows a complexity of the carbonate coat formation. (Figure 4D). With the increase in carbonate coat content, the roundness has a little variation (Figure 5A). The proportion mean values of carbonate coat covering the grain surface are from 4.9 to 7.7 (Table 2). EH1 and GH1 samples show low carbonate coat covering the grain surface, while other samples present high covering, especially the samples from the north of the lake (Figure 4C). The carbonate coat thickness of the samples present a mean value ranging from 11.4 to 18.8 μm (Table 2), and are mostly less than 20 μm (Figure 4E). Frontiers in Earth Science \| www.frontiersin.org Carbonate coat formation conditions When the hydrodynamics become weaker, the coarser sand deposit on the lake bed, but the ﬁne sand may be still suspended. So the ﬁne sand only remains a short time on the lake bed, which means that the ﬁne sand lack the resting and sleeping stages of the carbonate coat growth. Moreover, the carbonate coat growth needs to spend 95% of its life in the subsurface (Davies et al., 1978). In this area, the coarse sediment is mixed with ﬁne sediment; the critical shear stress to mobilize the coarse sediment decreases due to exposure effects (McCarron et al., 2019). At the weak wave condition, the coarse sediment may be stirred up, and the calcium carbonate crystal aggregates will precipitate on their surfaces. This may be the reason why the carbonate coats are often observed on medium- grained sand and rarely on ﬁne sand in this study area. This also could explain the maximum carbonate-coated grain (mean grain size of 372.3 μm, Table 2) occurring in the northeast lake shore February 2022 \| Volume 10 \| Article 824453 Frontiers in Earth Science \| www.frontiersin.org 6 Hao et al. Hao et al. Controlling Growth Factors of Ooids FIGURE 4 \| Results for grain and carbonate coat characteristics for the samples from the lake and lagoon. (A) Results for all grain sizes in the samples from the lake and lagoon. (B) Results for coated grain sizes in the samples from the lake and lagoon. (C) Results for coating integrity in the samples from the lake and lagoon. (D) Results for grain rounded in the samples from the lake and lagoon. (E) Results for coating thickness in the samples from the lake and lagoon. FIGURE 4 \| Results for grain and carbonate coat characteristics for the samples from the lake and lagoon. (A) Results for all grain sizes in the samples from the lake and lagoon. (B) Results for coated grain sizes in the samples from the lake and lagoon. (C) Results for coating integrity in the samples from the lake and lagoon. (D) Results for grain rounded in the samples from the lake and lagoon. (E) Results for coating thickness in the samples from the lake and lagoon. exposure effect, the relationship between the hydrodynamic force and the particle size will get more complex (McCarron et al., 2019). with the strongest waves (Zhang et al., 2018). Carbonate coat formation conditions In a unimodal mixture, coarse sediment requires a high critical shear stress to be mobilized, while the ﬁne unimodal sediment is readily mobile at a low critical shear stress (McCarron et al., 2019). If the lake shore is mainly composed of ﬁne sediments and has strong hydrodynamic force, it is likely that the carbonate coats will not be produced due to the lack of resting and sleeping stages. On the contrary, if the sediments are mainly composed of coarse sediments, and the hydrodynamic force is very weak, it is likely that the carbonate coats will not form as the grains cannot be suspended. Therefore, carbonate coats can be produced only when the hydrodynamic force and the particle size are well matched. Due to the hiding- Hydrodynamic effect on carbonate coat distribution The agitation and supersaturation are two important conditions in the formation of the carbonate coats, so which factor leads to the difference of the proportions of particles with carbonate coats? In the two lagoons, the proportions of particles with carbonate coats are similar values, but the hydrochemical conditions show great distinction presenting the Cl–Na type Frontiers in Earth Science \| www.frontiersin.org February 2022 \| Volume 10 \| Article 824453 7 Controlling Growth Factors of Ooids Hao et al. FIGURE 5 \| Correlation between coated grain content and carbonate coat characteristics. (A) Cross-plot of grain rounded versus coated grain content. (B) Cross- plot of coating integrity versus coated grain content. (C) Cross-plot of coating integrity versus grain rounded. (D) Cross-plot of coating thickness versus coated grain content. FIGURE 5 \| Correlation between coated grain content and carbonate coat characteristics. (A) Cross-plot of grain rounded versus coated grain content. (B) Cross- plot of coating integrity versus coated grain content. (C) Cross-plot of coating integrity versus grain rounded. (D) Cross-plot of coating thickness versus coated grain content. hydrodynamics, and particularly the waves, are interpreted to be major factors controlling the distribution of carbonate coats in a similar sedimentary environment. and HCO3–Na type (Figure 2andTables 1 and 2). For the lake, it shows a similar hydrochemical condition in the different lake shores, while the proportions of particles with carbonate coats present a difference (Figure 2andTables 1 and 2). Therefore, when the water in the different environments reaches carbonate supersaturation, the hydrochemical condition is not the major factor for controlling the distribution of carbonate coats. Frontiers in Earth Science \| www.frontiersin.org Hydrodynamic effect on carbonate coat integrity g The abundance of coated grains was compared with grain coating integrity (Figure 5B), which shows a strong correlation. This suggests that the stronger the hydrodynamic force is on the lake shore, the higher the integrity of coats. In observations from SEM, calcium carbonate crystal is generally found in the pit, on the low- coated covering grains (Figure 3B). Carbonate coats are initially precipitated in pits on the grain surfaces. When the pits are ﬁlled, the calcium carbonate crystal starts to cover other grain surfaces. Under weak hydrodynamic conditions, coarse particles are easy to suspend due to exposure effect, but there is a short time stay in the suspended stage due to their weight, which leads to the calcium carbon crystal only settling in small pits. In the lake shores with strong hydrodynamic forces, coarse particles can be constantly suspended, and the rate of calcium carbon microcrystalline precipitation on the grain surface is greater, leading to the particles being completely covered by calcium carbon crystal. In addition, the particles with a higher integrity of The distribution of carbonate coats could be strongly linked to the location of the hydrodynamics. The fact that the two lagoons have similar small waves could explain the similar low proportions of particles with carbonate coats (Table 2). Lakes with stronger hydrodynamic forces have more proportions of particles with carbonate coats than lagoons (Table 2). The strength of waves varies at different locations of the lake (Zhang et al., 2018; Li, 2020; Hu et al., 2021). The north of the lake has an open lake surface and strong winds, which can produce larger waves, so the proportions of particles with carbonate coats in the north lake shore except the sample HX1 are higher than those in the south lake shore (Table 2). Sample HX1 is located in the lake bay of the northern lake with weak waves, so it has a lower proportion of particles with carbonate coats than the other north lake shore samples. There is a good correlation between the hydrodynamics and the proportions of particles with carbonate coats. Therefore, the February 2022 \| Volume 10 \| Article 824453 Frontiers in Earth Science \| www.frontiersin.org 8 Hao et al. Controlling Growth Factors of Ooids found in different lake shores and lagoons, which show thin cortices and even some particles with discontinuous or patchy coatings. precipitated carbonate coats also have the better roundness (Figures 3Dand5C). Hydrodynamic effect on carbonate coat integrity Because the rounded particles are easy to be rolled in turbulent conditions, it is more conducive to the precipitation of microcrystalline carbonate in the suspension stage. 2. Carbonate-coated grain content presents variations in different locations in the lake. The hydrochemical condition is not the major factor but the hydrodynamics, and particularly, the wave characters seem to control the distribution of carbonate coats in the similar sedimentary environment. 2. Carbonate-coated grain content presents variations in different locations in the lake. The hydrochemical condition is not the major factor but the hydrodynamics, and particularly, the wave characters seem to control the distribution of carbonate coats in the similar sedimentary environment. Hydrodynamic effect on carbonate coat thickness Hydrodynamics is not only one of the important conditions for the formation of the carbonate coats but also has a signiﬁcant effect on carbonate grain coat distribution, integrity, thickness, and carbonate coat grain size. 5. Bacteria or EPS are not observed within ooid cortices. The bacteria are not the main factor, but hydrodynamic forces play a great role in the formation of ooids in this study area. 5. Bacteria or EPS are not observed within ooid cortices. The bacteria are not the main factor, but hydrodynamic forces play a great role in the formation of ooids in this study area. DATA AVAILABILITY STATEMENT The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors. FUNDING The study was supported by the National Natural Funding Science Foundation of China (Grant Nos. 41502142, 41772142) and the Second Tibetan Plateau Scientiﬁc Expedition and Research Program (STEP) (No. 2019QZKK0704). AUTHOR CONTRIBUTIONS LH contributed to the design of the study and wrote the manuscript. HT analyzed the result of the experiments. SL performed the statistical analysis and collected the samples. XM ﬁnished the experiments on the thin sections. HJ ﬁnished the experiments on the SEM. JQ ﬁnished the experiments on the chemical constituents. All authors contributed to the manuscript revision, and read and approved the submitted version. g Beach rock deposits exist in the shoreline of Qinghai Lake, and microorganisms are found in the carbonate cement of beach rock (Li, 2003; Chen et al., 2019). The grain cortices also develop in the beach rocks, which are disposed radially. Radial ooids are generally formed in quiet water with the bacteria (Davies et al., 1978). Therefore, microorganisms play an important role in the formation of cortices in this beach rocks. Recent studies have shown that microorganisms are found more and more important in the ooid formation (Diaz et al., 2015; Diaz et al., 2017). In this study, the ooids show tangential differences with those in the beach rocks, and bacteria or EPS are not observed within ooid cortices by SEM. Therefore, whether bacteria are involved in the formation of ooids needs more analytical methods to study in the future, but it is certain that hydrodynamic forces play a great role in carbonate grain coat distribution, integrity, thickness, and ooid grain size. Hydrodynamic effect on carbonate coat thickness The carbonate coat thickness has a weak correlation with the coated grain content, displaying that the low-coated grain content has a larger coat thickness (Figure 5D). On the lake shore with less-coated grain content, the carbonate is generally developed in pits of particles and covers less of the particle surface, and the thickness of the completely covered carbonate coats present is thin (Figures 3B, C). When measuring the carbonate coat thickness in the lake shore with the low-coated grain content, the depth of most pits was measured representing the thickness of the carbonate coats due to it rarely completely covering the grain surface, which leads to a large thickness of the carbonate coats in this lake shore. In the lake shore with more coated grain content, there are more particles completely covered with carbonate coats (Figure 3D), and the statistical thickness is mostly the thickness of the carbonate rings, not the depth of the pits. Different statistical methods lead to a higher carbonate coat thickness values in the lake shore with the low-coated grain content. If the thickness of the completely covered carbonate coats were only counted in all the samples, there should be a good positive correlation between the carbonate coat thickness and the coated grain content. The particles from the lake shore with strong waves can be stirred up many times and undergo several carbonate microcrystalline precipitation cycles, resulting in an increase in carbonate coat thickness. So, the hydrodynamic force on the lake shore can affect the thickness of the carbonate coats. When ooids are near a dynamic equilibrium size dependent upon precipitation and abrasion, ooid growth would cease (Trower et al., 2017). 3. The carbonate coats are often observed on medium-grained sand and rarely on ﬁne sand in this study area, and the maximum carbonate-coated grain occurred under the strongest waves. Carbonate coats can be produced only when the hydrodynamic force and particle size are well matched. 4. The strong hydrodynamic force on the lake shore induces higher integrity and thickness of sand coats. Hydrodynamics is not only one of the important conditions for the formation of the carbonate coats but also has a signiﬁcant effect on carbonate grain coat distribution, integrity, thickness, and carbonate coat grain size. 4. The strong hydrodynamic force on the lake shore induces higher integrity and thickness of sand coats. REFERENCES The Petrological Ch i i d F i M h i f L k B h k i L k Qi h i Li, X., Yang, X., Ma, Y., Hu, G., Hu, X., Wu, X., et al. (2018). Qinghai Lake Basin Critical Zone Observatory on the Qinghai-Tibet Plateau. Vadose Zone J. 17, 180069. doi:10.2136/vzj2018.04.0069 Bahama Banks. J. Geophys. Res. 71, 1575–1602. doi:10.1029/jz071i006p01575 Chen, J., Jiang, Z., Zhang, W., Liu, C., and Xu, W. (2019). The Petrological Characteristics and Formation Mechanism of Lake Beachrock in Lake Qinghai. J. Lake Sci. 31 (6), 1783–1796. doi:10.18307/2019.0623 Li, X. Y., Ma, Y. J., Huang, Y. M., Hu, X., Wu, X. C., Wang, P., et al. (2016). Evaporation and Surface Energy Budget over the Largest High-Altitude saline lake on the Qinghai-Tibet Plateau. J. Geophys. Res. Atmospheres 121 (18), 10470–10485. doi:10.1002/2016jd025027 Characteristics and Formation Mechanism of Lake Beachrock in Lake Qinghai. J. Lake Sci. 31 (6), 1783–1796. doi:10.18307/2019.0623 Davies, P. J., Bubela, B., and Ferguson, J. (1978). The Formation of Ooids. Sedimentology 25, 703–730. doi:10.1111/j.1365-3091.1978.tb00326.x Davies, P. J., Bubela, B., and Ferguson, J. (1978). The Formation of Ooids. Sedimentology 25, 703–730. doi:10.1111/j.1365-3091.1978.tb00326.x Li, X. Y. (2020). Qilian Mountains Integrated Observatory Network: Dataset of Qinghai Lake Integrated Observatory Network (An Observation System of Meteorological Elements Gradient of Yulei Station on Qinghai lake, 2019). Beijing: National Tibetan Plateau Data Center. doi:10.11888/Meteoro.tpdc. 270732 Davaud, E., and Girardclos, S. (2001). Recent freshwater ooids and oncoids from western Lake Geneva (Switzerland): Indications of a common organically mediated origin. J. Sediment. Res. 71, 423–429. doi:10.1306/2DC40950-0E47- 11D7-8643000102C1865D Davies, P. J., and Martin, K. (1976). Radial Aragonite Ooids, Lizard Island, Great Barrier Reef, Queensland, Australia. Geol 4, 120–122. doi:10.1130/0091- 7613(1976)4<120:raolig>2.0.co;2 Li, Y. C. (2003). Comparing Researchbeachrock in the South of Fujian Province and Lake Qinghai. J. Quanzhou Normal Univ. Nat. Sci. 21 (4), 43–47. doi:10. 16125/j.cnki.1009-8224.2003.04.011 Diaz, M. R., Eberli, G. P., Blackwelder, P., Phillips, B., and Swart, P. K. (2017). Microbially Mediated Organomineralization in the Formation of Ooids. Geology 45, 771–774. doi:10.1130/g39159.1 Liu, X., Chen, X., Tostevin, R., Yao, H., Han, K., Guo, H., et al. (2021). Post- depositional Modiﬁcation of Carbonate Ooids by Sulfate-Reducing Bacteria: Evidence from the Lower-Middle Jurassic, Tethyan Himalayas of Southern Tibet. Sediment. Geology. 426, 106027. doi:10.1016/j.sedgeo.2021.106027 Diaz, M. R., and Eberli, G. P. (2019). Decoding the Mechanism of Formation in marine Ooids: a Review. Earth-Science Rev. 190, 536–556. doi:10.1016/j. earscirev.2018.12.016 Lu, C., Li, F., Oehlert, A. REFERENCES Kump, L. R., and Hine, A. C. (1986). “Ooids as Sea-Level Indicators,” in Sea-Level Research: A Manual for the Collection and Evaluation of Data. Editor O. van de Plassche (Norwich: Geo Books), 175–193. doi:10.1007/978-94- 009-4215-8_7 An, Z., Colman, S. M., Zhou, W., Li, X., Brown, E. T., Jull, A. J. T., et al. (2012). Interplay between the Westerlies and Asian Monsoon Recorded in lake Qinghai Sediments since 32 Ka. Sci. Rep. 2, 619. doi:10.1038/srep00619 Li, F., Yan, J., Algeo, T., and Wu, X. (2013). Paleoceanographic Conditions Following the End-Permian Mass Extinction Recorded by Giant Ooids (Moyang, South China). Glob. Planet. Change 105, 102–120. doi:10.1016/j. gloplacha.2011.09.009 Ariztegui, D., Plée, K., Farah, R., Menzinger, N., and Pacton, M. (2012). Bridging the gap between Biological and Sedimentological Processes in Ooid Formation: Crystalizing F. A. Forel’s Vision. Arch. Sci. 65, 93–102. Li, J., and Fang, X. (1999). Uplift of the Tibetan Plateau and Environmental Changes. Chin. Sci. Bull. 44, 2117–2124. doi:10.1007/bf03182692 Ball, M. M. (1967). Carbonate Sand Bodies of ﬂorida and the bahamas. J. Sediment. Petrol. 37, 556–591. doi:10.1306/74d7171c-2b21-11d7-8648000102c1865d Li, X.-Y., Xu, H.-Y., Sun, Y.-L., Zhang, D.-S., and Yang, Z.-P. (2007). Lake-level Change and Water Balance Analysis at Lake Qinghai, West China during Recent Decades. Water Resour. Manage. 21, 1505–1516. doi:10.1007/s11269- 006-9096-1 Beukes, N. J. (1983). “Ooids and Oolites of the Proterophytic Boomplaas Formation, Transvaal Supergroup, Griqualand West, South Africa,” in Coated Grains. Editor T.M. Peryt (Berlin: Springer-Verlag), 199–214. doi:10.1007/978-3-642-68869-0_18 Broecker, W. S., and Takahashi, T. (1966). Calcium Carbonate Precipitation on the Bahama Banks. J. Geophys. Res. 71, 1575–1602. doi:10.1029/jz071i006p01575 Chen, J., Jiang, Z., Zhang, W., Liu, C., and Xu, W. (2019). The Petrological Characteristics and Formation Mechanism of Lake Beachrock in Lake Qinghai. Beukes, N. J. (1983). “Ooids and Oolites of the Proterophytic Boomplaas Formation, Transvaal Supergroup, Griqualand West, South Africa,” in Coated Grains. Editor T.M. Peryt (Berlin: Springer-Verlag), 199–214. doi:10.1007/978-3-642-68869-0_18 y p g g Broecker, W. S., and Takahashi, T. (1966). Calcium Carbonate Precipitation on the Bahama Banks J Geophys Res 71 1575–1602 doi:10 1029/jz071i006p01575 y p g g Broecker, W. S., and Takahashi, T. (1966). Calcium Carbonate Precipitation on the Bahama Banks. J. Geophys. Res. 71, 1575–1602. doi:10.1029/jz071i006p01575 Chen, J., Jiang, Z., Zhang, W., Liu, C., and Xu, W. (2019). REFERENCES M., Li, J., and Zou, H. (2020). Reconstructing Paleoceanographic Conditions during the Middle Ediacaran: Evidence from Giant Ooids in South China. Precambrian Res. 351, 105945. doi:10.1016/j. precamres.2020.105945 Diaz, M. R., Swart, P. K., Eberli, G. P., Oehlert, A. M., Devlin, Q., Saeid, A., et al. (2015). Geochemical Evidence of Microbial Activity within Ooids. Sedimentology 62, 2090–2112. doi:10.1111/sed.12218 LZIG (1979). General Investigation Report of Qinghai Lake. Beijing: Science Press, 1–270. Duguid, S. M. A., Kyser, T. K., James, N. P., and Rankey, E. C. (2010). Microbes and Ooids. J. Sediment. Res. 80, 236–251. doi:10.2110/jsr.2010.027 Dupraz, C., and Visscher, P. T. (2005). Microbial Lithiﬁcation in marine Stromatolites and Hypersaline Mats. Trends Microbiol. 13, 429–438. doi:10. 1016/j.tim.2005.07.008 McCarron, C. J., Van Landeghem, K. J. J., Baas, J. H., Amoudry, L. O., and Malarkey, J. (2019). The Hiding-Exposure Effect Revisited: A Method to Calculate the Mobility of Bimodal Sediment Mixtures. Mar. Geology. 410, 22–31. doi:10.1016/j.margeo.2018.12.001 Fan, J., Gijbels, I., and Ma, B. (1994). Evolution of Recent Environment in Qinghai Lake and its Prediction. Beijing: Science Press, 1–12. doi:10.1007/978-1-4899- 3150-4_1 Opdyke, B. N., and Wilkinson, B. H. (1990). Paleolatitude Distribution of Phanerozoic marine Ooids and Cements. Palaeogeogr. Palaeoclimatol. Palaeoecol. 78, 135–148. doi:10.1016/0031-0182(90)90208-o Halley, R. B. (1977). Ooid Fabric and Fracture in the Great Salt Lake and the Geologic Record. J. Sedim. Petrol. 47, 1099–1120. doi:10.1306/212f72ed-2b24- 11d7-8648000102c1865d Pacton, M., Ariztegui, D., Wacey, D., Kilburn, M. R., Rollion-Bard, C., Farah, R., et al. (2012). Going Nano: a New Step toward Understanding the Processes Governing Freshwater Ooid Formation. Geology 40, 547–550. doi:10.1130/ g32846.1 Hao, L., Tao, H., Guo, R., Mou, W., Tian, B., and Ma, X. (2019). Hydrodynamic Evolution from Quartz Microtextures of a beach ridge in Qinghai Lake, China. Sediment. Geology. 389, 13–25. doi:10.1016/j.sedgeo.2019.05.008 Piper, A. M. (1944). A Graphic Procedure in the Geochemical Interpretation of Water-Analyses. Trans. AGU 25 (6), 914–928. doi:10.1029/tr025i006p00914 Harris, P. M., Halley, R. B., and Lukas, K. J. (1979). Endolith Microborings and Their Preservation in Holocene-Pleistocene (Bahama-Florida) Ooids. Geol 7, 216–220. doi:10.1130/0091-7613(1979)7<216:ematpi>2.0.co;2 Plée, K., Ariztegui, D., Martini, R., and Davaud, E. (2008). Unravelling the Microbial Role in Ooid Formation–Results of an In Situ experiment in Modern Freshwater Lake Geneva in Switzerland. Geobiology 6, 341–350. doi:10.1111/j.1472-4669.2007.00140.x Harris, P. M., Purkis, S. J., and Ellis, J. (2011). Analyzing Spatial Patterns in Modern Carbonate Sand Bodies from Great Bahama Bank. J. Sediment. Res. 81, 185–206. doi:10.2110/jsr.2011.21 Popp, B. N., and Wilkinson, B. H. (1983). CONCLUSION We would like to thank Professor Chengjun Zhang, the Chief Editor, and the reviewers for their valuable comments. The Yl station dataset is provided by National Tibetan Plateau Data Center (http://data.tpdc.ac.cn). 1. Qinghai Lake has the hydrochemical and hydrodynamic conditions for the formation of ooids. Carbonate coats are February 2022 \| Volume 10 \| Article 824453 Frontiers in Earth Science \| www.frontiersin.org 9 Controlling Growth Factors of Ooids Hao et al. REFERENCES “Holocene Lacustrine Ooids from Pyramid Lake, Nevada,” in Coated Grain. Editor T. M. Peryt (Berlin: Springer- Verlag), 141–153. doi:10.1007/978-3-642-68869-0_12 Hu, G., Dong, Z., Zhang, Z., Yang, L., Hao, L., Hesp, P., et al. (2021). Wind Regime and Aeolian Landforms on the Eastern Shore of Qinghai Lake, Northeastern Tibetan Plateau, China. J. Arid Environments 188, 104451. doi:10.1016/j. jaridenv.2021.104451 Rankey, E. C., and Reeder, S. L. (2009). Holocene Ooids of Aitutaki Atoll, Cook Islands, South Paciﬁc. Geology 37, 971–974. doi:10.1130/g30332a.1 Jin, Z. D., Zhang, F., Li, F. C., Chen, L. M., Xiao, J., and He, M. Y. (2013). Seasonal and Interannual Variations of the lake Water Parameters and Particle Flux in Lake Qinghai: A Time-Series Sediment Trap Study (In Chinese). J. Earth Environ. 4, 1306–1313. doi:10.7515/JEE201303002 Rankey, E. C., Riegl, B., and Steffen, K. (2006). Form, Function and Feedbacks in a Tidally Dominated Ooid Shoal, Bahamas. Sedimentology 53, 1191–1210. doi:10. 1111/j.1365-3091.2006.00807.x Simonson, B. M., and Jarvis, D. G. (1993). “Microfabrics of Oolites and Pisolites in the Early Precambrian Carawine Dolomite of Western Australia,” in Carbonate Microfabrics. Editors R. Rezak and D. Lavoieeds (Heidelberg: Springer-Verlag), 227–237. doi:10.1007/978-1-4684-9421-1_17 Kahle, C. F. (1974). Ooids from Great Salt Lake, Utah, as an Analogue for the Genesis and Diagenesis of Ooids in marine Limestones. J. Sedim. Petrol. 44, 30–39. doi:10.1306/74d7296e-2b21-11d7-8648000102c1865d February 2022 \| Volume 10 \| Article 824453 Frontiers in Earth Science \| www.frontiersin.org 10 Hao et al. Controlling Growth Factors of Ooids Zhang, Y., Hu, C., Wang, M., Ma, M., Wang, X., and Jiang, Z. (2018). A Quantitative Sedimentary Model for the Modern Lacustrine beach Bar (Qinghai Lake, Northwest China). J. Paleolimnol. 59, 279–296. doi:10.1007/ s10933-016-9930-2 Sipos, A. A., Domokos, G., and Jerolmack, D. J. (2018). Shape Evolution of Ooids: a Geometric Model. Sci. Rep. 8, 1758. doi:10.1038/s41598-018- 19152-0 Sumner, D. Y., and Grotzinger, J. P. (1993). Numerical Modeling of Ooid Size and the Problem of Neoproterozoic Giant Ooids. J. Sediment. Petrol. 63, 974–982. doi:10.1306/d4267c5d-2b26-11d7-8648000102c1865d 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. Trower, E. J., Lamb, M. P., and Fischer, W. W. (2017). Experimental Evidence that Ooid Size Reﬂects a Dynamic Equilibrium between Rapid Precipitation and Abrasion Rates. Earth Planet. Sci. Lett. 468, 112–118. doi:10.1016/j.epsl.2017. Frontiers in Earth Science \| www.frontiersin.org February 2022 \| Volume 10 \| Article 824453 REFERENCES 04.004 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. Tucker, M. E., and Wright, V. P. (1990). Carbonate Sedimentology. Oxford: Blackwell Science, 482. Wanless, H. R., and Tedesco, L. P. (1993). “Comparison of Oolitic Sand Bodies Generated by Tidal vs Wind-Wave Agitation,” in Mississippian Oolites and Modern Analogs. 35. Editors B.D. Keith and C.W. Zuppann (Tulsa, Oklahoma: AAPG Stud. Geol), 199–225. doi:10.1306/st35571c15 Copyright © 2022 Hao, Tao, Li, Ma, Ji and Qiu. 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. Wilkinson, B. H., Pope, B. N., and Owen, R. M. (1980). Nearshore Ooid Formation in a Modern Temperate Region Marl Lake. J. Geology. 88, 697–704. doi:10.1086/ 628555 Yuan, Z., Zhong, X. G., and Ye, S. J. (1990). The Core Evolution of a Globular Cluster Containing Massive Black Holes. Astrophys Space Sci. 168, 233–241. doi:10.1007/bf00636869 February 2022 \| Volume 10 \| Article 824453 Frontiers in Earth Science \| www.frontiersin.org 11
https://openalex.org/W2971578213	https://equityhealthj.biomedcentral.com/track/pdf/10.1186/s12939-019-1032-0	English	null	Salutogenic health promotion program for migrant women at risk of social exclusion	International journal for equity in health	2,019	cc-by	8,494	© 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. Abstract Background: Migrant women at risk of social exclusion often experience health inequities based on gender, country of origin or socioeconomic status. Traditional health promotion programs designed for this population have focused on covering their basic needs or modifying lifestyle behaviors. The salutogenic model of health could offer a new perspective enabling health promotion programs to reduce the impact of health inequities. This study evaluated the effectiveness of a salutogenic health promotion program focused on the empowerment of migrant women at risk of social exclusion. Methods: A four-session salutogenic health promotion program was conducted over a period of 6 months. In a quasi-experimental pre-test post-test design, an ad hoc questionnaire was administered to 26 women to collect sociodemographic data, together with 5 validated instruments: Antonovsky’s Sense of Coherence (SOC-13), Duke- UNC-11 (perceived social support), Quality of Life Short Form-36 (SF-36), Rosenberg’s Self-Esteem Scale, and the Cohen et al. Perceived Stress Scale (PSS-10). Descriptive analysis and multiple linear regression models were performed. Statistical tests were considered significant with a two-tailed p value < 0.05. Results: Participants had a low initial SOC-13 score (60.36; SD 8.16), which did not show significant change after the health promotion program. Perceived social support (37.07; SD 6.28) and mental quality of life also remained unchanged, while physical quality of life increased from 50.84 (SD 4.60) to 53.08 (SD 5.31) (p = 0.049). Self-esteem showed an increasing trend from 30.14 (SD 4.21) to 31.92 (SD 4.38) (p = 0.120). Perceived stress decreased from 20.57 (SD 2.91) to 18.38 (SD 3.78) (p = 0.016). A greater effect was observed at the end of the program in women with lower initial scores for SOC-13 and quality of life and higher initial scores of perceived stress. Conclusions: The health promotion program reduced perceived stress, increased physical quality of life and showed a trend toward increased self-esteem, especially among migrant women with multiple vulnerability factors. The salutogenic model of health should be considered as a good practice to apply in health promotion programs and to be included in national policies to reduce health inequity in migrant populations. Keywords: Salutogenesis, Migrant women, Health promotion, Self-esteem, Perceived stress, Quality of life heterogeneous group with a great diversity of life experi- ences closely related to the migration process [2]. Open Access Open Access Salutogenic health promotion program for migrant women at risk of social exclusion M. C. Malagón-Aguilera1,2 , S. Gelabert-Vilella1 , C. Bosch-Farré1,2 , L. Vaandrager3,4, D J à C l2 7 M. M. García-Gil5,6 and D. Juvinyà-Canal2,7 * Correspondence: anna.bonmati@udg.edu 1Faculty of Nursing, University of Girona, Emili Grahit, 77, 17071 Girona, Spain 2Health and Health Care Research Group, University of Girona, Emili Grahit, 77, 17071 Girona, Spain Full list of author information is available at the end of the article Bonmatí-Tomas et al. International Journal for Equity in Health (2019) 18:139 https://doi.org/10.1186/s12939-019-1032-0 Bonmatí-Tomas et al. International Journal for Equity in Health (2019) 18:139 https://doi.org/10.1186/s12939-019-1032-0 Bonmatí-Tomas et al. International Journal for Equity in Health (2019) 18:139 https://doi.org/10.1186/s12939-019-1032-0 (2019) 18:139 Stressors of migration Migration can negatively affect physical, mental and emo- tional health [6, 7]. It has a stressful effect on people be- cause it begins with the hope of a new and better life but experiences at the new destination often cause unexpected loss and culture shock [8, 9]. Migrants must cope with these stressors until they can regain a sense of balance or adapt to the new situation [9, 10]. For these reasons, mi- grants are considered a vulnerable population [11]. Antonovsky’s salutogenic model is based on a health- disease continuum between dis-ease and ease. Saluto- genesis focuses on the origins of what creates health (strengths and resources) and focuses attention toward the development of health [34]. In contrast, the usual pathogenic approach considers health and disease as two mutually exclusive states and focuses on the risk factors associated with disease [35]. Salutogenesis suggests that stressors are common in daily life, generating tensions that can cause a landslide of health consequences if the individual succumbs to them, or a positive health effect if the tensions are overcome [34]. Although migrant populations are among those most af- fected by health inequities, few health programs and pol- icies focus specifically on reducing inequities in these vulnerable populations [12–14]. Migrant women are espe- cially at risk due to gender discrimination, stigmatization, and lack of resources [15]. Gender is a social determinant of health that triggers differences in the socialization of women and men. This implies distinctive values, attitudes, and behaviors that lead to gender-based differences in ac- cess to resources and jobs [16]. Moreover, migrant women usually work at home, so their work is unpaid, ignored, and not recognized by others [17]. The key elements in Antonovsky’s theory of salutogenesis are generalized resistance resources (GRRs) and sense of coherence (SOC). GRRs are defined as “a property of a per- son, a collective or a situation which, as evidence or logic has indicated, facilitates successful coping with the inherent stressors of human existence”. SOC is defined as “the ability to understand the overall situation and the capacity to use available resources” [35]. Antonovsky described three SOC dimensions: comprehensibility, manageability and meaning- fulness. Comprehensibility represents the person’s capacity to transform stimuli into information about what is being experienced or perceived as meaningful, orderly, consistent, structured, and clear. Manageability refers to the individ- ual’s perception of the available resources and their ad- equacy to meet daily demands. Stressors of migration Meaningfulness indicates the extent to which a person feels that life makes sense emotionally and is sufficiently motivated to put effort into confronting problems and difficulties [36]. In addition, they suffer from the stigmatization or dis- crimination [18, 19] to which the migrant population is especially vulnerable [20]. Migrant individuals are often targeted for their differentiating features, ethnic traits or cultural behaviors [21]. It is known that self-esteem helps to cope with stigmatization [22] and that a high level of self-esteem and self-confidence help to reduce perceived stress, allowing the individual to choose the most effective coping strategies [23]. A high level of self- esteem together with strong social support reduces vulnerability to stressors [24, 25], and social support is a protective determinant for adults to adapt to a new en- vironment [26] and to cope with stressful situations [27]. The aim of health promotion programs with a salutogenic model of health is to increase the internal and external awareness of the GRRs and enhance the ability to use them in daily life [37] in order to improve health. These programs are focused not only on minimizing (disease) risks, but also on strengthening the existing GRRs of each participant, thus facilitating the participant’s progress towards health as de- scribed by Antonovsky. This study explored the effectiveness of a salutogenic health promotion program for migrant women at risk of social exclusion. Finally, lack of resources in migrant populations causes difficult life conditions and limits access to opportun- ities. In Spain, the unemployment rate is 19% in the native population, while it is 35% among migrants [28]. Background Migration is a dynamic individual or group process that affects personal relationships at the point of origin and the destination. It is therefore not merely a spatial or temporary movement of people, but a process of neces- sary cultural readaptation in the new environment [1]. The migrant population can be described as a very In recent decades, global migration has increased con- siderably [3]. According to Eurostat data, 36.9 million of the migrants to Europe in 2017 were born outside of EU-28 countries. During that same year, Spain counted approximately 4.4 million migrants [4]. In Catalonia, for- eign-born legal residents were less than 2.5% of the population in 2001; in 2018, they constituted 13.78% of the Catalan population (approximately 1 million people). * Correspondence: anna.bonmati@udg.edu 1Faculty of Nursing, University of Girona, Emili Grahit, 77, 17071 Girona, Spain 2Health and Health Care Research Group, University of Girona, Emili Grahit, 77, 17071 Girona, Spain Full list of author information is available at the end of the article Bonmatí-Tomas et al. International Journal for Equity in Health (2019) 18:139 Page 2 of 9 Bonmatí-Tomas et al. International Journal for Equity in Health (2019) 18:139 Page 2 of 9 In the region of Girona, the number of migrants in 2017 was about 140,000, almost 18% of the population. Among these, 51.8% were between 24 and 50 years old. The mi- grant population consisted of 48.2% women. In three spe- cific villages, the percentage of migrants is even higher, around 40% of the population. The majority of the migrant communities in the region are Moroccan (36,467 people, almost 25% of the total population), followed by Hondurans (9595 people, 6.8%) and Gambians (7155, 5%) [5]. [30, 31]. Although necessary, these programs are not suffi- cient to overcome health inequities. New approaches and new strategies are needed. In Spain, the strategic goals of the national program in prevention and health promotion in- clude a commitment to tackle health inequity and to facili- tate the health empowerment of individuals [32]. Moreover, the Spanish Commission to Reduce Social Inequalities in Health was created in 2008 specifically to develop a nation- wide proposal for interventions to reduce health inequities [33]. In accordance with national policy and strategies, the present study introduced the salutogenic model developed by Aaron Antonovsky into a health promotion program in an effort to explore new approaches that can be effective in reducing health inequities. Study design SESSION 2: Identifying community GRRs, including self-recognition of one’s own role in the family and family social support. Two activities were developed. The first consisted of participants drawing a picture of their families. In the second activity, different situations were presented to enable participants to recognize their role and their available support in the family (e.g. who takes care of family members when they are ill? Who is in charge of the household shopping? Who attends the parent-teacher meetings? Who is in charge of the paperwork, of paying the household bills? In case you get ill, who would you ask for help? Who do you think would help you? In case of economic hardship, to whom would you explain it? Who do you think would help you?). A quasi-experimental pre-test post-test study was de- signed. The intervention consisted of a salutogenic health promotion program, carried out at a job placement pro- gram of Caritas, the international Catholic Charities organization that works with people who are unemployed, homeless and/or who are migrants. The study took place in Girona (Spain), beginning in January 2015 with partici- pant recruitment and ending in February 2016 with post- test data collection. Description of the salutogenic health promotion program The main objective of this salutogenic health promotion program was to improve awareness of GRRs and in- crease self-esteem, physical and mental quality of life (QL), and SOC in the study population, and decrease their perception of stress. For this reason, program activ- ities were planned with the aim of raising questions and encouraging the participants to help each other identify their own resources through the contributions of the group discussions. The role of the facilitator of the ses- sions was to enable the women to discover their GRRs. SESSION 3: Exploring the role of each participant in her community; further identifying community GRRs. The participants were asked to take pictures that characterized what was important in their daily life as members of a community. These pictures were presented and explained to the rest of the group. This program consisted of 4 group sessions, each last- ing 2 h for a total of 8 h, a duration similar to analogous programs in other studies [38] (Fig. 1). The specific ac- tivities carried out in each session were as follows: SESSION 4: Building individual capacity to complete a personal project. Study design A text about a woman entrepreneur was read and the meaning of entrepreneurship was discussed. In the end, each participant explained her own goals for the short, medium and long-term future. SESSION 1: Improving self-knowledge of individual GRRs. As an individual activity, each participant was encouraged to describe 5 qualities about herself. Health promotion programs based on the salutogenic model of health The aim of health promotion programs for migrant individ- uals are generally focused on covering their basic needs, such as food and housing [29], or on modifying lifestyle behaviors Page 3 of 9 Page 3 of 9 Bonmatí-Tomas et al. International Journal for Equity in Health (2019) 18:139 (2019) 18:139 Methods Afterwards these qualities were shared with the rest of the group. Fig. 1 Description of the health promotion program and its relation with the salutogenic model of health Fig. 1 Description of the health promotion program and its relation with the salutogenic model of health Page 4 of 9 Bonmatí-Tomas et al. International Journal for Equity in Health (2019) 18:139 (2019) 18:139 family members, proportion of time dedicated to the family. Factors such as limited command of the language, low literacy level and the different cultural backgrounds of the participants created difficulties during the develop- ment of the study, both in the data collection phase and in designing the health promotion program. In order to reduce these difficulties, a facilitator with training in questionnaire administration was included on the re- search team. In addition, a good practice guide was cre- ated together with the technical team of Caritas to help both the facilitator of the questionnaire process and the session leader. As needed, participants were given more time to complete the questionnaire and the duration of each session was extended. family members, proportion of time dedicated to the family. General data: current employment status, self- perception of the current socioeconomic situation, importance of spirituality in her life. – Dependent variables: Sense of coherence (SOC-13), using the Spanish version of a scale created by Aaron Antonovsky [39] that has been validated for use in the population of Spain [40, 41]. Duke-UNC-11, assessing perceived social support using the Spanish version of the instrument developed by Bellon Saameno et al. [42]. Quality of Life, Short Form-36 (SF-36 v.2), adapted and validated by Vilagut et al. [43]. Study population Rosenberg’s Self-Esteem Scale, using the validated Spanish version of Martiín-Albo [44]. Participants in the study were migrant women at risk of social exclusion who were participating in the Ecosol and Avancem Santa Clara programs of Caritas Diocesana. The 6-month programs focus on strengthening profes- sional skills through a personalized itinerary, with the aim of helping these women join the job market. Partici- pants were divided into 4 groups of 7 women each. The groups were intentionally heterogeneous, with partici- pants of different ethnic origins, religious and cultural backgrounds, and languages. The validated Spanish version of the Perceived Stress Scale (PSS 10) developed by Cohen et al [4 The validated Spanish version of the Perceived The validated Spanish version of the Perceived Stress Scale (PSS-10) developed by Cohen et al. [45]. Stress Scale (PSS-10) developed by Cohen et al. [45]. Data analysis A descriptive analysis of the sociodemographic variables was carried out. The binary relationship between vari- ables was calculated using Pearson’s chi-square test/ Pearson’s correlation, Student t-test and one-way ANOVA test. The bivariate effectiveness of the program was evaluated by Student paired t-test or Wilcoxon test. Normal distribution was assumed with the Kolmogorov- Smirnov and Shapiro-Wilk tests. All participants met the following inclusion criteria and consented to participate in the study: migrant women, older than 18 years, and at risk of social exclu- sion. Risk of social exclusion was assessed by the Caritas Institution according to the following criteria: communi- cation difficulties, lack of work experience, lack of school education, single parenthood, family breakdown, subsist- ence or precarious employment, poverty, deprivation of freedom, lack of legal documents, isolation, cultural dis- tance, illness, disability, lack of motivation, work habits not established, substandard housing, difficulties in hav- ing geographic and work-schedule flexibility, and un- employment. Participants had to meet at least 6 of these criteria to be considered at risk of social exclusion. A sequential multiple regression analysis was used to predict the factors associated with the effectiveness of the program. Initially, the model included as independ- ent variables the factors with published evidence of sta- tistically significant bivariate association with the selected program. The Statistical Package for the Social Sciences (SPSS) v.20.0 for Windows was used for data processing and analysis. The level of significance (p) was established as p < 0.05. Independent variables and study outcomes In contrast, the younger women reported a decrease in the mean quality of men- Table 1 Socio-demographic profile of the participants TOTAL (n=26) % Age 18-35 year old 11 42.3 > 35 year old 15 57.7 Country/Region of birth Morocco 10 38.5 Sub-Saharan Africa 10 38.5 Latin American Countries 6 23.0 Rural/urban origin Rural 4 15.4 Urban 22 84.6 Years outside the country of birth 0-10 years 12 46.2 > 10 years 14 53.8 Residency Background Migrated directly to Spain 23 88.5 Migrated to other countries 3 11.5 Years of schooling < 7 years 6 23.1 7-11 years 13 50.0 > 11 years 7 26.9 Importance of spirituality Low (0-7) 4 15.4 High (>7) 22 84.6 Marital status Single/Separated/Divorced 8 30.8 Married 18 69.2 Number of children 0 2 7.7 1 and 2 children 9 34.6 3 or more children 15 57.7 Current cohabitation status With family or relatives 22 84.6 Without family nor relatives 4 15.4 Dependant family members No 1 03.9 Yes 25 96.1 Daily proportion of time dedicated to family None to quite a lot (0-5) 13 50.0 Much time to all my time (5-10) 13 50.0 Current employment status Paid work outside the home a 20 76.9 Unemployment 6 23.1 Table 1 Socio-demographic profile of the participants (Continued) TOTAL (n=26) % Perceived socioeconomic status Good 10 38.5 Bad or very bad 16 61.5 NOTE: The variables are expressed with absolute frequency and the percentage aThis remuneration was the monetary contribution they received monthly from Caritas Institution for the work done in the programs in which they participate, which did not exceed in any case the 300€ Table 1 Socio-demographic profile of the p TOTA Age 18-35 year old 11 > 35 year old 15 Country/Region of birth Morocco 10 Sub-Saharan Africa 10 Latin American Countries 6 Rural/urban origin Rural 4 Urban 22 Years outside the country of birth 0-10 years 12 > 10 years 14 Residency Background Migrated directly to Spain 23 Migrated to other countries 3 Years of schooling < 7 years 6 7-11 years 13 > 11 years 7 Importance of spirituality Low (0-7) 4 High (>7) 22 Marital status Single/Separated/Divorced 8 Married 18 Number of children 0 2 1 and 2 children 9 3 or more children 15 Current cohabitation status With family or relatives 22 Without family nor relatives 4 Dependant family members No 1 Yes 25 Daily proportion of time dedicated to family None to quite a lot (0-5) 13 Much time to all my time (5-10) 13 Current employment status Paid work outside the home a 20 Unemployment 6 After their participation in the health promotion program, participants showed a remarkable decrease in the SOC di- mension of comprehensibility and in their perceived stress [see Table 2]. Independent variables and study outcomes From the 30 women enrolled in the programs Ecosol and Avancem Santa Clara offered by Caritas Diocesana in Girona, 28 started the health promotion program. One of them quit after the second session and another missed the final session. The sociodemographic data of the 26 participants who completed the study are sum- marized in Table 1. The majority of them migrated dir- ectly to Spain, are married, have children, and are living with family or relatives who are dependent in some way. They have a basic level of education, place high value on spirituality or religious faith in their daily life, and report a bad or very bad perception of their economic situation and a high level of spirituality. Independent variables and study outcomes The effectiveness of the intervention was measured by comparing the results of a questionnaire before and after the 6-month salutogenic health promotion program. The questionnaire included the following information: – Independent variables: Sociodemographic data: age, country of birth, background, geographic area, years of education. Migration history and current situation: number of years away from home country, number of years living in Spain, marital status, number of offspring, current cohabitation status, dependent Bonmatí-Tomas et al. International Journal for Equity in Health (2019) 18:139 Page 5 of 9 Associations between dependent variables and sociodemographic data The results of the pretest questionnaire according to the social demographic data are shown in Additional file 1. On one hand, women from Sub-Saharan African coun- tries had higher self-esteem compared to women from Morocco and Latin-American countries. On the other hand, participants who reported their socioeconomic situation as “bad” or “very bad” had higher levels of per- ceived stress than participants who considered their so- cioeconomic status to be “good”. Furthermore, participants from rural environments had higher scores in the mental dimension of quality of life, compared to those from urban areas. Effectiveness of the salutogenic health promotion program After their participation in the health promotion program, participants showed a remarkable decrease in the SOC di- mension of comprehensibility and in their perceived stress [see Table 2]. They also had an increased physical QL and showed a trend toward increased self-esteem. Changes in the dependent variables between the pre- test and posttest in relationship with the sociodemo- graphic data are shown in Additional file 2. Independent variables and study outcomes A greater increase in SOC can be observed in the women from Latin-American countries who were not living within a family setting at the moment of the study. Mean values for self-esteem increased more among participants from Latin-American countries, compared to those from Sub- Saharan African countries. In contrast, a decrease in self-esteem was observed among the women from Morocco. Participants with responsibility for dependent family members experienced a decrease in the mean value of perceived stress. Women younger than 35 years increased their mean physical QL score, while the older participants showed a decrease. In contrast, the younger women reported a decrease in the mean quality of men- tal health, while participants older than 35 years reported an increase. % 42.3 57.7 38.5 38.5 23.0 15.4 84.6 46.2 53.8 88.5 11.5 23.1 50.0 26.9 15.4 84.6 30.8 69.2 7.7 34.6 57.7 84.6 15.4 03.9 96.1 50.0 50.0 76.9 23.1 Table 1 Socio-demographic profile of the participants (Continued) TOTAL (n=26) % Perceived socioeconomic status Good 10 38.5 Bad or very bad 16 61.5 NOTE: The variables are expressed with absolute frequency and the percentage aThis remuneration was the monetary contribution they received monthly from Caritas Institution for the work done in the programs in which they participate, which did not exceed in any case the 300€ Associations between dependent variables and sociodemographic data The results of the pretest questionnaire according to the social demographic data are shown in Additional file 1. On one hand, women from Sub-Saharan African coun- tries had higher self-esteem compared to women from Morocco and Latin-American countries. On the other hand, participants who reported their socioeconomic situation as “bad” or “very bad” had higher levels of per- ceived stress than participants who considered their so- cioeconomic status to be “good”. Furthermore, participants from rural environments had higher scores in the mental dimension of quality of life, compared to those from urban areas. Effectiveness of the salutogenic health promotion program After their participation in the health promotion program, participants showed a remarkable decrease in the SOC di- mension of comprehensibility and in their perceived stress [see Table 2]. They also had an increased physical QL and showed a trend toward increased self-esteem. Changes in the dependent variables between the pre- test and posttest in relationship with the sociodemo- graphic data are shown in Additional file 2. Independent variables and study outcomes A greater increase in SOC can be observed in the women from Latin-American countries who were not living within a family setting at the moment of the study. Mean values for self-esteem increased more among participants from Latin-American countries, compared to those from Sub- Saharan African countries. In contrast, a decrease in self-esteem was observed among the women from Morocco. Participants with responsibility for dependent family members experienced a decrease in the mean value of perceived stress. Women younger than 35 years increased their mean physical QL score, while the older participants showed a decrease. Multiple regression analysis Multiple regression analysis was carried out to examine the relationship between the change in each construct and the various potential predictive factors. Additional file 4 summarizes the initial model (model 1), which compiled all independent variables, and the final model (model 2), which assembled the variables having a statistically signifi- cant correlation with the change in each construct after the health promotion program [see Fig. 2]. The activities of the health promotion program enabled participants to self-recognize not only their internal GRRs, but also those in their community and broader environment (external GRRs). In this sense, awareness of the GRRs promotes increased self-esteem, as previously reported [35]. Our results are consistent with the findings of an Australian study using a health promotion program and objectives similar to the present study [49]. The health promotion program contributed to a greater increase in SOC in participants with a higher so- cial support score and lower SOC at baseline, compared to the mean. The greatest change in perceived social support was observed in participants with a low initial score who were living within a family setting. In contrast, self-awareness of GRRs did not support an increase in participants’ SOC, which might be attributed to two explanations. The first, as suggested by other au- thors, is that the SOC remains constant for people older than 30 years [36]. The second is that this specific health promotion program did not include activities that de- velop the ability to use the GRRs in daily life. Higher perceived social support and mental QL at baseline were correlated with an increase in self-esteem at follow-up. In contrast, the higher the initial self-es- teem values, the less change was observed. Eventually, a greater change in perceived stress was noted in partici- pants with lower initial values for perceived stress, men- tal QL, and social support. Apart from this, the lower the initial parameters of perceived stress, self-esteem, mental and physical QL, and social support, the higher the observed changes in these parameters. This suggests greater effectiveness of salutogenic health promotion programs among women living in situations of greater vulnerability. Consequently, such programs would be especially effective in reducing health inequities among the most vulnerable population In reference to the quality of mental and physical health, greater change in physical QL was detected in younger women (< 35 years old) and in participants with lower initial physical QL scores. Multiple correlations The salutogenic health promotion program was associ- ated with a significant reduction in perceived stress, an increase in physical QL, and a tendency to increase self- esteem of the participants. Furthermore, this type of health promotion programs was more effective in women with lower initial scores in SOC, perceived stress, and mental and physical QL. Findings presented in Additional file 3 showed that all var- iations in mean values were inversely related to partici- pants’ own initial value. In addition, an inverse relationship between the change in perceived stress and initial mental health QL was observed (−0.426; p < 0.05). Thus, the decline in perceived stress was greater in women who initially reported a lower mental QL. On the other hand, the initial physical QL scores (0.466; p < 0.05) were positively correlated with changes in self-esteem. The lower scores in mental QL [46] and self-esteem detected in the study participants, compared with the mean for women born in Spain, are consistent with the results of other studies [47]. Some authors have con- nected these findings with the triple discrimination that migrant women at risk of social exclusion experience due to their gender, cultural, and social status [48]. Independent variables and study outcomes They also had an increased physical QL and showed a trend toward increased self-esteem. 69.2 7.7 34.6 Changes in the dependent variables between the pre- test and posttest in relationship with the sociodemo- graphic data are shown in Additional file 2. A greater increase in SOC can be observed in the women from Latin-American countries who were not living within a family setting at the moment of the study. Mean values for self-esteem increased more among participants from Latin-American countries, compared to those from Sub- Saharan African countries. In contrast, a decrease in self-esteem was observed among the women from Morocco. Participants with responsibility for dependent family members experienced a decrease in the mean value of perceived stress. Women younger than 35 years increased their mean physical QL score, while the older participants showed a decrease. In contrast, the younger women reported a decrease in the mean quality of men- tal health, while participants older than 35 years reported an increase. 57.7 84.6 15.4 03.9 96.1 50.0 50.0 76.9 23.1 Bonmatí-Tomas et al. International Journal for Equity in Health (2019) 18:139 Page 6 of 9 Table 2 Mean differences and distribution of the variables of interest before and after the health promotion program Mean PRE SD Mean POST SD Δ mean Post-Pre p Kolmo-gorov-Smirnov p Shapiro- Wilk p Student T SOC 60.36 8.16 59.81 8.16 -0.55 0.2 0.876 0.850 Comprehensibility 22.71 4.28 21.15 3.98 -1.56 0.000 0.000 0.018* Manageability 18.18 3.76 18.85 3.08 0.67 0.200 0.209 0.498 Meaningfulness 19.46 4.09 19.81 3.73 0.35 0.023 0.147 0.569* Self-esteem 30.14 4.21 31.92 4.38 1.78 0.200 0.543 0.120 Perceived Stress 20.57 2.91 18.38 3.78 -2.19 0.200 0.394 0.016 Social Support 37.07 6.28 37.08 5.56 0.01 0.200 0.375 0.782 Physical QL 50.84 4.60 53.08 5.31 2.24 0.200 0.216 0.049 Mental QL 46.00 5.90 45.62 7.13 -0.38 0.200 0.589 0.697 NOTE: Quantitative variables are expressed as mean and standard deviation (SD) Multiple regression analysis In contrast, greater change in mental QL was observed in older women and participants coming from Latin-American countries. Bonmatí-Tomas et al. International Journal for Equity in Health (2019) 18:139 Bonmatí-Tomas et al. International Journal for Equity in Health (2019) 18:139 Page 7 of 9 Page 7 of 9 Bonmatí-Tomas et al. International Journal for Equity in Health (2019) 18:139 Fig. 2 Variables showed relationship with the change of SOC, Self esteem, Perceived Stress, Social Support, Physical QL and mental QL Fig. 2 Variables showed relationship with the change of SOC, Self esteem, Perceived Stress, Social Support, Physical QL and mental QL to compare the results achieved by this intervention with other types of interventions. groups. This is especially relevant because health inter- ventions that are not specifically designed to benefit vul- nerable populations may actually increase inequities [50]. In addition, this program was aligned with current national policy guidelines in Spain [33] that prioritize this strategic goal for health programs designed for the most vulnerable populations. Despite these limitations, the present study has some strengths. One of them is the successful administration of a quantitative longitudinal questionnaire in a popula- tion of migrant women with high rates of illiteracy and general difficulties in communicating. These characteris- tics contribute to their exclusion from most quantitative studies. In turn, this exclusion reinforces the lack of ef- fective health promotion programs for this population, thus perpetuating health inequities. The significant decrease in perceived stress we observed is consistent with the findings in other studies [51]. The program activities were focused on awareness of stress protectors such as social support and knowing one’s role in the family [52], which could explain these results. Another strength of this study is the introduction of the salutogenic model into the program design. This new model of health takes into consideration the cap- acity of people to empower themselves using their own GRRs, which often go unrecognized by themselves and by their environment. This new approach may enable a new societal perspective on vulnerable populations, a vi- sion that treats the collective of vulnerable people as in- dividuals with abilities and resources even though they temporarily have some needs to be met. Another aspect to take into account, given our study re- sults, is the paradoxical increase in the physical but not in the mental QL of the participants after the program. References O d 1. Omidina P. Aging and family in an afghan refugee community. New York: Taylor&Francis Inc; 1996. Conclusions The salutogenic health promotion program for migrant women at risk of social exclusion significantly reduced per- ceived stress, increased physical QL, and tended to increase self-esteem. The program was more effective in women with lower initial scores for SOC and QL and higher scores for perceived stress. Perceived social support is also a key factor in the empowerment of these migrant women. The salutogenic model of health should be considered as a good practice to apply in health promotion pro- grams and to be included in national policies to reduce health inequity in migrant populations. Received: 1 December 2018 Accepted: 13 August 2019 Received: 1 December 2018 Accepted: 13 August 2019 Funding There was no external funding for this research. 12. Fernandez-Gutierrez M, Bas-Sarmiento P, Albar-Marin MJ, Paloma-Castro O, Romero-Sanchez JM. Health literacy interventions for immigrant populations a systematic review. Int Nurs Rev. 2018;65:54–64. https://doi.org/10.1111/ inr.12373. Author details 1 1Faculty of Nursing, University of Girona, Emili Grahit, 77, 17071 Girona, Spain. 2Health and Health Care Research Group, University of Girona, Emili Grahit, 77, 17071 Girona, Spain. 3European Training Consortium-Public Health and Health Promotion, Emili Grahit, 77, 17071 Girona, Spain. 4Department of Social Sciences, Heath and Society, Wageningen University & Research, Hollandseweg 1, Wageningen, KN 6706, The Netherlands. 5Vascular Health Research Group, Unitat de Suport a la Recerca Girona, Institut Universitari d’Investigació en Atenció Primària Jordi Gol (IDIAPJGol), Girona, Spain. 6Information System for Research in Primary Care (SIDIAP), Institut Universitari d’Investigació en Atenció Primària Jordi Gol (IDIAPJGol), Barcelona, Spain. 7Chair of Health Promotion, University of Girona, Emili Grahit, 77, 17071 Girona, Spain. Acknowledgements Elaine Lilly, Ph.D., provided guidance in the translation and assistance with final English language revision of the article. 8. Bhugra D. Migration and mental health. Acta Psiquiatr Scan. 2004;109(4): 243–58. https://doi.org/10.1046/j.0001-690X.2003.00246.x. 8. Bhugra D. Migration and mental health. Acta Psiquiatr Scan. 2004;109(4): 243–58. https://doi.org/10.1046/j.0001-690X.2003.00246.x. 9. Dow HD. An overview of stressors faced by immigrants and refugees: a guide for mental health practitioners. Home Health Care Manag Pract. 2001; 23(3):210–7. https://doi.org/10.1177/1084822310390878. 9. Dow HD. An overview of stressors faced by immigrants and refugees: a guide for mental health practitioners. Home Health Care Manag Pract. 2001; 23(3):210–7. https://doi.org/10.1177/1084822310390878. Abbreviations GRR G li GRRs: Generalized Resistance Resources; QL: Quality of Life; SOC: Sense of Coherence; SPSS: Statistical Package for the Social Science 7. Bregant T, Torosyan M, Shriwise A, Balwicki L, Tulchinsky T. Migrant and minority health in Europe: the way forward report on the Salzburg workshop on migrant and minority health, 3–9 April 2016. Public Health Rev. 2016;37:26. https://doi.org/10.1186/s40985-016-0045-0. Additional files No real progress towards equity: health of migrants and ethnic minorities on the eve of the year 2000. Soc Sci Med. 1995; 41:819–28. 6. Bollini P, Siem H. No real progress towards equity: health of migrants and ethnic minorities on the eve of the year 2000. Soc Sci Med. 1995; 41:819–28. Competing interests Competing interests The authors declare that they have no competing interests. p g The authors declare that they have no competing interests. Authors’ contributions Design of study: BTA, GGM, JCD. Design health program: BTA, MAMC, GVS, BFC. Health program realization: BTA. Analyses: BTA. Drafting manuscript: BTA and VL. Critical revisions: All authors. Final approval: All authors. Design of study: BTA, GGM, JCD. Design health program: BTA, MAMC, GVS, BFC. Health program realization: BTA. Analyses: BTA. Drafting manuscript: BTA and VL. Critical revisions: All authors. Final approval: All authors. 10. Lipson J, Melies A. Issues in health care of middle eastern patients in cross culture medicine. West J Med. 1983;139(6):854–61. 11. Flaskerud JH, Winslow BJ. Conceptualizing vulnerable populations health- related research. Nurs Res. 1998;47(2):69–78. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. 13. Urbanos-Garrido RM. Social inequalities in health: measuring the contribution of housing deprivation and social interactions for Spain. Int J Equity Health. 2012;11:77. https://doi.org/10.1186/1475-9276-11-77. 13. Urbanos-Garrido RM. Social inequalities in health: measuring the contribution of housing deprivation and social interactions for Spain. Int J Equity Health. 2012;11:77. https://doi.org/10.1186/1475-9276-11-77. Multiple regression analysis Two factors might explain this observation: differing cultural concepts of “health” and the complexity and multidimen- sional definition of QL. In some cultures, mental health problems stigmatize the affected person [53]. As a result, people tend to “unconsciously hide” their mental discom- fort, with a somatization into physical discomfort [54]. In this sense, the increased physical QL perceived by young adults such as our participants could be attributed to their improvement in self-esteem and reduction of stress, which they perceived as an improvement in their physical QL. Second, the inclusion of physical, psychic and social as- pects, and also subjective and objective facets of each par- ticipant, in defining QL [55] makes it particularly difficult to assess the changes in each dimension separately. The present study results have important implications for policymakers. Based on previous evidence [56] that new models of health empowerment for migrants are needed to improve their long-term health and well-being, applying this new approach that encourages the abilities of disadvan- taged individuals would go far beyond covering their basic needs. This would certainly reduce the stigmatization that vulnerable people experience today. In Spain, although health policy considers the reduction of inequities a priority in the political agenda, few health promotion initiatives have successfully integrated this new emphasis [33]. This study had several limitations. The small sample size affected the statistical power of the analysis. Furthermore, the use of a convenience sample limited the external validity of the results; this bias was diminished with the in- clusion of participants of different ages, cultures, and back- grounds. Another limitation was the quasi-experimental design without a control group, which made it impossible Further work is needed on long-term studies of vulner- able populations such as migrants. To study the effective- ness of the salutogenic health promotion programs, more longitudinal studies and interventional studies with Bonmatí-Tomas et al. International Journal for Equity in Health (2019) 18:139 Page 8 of 9 Page 8 of 9 Page 8 of 9 (2019) 18:139 representative samples are needed to support the adoption of policies to reduce health inequities. Additionally, new health promotion programs with the salutogenic model of health should be developed to enable participants not only to become aware of their own GRRs but also to use them in their daily life. Consent for publication Not applicable. Additional files 2. Nesterko Y, Braehler E, Grande G, Glaesmer H. Life satisfaction and health- related quality of life in immigrants and native-born Germans: the role of immigration-related factors. Qual Life Res. 2013;22(5):1005–13. https://doi. org/10.1007/s11136-012-0239-y. Additional file 1: Initials SOC, social support, self-esteem, perceived stress and quality of life by socio-demographic data. (XLSX 16 kb) Additional file 2: Variations of SOC, social support, self-esteem, perceived stress and quality of life by socio-demographic data. (XLSX 15 kb) Additional file 3: Correlations between the variations of the variables before and after the intervention. (XLSX 10 kb) Additional file 4: Models of the variation of SOC, social support, self- esteem, stress, physical quality of life and mental quality of life according to criteria of variables selection (n = 26). (XLSX 15 kb) Additional file 1: Initials SOC, social support, self-esteem, perceived stress and quality of life by socio-demographic data. (XLSX 16 kb) Additional file 2: Variations of SOC, social support, self-esteem, perceived stress and quality of life by socio-demographic data. (XLSX 15 kb) Additional file 3: Correlations between the variations of the variables before and after the intervention. (XLSX 10 kb) Additional file 4: Models of the variation of SOC, social support, self- esteem, stress, physical quality of life and mental quality of life according to criteria of variables selection (n = 26). (XLSX 15 kb) Additional file 1: Initials SOC, social support, self-esteem, perceived stress and quality of life by socio-demographic data. (XLSX 16 kb) 3. Bak-Klimek A, Karatzias T, Elliott L, Maclean R. The determinants of well- being among international economic immigrants: a systematic literature review and meta-analysis. Appl Res Qual Life. 2010;10(1):161–88. https://doi. org/10.1007/s11482-013-9297-8. Additional file 2: Variations of SOC, social support, self-esteem, perceived stress and quality of life by socio-demographic data. (XLSX 15 kb) Additional file 3: Correlations between the variations of the variables before and after the intervention. (XLSX 10 kb) 4. Eurostat. Migration and migrant population statistics. Luxemburg: European Union; 2018. https://ec.europa.eu/eurostat/data/database. Accessed 4 Sept 2018 Additional file 4: Models of the variation of SOC, social support, self- esteem, stress, physical quality of life and mental quality of life according to criteria of variables selection (n = 26). (XLSX 15 kb) 5. Idescat. Anuari estadístic de Catalunya. Barcelona: Generalitat de Catalunya; 2018. http://www.idescat.cat/pub/?id=aec&n=996. Accessed 11 Nov 2018 5. Idescat. Anuari estadístic de Catalunya. Barcelona: Generalitat de Catalunya; 2018. http://www.idescat.cat/pub/?id=aec&n=996. Accessed 11 Nov 2018 6. Bollini P, Siem H. Ethics approval and consent to participate The study was carried out in accordance with the Declaration of Helsinki and its later amendments and with the Council for International Organizations of Medical Sciences CIOMS guidelines for research in vulnerable populations. It was approved by the Committee on Clinical Research Ethics of Doctor Josep Trueta University Hospital in Girona (Spain) (Code 2014/029). All participants gave signed informed consent and agreed to maintain the confidentiality of everything that was said during the sessions of the health promotion program. The study was carried out in accordance with the Declaration of Helsinki and its later amendments and with the Council for International Organizations of Medical Sciences CIOMS guidelines for research in vulnerable populations. It was approved by the Committee on Clinical Research Ethics of Doctor Josep Trueta University Hospital in Girona (Spain) (Code 2014/029). All participants gave signed informed consent and agreed to maintain the confidentiality of everything that was said during the sessions of the health promotion program. 14. De Vito C, Massimi A, Di Thiene D, Rosso A, D’Andrea E, Vacchio MR, Villari P, Marzuillo C. Low level of attention to health inequalities in prevention planning activities of the Italian regions. Int J Equity Health. 2016;19:15–28. https://doi.org/10.1186/s12939-016-0318-8. 14. De Vito C, Massimi A, Di Thiene D, Rosso A, D’Andrea E, Vacchio MR, Villari P, Marzuillo C. Low level of attention to health inequalities in prevention planning activities of the Italian regions. Int J Equity Health. 2016;19:15–28. https://doi.org/10.1186/s12939-016-0318-8. 15. Malmusi D, Borrell C, Benach J. Migration-related health inequalities: showing the complex interactions between gender, social class and place of origin. Soc Sci Med. 2010;71(9):1610–9. https://doi.org/10.1016/j. socscimed.2010.07.043. Page 9 of 9 Page 9 of 9 Bonmatí-Tomas et al. International Journal for Equity in Health (2019) 18:139 Page 9 of 9 (2019) 18:139 16. Comisión para Reducir las Desigualdades Sociales en Salud en España. Avanzando hacia la equidad: propuesta de Políticas e intervenciones para reducir las desigualdades Sociales en salud en España. Madrid: Ministerio de Sanidad, Servicios Sociales e Igualdad; 2015. 16. Comisión para Reducir las Desigualdades Sociales en Salud en España. Avanzando hacia la equidad: propuesta de Políticas e intervenciones para reducir las desigualdades Sociales en salud en España. Madrid: Ministerio de Sanidad, Servicios Sociales e Igualdad; 2015. 39. Antonovsky A. The structure and properties of the sense of coherence scale. Soc Sci Med. 1993;36:725–33. 40. Virués-Ortega J, Martínez-Martín P, del Barrio JL, Lozano LM. Ethics approval and consent to participate 1990;25(5):235–43. 47. Bhugra D, Becker M. Migration, cultural bereavement and cultural identity. World Psychiatry. 2005;4(1):18–24. 25. Rutter M. Psychological resilience and protective mechanisms. In: Rolf JA, Masten D, Cichetti K, Nuechterlein SW, editors. Risk and Protective Factors in the Development of Psychopathology. Cambridge: Cambridge University Press; 1993. p. 181–214. 48. Berjot S, Gillet N. Stress and coping with discrimination and stigmatization. Front Psychol. 2011;2(33):1–13. https://doi.org/10.3389/fpsyg.2011.00033. 49. Spry N, Marchant T. How a personal development program enhances social connection and mobilises women in the community. Aust J Adult Learn. 2014;54(2):32–53. 26. Trickett E, Buchanan R. The role of personal relationships in transitions: contributions of an ecological perspective. In: Sarason B, Duck S, editors. Personal relationships: implications for clinical and community psychology. Chichester: Wiley; 2001. p. 141–57. 50. Frohlich KL, Potvin L. The inequality paradox: the population approach and vulnerable populations. Am J Public Health. 2008;98:216–21. 51. Van der Waerden JEB, Jansen M, Hoefnagels C, Hosman C. Do disavadvantaged women appreciate a synergetic exercise and psychoeducative program? Rationale process evaluation of the exercise without worries course. Int J Pers Cent Med. 2011;17:180–99. 27. Gracia E, Herrero J. Personal and situational determinants of relationship- specific perceptions of social support. Soc Behav Pers. 2004;32(5):459–76. https://doi.org/10.2224/sbp.2004.32.5.459. 28. UCL Institute of Health Equity. Review of social determinants and the health divide in the WHO European Region: Executive Summary. 2013. http://www. instituteofhealthequity.org/resources-reports/review-of-social-determinants- and-the-health-divide-in-the-who-european-region-final-report 52. Golding J. Role occupancy and role-specific stress and social support and predictors of depression. Basic Appl Soc Psych. 1989;10(2):173–95. https:// doi.org/10.1207/s15324834basp1002_7. 53. Gary FA. Stigma: Barrier to mental health care among ethnic minorities. Issues Ment Health Nurs. 2005;26(10):979–99. https://doi.org/10.1080/01612 840500280638. 29. Maxwell AE, Young S, Crespi CM, Vega RR, Cayetano RT, Bastani R. Social determinants of health in the Mixtec and Zapotec community in Ventura County, California. Int J Equity Health. 2015;14:16. https://doi.org/10.1186/s12 939-015-0148-0. 54. Bekhuis E, Boschloo L, Rosmalen JGM, Schoevers RA. Differential associations of specific depressive and anxiety disorders with somatic symptoms. J Psychosom Res. 2015;78:116–22. https://doi.org/10.1016/j.jpsychores.2014.11.007. 30. Capewell S, Graham H. Will cardiovascular disease prevention widen health inequalities? PLoS Med. 2010;7:e1000320. 55. Costanza R, Fisher B, Ali S, Beer C, Bond L, Boumans R, Louis N, et al. Quality of life: an approach integrating opportunities, human needs, and subjective well-being. Ecol Econom. 2007;61(2–3):267–76. https://doi.org/10.1016/j. ecolecon.2006.02.023. 31. Bambra C, Gibson M, Sowden A, Wright K, Whitehead M, Petticrew M. Ethics approval and consent to participate Tackling the wider social determinants of health and health inequalities: evidence from systematic reviews. J Epidemiol Public Health Rev. 2010;64:284–91. 32. Ministerio de Sanidad, Consumo y Bienestar Social. Estrategia de Promoción de la Salud y Prevención en el Sistema Nacional de Salud. Memoria Buenas Prácticas. 2017. http://www.mscbs.gob.es/profesionales/saludPublica/ prevPromocion/Estrategia/docs/MemoriaBuenasPracticasEstrategia_2017.pdf 56. Wong W, Ho P, Liang J, Holroyd E, Lam C, Pau A. Road to better health and integration: a Delphi study on health service models for Hong Kong migrants. Int J Equity Health. 2014;13:127. https://doi.org/10.1186/s12939- 014-0127-x. 56. Wong W, Ho P, Liang J, Holroyd E, Lam C, Pau A. Road to better health and integration: a Delphi study on health service models for Hong Kong migrants. Int J Equity Health. 2014;13:127. https://doi.org/10.1186/s12939- 014-0127-x. 33. Comisión para Reducir las Desigualdades Sociales en Salud en España. Propuesta de políticas e intervenciones para reducir las desigualdades sociales en salud en España. Gac Sanit. 2012;26(2):182–9. https://doi.org/10.1 016/j.gaceta.2011.07.024. Ethics approval and consent to participate Validación transcultural de la Escala de Sentido de Coherencia de Antonovsky (OLQ- 13) en ancianos mayores de 70 años. Med Clín. 2007;128(13):486–92. https:// doi.org/10.1157/13100935. 17. Ahonen E, López-Jacob M, Vázquez M, Porthé V. Gil- González D, García a. invisible work, unseeen hazards: the health of women immigrant household service workers in Spain. Am J Ind Med. 2010;53(4):405–16. 41. Malagón-Aguilera M, Juvinyà Canal D, Bonmatí-Tomàs A, Fernández-Peña R, Bosch-Farré C, Bertran Noguer C, Suñer-Soler R. Sentido de coherencia de las enfermeras y validación del cuestionario SOC-13. Metas Enferm. 2012; 15(9):27–31. 18. Clark R, Anderson N, Clark V, Williams D. Racism as a stressor for African Americans: a biopsychosocial model. Am Psychol. 1999;54(10):805–16. Americans: a biopsychosocial model. Am Psychol. 1999;54(10):805–16. 19. Miller C, Kaiser C. A theoretical perspective on coping with stigma. J Soc Issues. 2001;57(1):73–92. https://doi.org/10.1111/0022-4537.00202. 19. Miller C, Kaiser C. A theoretical perspective on coping with stigma. J Soc Issues. 2001;57(1):73–92. https://doi.org/10.1111/0022-4537.00202. 42. Bellon Saameno JA, Delgado SA, Luna del Castillo JD, Lardelli CP. Validez y fiabilidad del cuestionario de apoyo social funcional Duke-UNC-11. Atenc Prim. 1996;18(4):153–63. 20. Aguadelo-Suárez A, Gil-González D, Ronda-Pérez E, Porthé V, Paramio-Pérez G, García A, Garí A. Discrimination, work and health in immigrant populations in Spain. Soc Sci Med. 2009;68(10):1866–74. 43. Vilagut G, Valderas J, Ferrer M, Garín O, Lopez-Garcia E, Alonso J. Interpretación de los cuestionarios de salud SF-36 y SF-12 en España: componentes físico y mental. Med Clín. 2008;130(19):726–35. https://doi.org/10.1157/13121076. 21. Major B, O’Brien L. The social psychlogy of stigma. Annu Rev Clin Psychol. 2005;56:393–421. https://doi.org/10.1146/annurev.psych.56.091103.070137. 44. Martín-Albo J, Núñiez JL, Navarro JG, Grijalvo F. The Rosenberg self-esteem scale: translation and validation in university students. Span J Psychol. 2007; 10(2):458–67. https://doi.org/10.1017/S1138741600006727. 22. Corker E, Brown J, Henderson C. How does self stigma differ across people with psychiatric diagnoses and rheumatoid arthritis, and how does it impact on self-esteem and empowerment? Psychol Health Med. 2016;85:1– 13. https://doi.org/10.1080/13548506.2016.1139139. 45. Remor E. Psychometric properties of a European Spanish version of the perceived stress scale (PSS). Span J Psychol. 2006;9(1):86–93. 23. Mann M, Hosman CMH, Schaalma HP, de Vries NK. Self-esteem in a broad- spectrum approach for mental health promotion. Health Educ Res. 2004; 19(4):357–72. https://doi.org/10.1093/her/cyg041. 46. Bayram N, Thorburn D, Demirhan H, Bilgel N. Quality of life among Turkish immigrants in Sweden. Qual Health Res. 2007;16(8):1319–33. https://doi. org/10.1007/s11136-007-9249-6. 24. Brown GK, Bifuco A, Andrews B. Self esteem and depression III. Aetiological issues. Soc Psychiatry Psychiatr Epidemio. 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. 34. Antonovsky A. Health, stress and coping. San Francisco: Jossey-Bass; 1979. 35. Eriksson M, Lindström B. Validity of Antonovsky’s sense of coherence scale: a systematic review. J Epidemiol Public Health Rev. 2005;59(6):460–6. https:// doi.org/10.1136/jech.2003.018085. 36. Antonovsky A. Unraveling the mystery of health. San Francisco: Jossey-Bass; 1987. 37. Langeland E, Riise T, Hanestad BR, Nortvedt MW, Kristoffersen K, Wahl AK. The effect of salutogenic treatment principles on coping with mental health problems. A randomised controlled trial. Patient Educ Couns. 2006;62(2): 212–9. https://doi.org/10.1016/j.pec.2005.07.004. 38. Van der Waerden JEB, Hoefnagels C, Hosman CH, Souren PM, Jansen MWJ. A randomized controlled trial of combined exercise and psycho-education for low-SES women: short-and long-term outcomes in the reduction of stress and depressive symptoms. Soc Sci Med. 2013;91:84–93. https://doi. org/10.1016/j.socscimed.2013.05.015.
https://openalex.org/W4225294459	https://www.eurosurveillance.org/deliver/fulltext/eurosurveillance/27/17/eurosurv-27-17-5.pdf?itemId=%2Fcontent%2F10.2807%2F1560-7917.ES.2022.27.17.2200155&mimeType=pdf&containerItemId=content/eurosurveillance	English	null	Public health considerations for transitioning beyond the acute phase of the COVID-19 pandemic in the EU/EEA	Euro surveillance/Eurosurveillance	2,022	cc-by	3,742	Public health considerations for transitioning beyond the acute phase of the COVID-19 pandemic in the EU/ EEA Jonathan E Suk1 , Anastasia Pharris¹ , Julien Beauté¹ , Edoardo Colzani¹ , Howard Needham¹ , John Kinsman¹ , Rene Niehus¹ , Rok Grah¹ , Ajibola Omokanye¹ , Diamantis Plachouras¹ , Agoritsa Baka¹ , Bastian Prasse¹ , Frank Sandmann¹ , Ettore Severi¹ , Erik Alm¹ , Emma Wiltshire¹ , Bruno Ciancio¹ , , 1. European Centre for Disease Prevention and Control (ECDC), Stockholm, Sweden Correspondence: Jonathan E Suk (jonathan.suk@ecdc.europa.eu) Citation style for this article: Suk Jonathan E, Pharris Anastasia, Beauté Julien, Colzani Edoardo, Needham Howard, Kinsman John, Niehus Rene, Grah Rok, Omokanye Ajibola, Plachouras Diamantis, Baka Agoritsa, Prasse Bastian, Sandmann Frank, Severi Ettore, Alm Erik, Wiltshire Emma, Ciancio Bruno. Public health considerations for transitioning beyond the acute phase of the COVID-19 pandemic in the EU/EEA. Euro Surveill. 2022;27(17):pii=2200155. https://doi.org/10.2807/1560-7917. ES 2022 27 17 2200155 Article submitted on 10 Feb 2022 / accepted on 24 Mar 2022 / published on 28 Apr 2022 Many countries, including some within the EU/EEA, are in the process of transitioning from the acute pan- demic phase. During this transition, it is crucial that countries’ strategies and activities remain guided by clear COVID-19 control objectives, which increas- ingly will focus on preventing and managing severe outcomes. Therefore, attention must be given to the groups that are particularly vulnerable to severe out- comes of SARS-CoV-2 infection, including individuals in congregate and healthcare settings. In this phase of pandemic management, a strong focus must remain on transitioning testing approaches and systems for targeted surveillance of COVID-19, capitalising on and strengthening existing systems for respiratory virus surveillance. Furthermore, it will be crucial to focus on lessons learned from the pandemic to enhance preparedness and to enact robust systems for the pre- paredness, detection, rapid investigation and assess- ment of new and emerging SARS-CoV-2 variants. Filling existing knowledge gaps, including behavioural insights, can help guide the response to future resur- gences of SARS-CoV-2 and/or the emergence of other pandemics. Finally, ‘vaccine agility’ will be needed to respond to changes in people’s behaviours, changes in the virus, and changes in population immunity, all the while addressing issues of global health equity. generally high, it is expected that at the end of the ongoing Omicron wave the vast majority of the EU/EEA population will have built a certain degree of immuno- logical memory against SARS-CoV-2. This could result in a reduced severity of coronavirus disease (COVID-19) in the population going forward. Public health considerations for transitioning beyond the acute phase of the COVID-19 pandemic in the EU/ EEA Such a situation would enable countries to more efficiently manage the pan- demic [1]. However, it is generally expected that SARS- CoV-2 will become endemic [2], and this may involve repeated reinfections, some degree of seasonality in temperate regions, and/or unanticipated upsurges in cases. For example, following what appeared to be the end of a SARS-CoV-2 Omicron wave and follow- ing the accompanying relaxation of measures in many countries, COVID-19 case notification rates started to increase again in 14 of 30 EU/EEA countries by 18 March 2022, impacting hospitalisation and death rates in some of these settings [3]. Following a few weeks of an increase, most countries are now observing declining rates again as of the week ending 17 April 2022, with hospitalisation and death rates similar to pre-Omicron levels. Meanwhile, high levels of global SARS-CoV-2 circulation leads to an elevated risk of viral mutation, and it should not be assumed that each subsequent SARS-CoV-2 variant will lead to less severe disease than its predecessor [4]. Citation style for this article: Suk Jonathan E, Pharris Anastasia, Beauté Julien, Colzani Edoardo, Needham Howard, Kinsman John, Niehus Rene, Grah Rok, Omokanye Ajibola, Plachouras Diamantis, Baka Agoritsa, Prasse Bastian, Sandmann Frank, Severi Ettore, Alm Erik, Wiltshire Emma, Ciancio Bruno. Public health considerations for transitioning beyond the acute phase of the COVID-19 pandemic in the EU/EEA. Euro Surveill. 2022;27(17):pii=2200155. https://doi.org/10.2807/1560-7917. ES.2022.27.17.2200155 www.eurosurveillance.org Perspective Perspective Towards a pandemic transition phase p p Relaxing NPI mitigation measures while SARS-CoV-2 continues to transmit globally implies a shift in focus, from limiting both disease spread and severe outcomes to primarily focusing on managing severe outcomes. It is instructive to view the current context as the begin- ning of a pandemic transition phase, which cannot be time-stamped but may last for 1–2 years. During this time, healthcare systems will seek to recover from the pandemic strain and make strategic decisions to adapt public health preparedness, response, and sur- veillance systems capable of managing COVID-19 and other future pandemics over the long term. During this pandemic transition phase, many public health issues will need to be considered, the most important being that countries focus on enhancing pandemic prepared- ness and response, adapting testing and surveillance strategies, protecting vulnerable groups, expand- ing capabilities for conducting behavioural research, ensuring ‘vaccine agility’, strengthening global health, and addressing critical knowledge gaps. Adapting testing strategies and surveillance Due to reduced severity associated with the Omicron variant and its higher transmissibility making iso- lation and quarantine less practical, countries are contemplating a shift in testing approach away from diagnosis and case detection and towards routine indicator-based surveillance [10]. Until recently, in most countries, COVID-19 surveillance systems relied on comprehensive collection of testing data. Testing rates (10,000 tests per 100,000 population for week 2022–02 at EU/EEA level) may not be sustainable and their cost-effectiveness is unclear. However, changing testing strategies impact testing indicators and may distort surveillance data in unpredictable ways [11]. Background h The extensive range of non-pharmaceutical interven- tions (NPIs) implemented in the EU/EEA over the past 2 years has contributed to reductions in COVID-19-related hospitalisations and mortality, particularly before vac- cination uptake reached very high levels [5]. However, NPIs have also exacted heavy societal and economic costs. Decisionmakers will need to continue to weigh the effectiveness and societal impact of COVID-19 con- trol measures against the observed severity of COVID- 19 at the population level, and information around The emergence and rapid global transmission of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Omicron variant of concern (VOC) serves as both a reason for cautious optimism and a cau- tionary tale. Given that the attack rate of the Omicron VOC in the population has been very high, that 70% of the European Union/European Economic Area (EU/ EEA) population has completed its primary vaccination course, and that the uptake of booster doses has been www.eurosurveillance.org 1 key variables, including COVID-19 incidence and severity, will continue to vary across time and space. Furthermore, as widely observed up to this point in the COVID-19 pandemic, decisions regarding COVID-19 con- trol measures also occur in political spheres [6], where factors beyond public health evidence often influence decision making [7]. most effective, cost-effective, and socially tolerable measures when needed. Thus, when NPIs are imple- mented, they should be accompanied by assessments of their effectiveness, cost-effectiveness and public acceptance. In anticipation of future outbreaks, national prepared- ness and response plans should be revised and tested routinely, and protocols for quickly assessing the transmissibility and severity of new variants should be put in place. Networks of teams that can be quickly deployed to outbreak situations (whether for respira- tory or other types of disease) should be expanded and reinforced, as is envisioned by establishment of the EU Health Task Force [9], to provide more coordinated and operational responses to outbreak settings across Europe. www.eurosurveillance.org Protecting vulnerable groups and expanding behavioural research Beyond spring 2022, there will likely remain population groups that are vulnerable to unfavourable outcomes, such as elderly people, people with underlying con- ditions, as well as immunocompromised and immu- nologically-naive people. In addition, because of the dynamically changing immunity landscape, immunity is also likely to wane for a larger share of the currently protected population during 2022. Although one can expect the protection to last longer against severe out- comes, individuals may underestimate their personal risk should their immunity have waned [14]. Furthermore, future efforts need to consider the speed at which vaccines can be distributed and administered to citizens at scale. This will require substantial global investments to develop and sustain such capacities, with resources to additionally monitor vaccine accept- ance and reach the most vulnerable and at-risk popula- tion groups [15]. Two years into the pandemic, there is still insufficient understanding of the behavioural, cultural and societal drivers that either facilitate or inhibit population accept- ance and adherence to public health interventions or on effective risk communication of these issues and how and why these drivers can change over time. To this end, a range of specific issues needs to be inves- tigated using behavioural insights research methods. These methods include providing in-depth and action- able understanding about (i) how to continue to effec- tively promote COVID-19 vaccination acceptance and uptake rates (both for the primary vaccination course and additional booster doses), in particular in popula- tions where there is active resistance to vaccination [15] and, (ii) within a context of pandemic fatigue [16], how to facilitate population acceptance of and adher- ence to any future NPIs should these become epide- miologically necessary. In relation to both these areas, it will also be necessary to systematically monitor and respond to misinformation that can reduce people’s willingness and motivation to undertake the respec- tive public health measures [17]. To accomplish these goals, strengthening of behavioural insights research capacity is needed in many countries [18]. It is important to be clear about the objectives of future revaccinations of the general population and of vulner- able groups. Long-term vaccination strategies should align with public health priorities for managing COVID- 19 burden as the situation (and population immunity) evolves, addressing immunity gaps and protecting the most at-risk populations. Protecting vulnerable groups and expanding behavioural research Depending on the objectives and the priorities as well as on the actual added ben- efit that can be obtained from additional doses, dif- ferent needs for COVID-19 vaccine development and deployment may arise [22]. Ensuring ‘vaccine agility’ Ensuring ‘vaccine agility’ who have contact with vulnerable populations such as healthcare workers in acute and long-term care set- tings. Since groups that are likely to have mild disease (i.e., young and healthy individuals) are also those more likely to be the first infected in an upsurge, it is impor- tant to keep a representative sample of these groups to be tested for surveillance purposes. Screening test- ing (i.e., testing individuals without symptoms) should be restricted to high-risk settings. Although the cost- effectiveness of community screening to prevent future epidemics remains unclear (particularly before large gatherings and festive holidays), community screen- ing should be assessed in comparison to the costs of population level NPIs. As new variants of concern may emerge at unpredicta- ble times and with unpredictable characteristics, these variants could affect the effectiveness of the immune response to current vaccine formulations. However, cell-mediated immunity from current vaccines, which is important for protection against severe disease, appears to show relatively good cross-protection against different variants [19]. Efficiency improvements are required for the full vac- cine development cycle, from processes for selecting updated vaccine targets through to manufacturing. This might be based on lessons learned from a sea- sonal influenza model and should be based on a gov- ernance mechanism for strain selection. It should also include consideration of alternative vaccine strate- gies such as developing vaccines targeting conserved SARS-CoV-2 antigens that may offer broader protec- tion against future variants or vaccines inducing strong mucosal immunity [20] and consideration of alternative vaccine strategies such as developing vaccines tar- geting conserved SARS-CoV-2 antigens that may offer broader protection against future variants or vaccines inducing strong mucosal immunity [20,21]. Another challenge for surveillance is its ability to assess the severity of an emerging variant, especially if the levels of both natural-acquired and vaccine- induced immunity are very different across countries. www.eurosurveillance.org Enhancing pandemic preparedness and response The COVID-19 pandemic has had many long-ranging impacts on public health as well as on the larger global economic and social sphere in which public health operates. It is imperative that innovations and good practices that emerged during the pandemic are safe- guarded. Structured after-action reviews and lessons learned exercises, with sufficient high-level buy-in to ensure that lessons are acted on, should be an impor- tant activity area during the pandemic transition phase [8]. ECDC published a COVID-19 surveillance guidance encouraging countries to transition from emergency surveillance for COVID-19 to more sustainable, objec- tive-driven surveillance systems [12]. These systems should allow for integrated surveillance of COVID-19, influenza and other respiratory pathogens that are likely to co-circulate in the population. Such a transi- tion will require adjusting and enhancing existing sen- tinel schemes used for seasonal influenza surveillance and implementing SARI (severe acute respiratory infec- tion) surveillance. A key short- and long-term objective for pandemic pre- paredness will be to ensure the rapid identification, investigation and assessment of key epidemiological parameters for each new variant of interest or of con- cern. Arrangements to ensure the timely information sharing from scientific or public health communities on new variants are vital to ensure a globally coordi- nated response to the pandemic. Operational study protocols, where not already in place, must be devel- oped and implemented so that secondary attack and viral growth rates for newly circulating variants can be assessed and neutralisation studies performed. The contact patterns between individuals are chang- ing over time. The paths of SARS-CoV-2 transmission in the population are determined by the heterogeneous and time-varying physical contacts between individu- als, ranging from superspreading events by individuals with many contacts and/or particularly high viral shed- ding to isolated individuals with only few contacts [13]. Accurate data on temporal contact patterns between individuals for different regions, vaccination status and age will be important for predicting the future course of COVID-19. As stringent NPIs have many collateral socioeconomic impacts, several population-level NPIs may princi- pally be viewed as a last resort, to be implemented only during high-impact outbreaks. However, it will be important to maintain the possibility to implement the The focus of diagnosis testing will increasingly shift to the timely testing of symptomatic people with underly- ing risk factors for severe COVID-19 who may benefit from early antiviral treatment and the testing of people www.eurosurveillance.org 2 Addressing knowledge gaps i i l i h All authors contributed to the design, content, and reviewing of this manuscript. It is essential to recognise the many uncertainties that currently exist around factors that could have profound impact on the trajectory of the COVID-19 pandemic. Some uncertainties are intrinsic to the evolving epi- demiological situation and are inherently unpredict- able, such as the emergence of new variants or the negative impact on quality of life and economic pro- ductivity potentially arising from post-acute COVID. In addition, and in spite of the impressive scientific research effort that continues to be directed at COVID- 19, there remains a lack of detailed understanding on key clinical aspects including susceptibility, mecha- nisms and duration of immunity, predictors of severity and correlates of protection. The multifaceted public heath response and increasingly divergent immuno- logical status following multiple rounds of natural- and vaccine-induced exposure also makes assessment of the relative effectiveness of specific interventions ever more challenging. All of these factors are in principle amendable to research study, and there is a need for further and sustained investment and innovation to inform current and future public health policy. References 1. Murray CJL. COVID-19 will continue but the end of the pandemic is near. Lancet. 2022;399(10323):417-9. https://doi. org/10.1016/S0140-6736(22)00100-3 PMID: 35065006 2. Emanuel EJ, Osterholm M, Gounder CR. A National Strategy for the “New Normal” of Life With COVID. JAMA. 2022;327(3):211-2. https://doi.org/10.1001/jama.2021.24282 PMID: 34989789 3. European Centre for Disease Prevention and Control (ECDC). Country overview report: week 15 2022. Stockholm: ECDC; 2022. Available from: https://www.ecdc.europa.eu/en/ covid-19/country-overviews 4. Katzourakis A. COVID-19: endemic doesn’t mean harmless. Nature. 2022;601(7894):485. https://doi.org/10.1038/d41586- 022-00155-x PMID: 35075305 4. Katzourakis A. COVID-19: endemic doesn’t mean harmless. Nature. 2022;601(7894):485. https://doi.org/10.1038/d41586- 022-00155-x PMID: 35075305 4. Katzourakis A. COVID-19: endemic doesn’t mean harmless. Nature. 2022;601(7894):485. https://doi.org/10.1038/d41586- 022-00155-x PMID: 35075305 5. Vardavas CI, Nikitara K, Aslanoglou K, Hilton-Boon M, Phalkey R, Leonardi-Bee J, et al. Effectiveness of non-pharmaceutical measures (NPIs) on COVID-19 in Europe: A systematic literature review. medRxiv. 2021:2021.11.11.21266216 . https://doi.org/10 .1101/2021.11.11.21266216 6. Lee CT, Buissonnière M, McClelland A, Frieden TR. Association Between Preparedness and Response Measures and COVID-19 Incidence and Mortality. medRxiv. 2021:2021.02.02.21251013 . https://doi.org/10.1101/2021.02.02.21251013 7. Salajan A, Tsolova S, Ciotti M, Suk JE. To what extent does evidence support decision making during infectious disease outbreaks? A scoping literature review. Evid Policy. 2020;16(3):453-75. https://doi.org/10.1332/17442642 0X15808913064302 We would like to thank the ECDC stakeholders that comment- ed upon an earlier version of this document. In addition, we would like to thank Mike Catchpole, Thomas Hoffmann, and Vicky Lefevre for their contributions to this work. Strengthening global health g g g The SARS-CoV-2 Omicron variant has intensified exist- ing discussions surrounding global health equity and has demonstrated yet again that no country is safe until all countries are safe. Strategic decisions are required around enhanced investments in global health infrastructure and in bolstering availability of COVID-19 vaccines globally. In addition, there is a need to support all countries to strengthen their pandemic preparedness and to develop the capacity to conduct epidemiologic investigations [23]. Such investments would pay dividends beyond COVID-19. Meanwhile, better understanding of possible path- ways for viral evolution is needed. During the COVID-19 www.eurosurveillance.org 3 pandemic, there have been instances of SARS-CoV-2 transmission among a range of mammalian species, including mink in Europe or deer in North America [24], and there is a risk that the novel variants of con- cern (VOC) could emerge through zoonotic events. Therefore, greater emphasis on One Health aspects should be given at the global level. Conclusion In the future, a new pandemic will occur, whether due to a very different new variant of SARS-CoV-2 or another pathogen altogether. Until then, it is essential to take advantage of the upcoming post-acute phase of the pandemic to foster recovery by identifying lessons from the pandemic and using these to strengthen pandemic preparedness and to design public health systems to efficiently manage COVID-19 over the long-term. 8. European Centre for Disease Prevention and Control (ECDC). Conducting in-action and after-action reviews of the public health response to COVID-19. Stockholm: ECDC; 2020. Available from: https://www.ecdc.europa.eu/en/publications- data/conducting-action-and-after-action-reviews-public-health- response-covid-19 9. European Commission. European Health Union: Commission welcomes agreement on a stronger European Centre for Disease Prevention and Control. Brussels: European Commission; 30 Nov 2021. Available from: https://ec.europa. eu/commission/presscorner/detail/en/IP_21_6435 10. Peeling RW, Heymann DL, Teo Y-Y, Garcia PJ. Diagnostics for COVID-19: moving from pandemic response to control. Lancet. 2022;399(10326):757-68. https://doi.org/10.1016/S0140- 6736(21)02346-1 PMID: 34942102 There are multiple uncertainties and conflicting factors currently at play. While individual and societal fatigue cannot be ignored, there is also the need for contin- ued vigilance to the threats posed by COVID-19. Public health decisionmakers will need to be attentive to this dynamic while advocating for further public health work to reduce scientific uncertainties and to mini- mise the overall societal burden of COVID-19. It is also imperative to help citizens and communities to recover from the pandemic and to build societal resilience for future pandemics. 11. Beauté J, Adlhoch C, Bundle N, Melidou A, Spiteri G. Testing indicators to monitor the COVID-19 pandemic. Lancet Infect Dis. 2021;21(10):1344-5. https://doi.org/10.1016/S1473- 3099(21)00461-8 PMID: 34450053 12. European Centre for Disease Prevention and Control (ECDC). COVID-19 surveillance guidance. Transition from COVID-19 emergency surveillance to routine surveillance of respiratory pathogens. Stockholm: ECDC; 2021. Available from: https://www.ecdc.europa.eu/en/publications-data/ covid-19-surveillance-guidance 13. Verelst F, Hermans L, Vercruysse S, Gimma A, Coletti P, Backer JA, et al. SOCRATES-CoMix: a platform for timely and open- source contact mixing data during and in between COVID-19 surges and interventions in over 20 European countries. BMC Med. 2021;19(1):254. https://doi.org/10.1186/s12916-021- 02133-y PMID: 34583683 Acknowledgements Acknowledgements 14. Bhattacharyya RP, Hanage WP. Challenges in Inferring Intrinsic Severity of the SARS-CoV-2 Omicron Variant. N Engl J Med. 2022;386(7):e14. https://doi.org/10.1056/NEJMp2119682 PMID: 35108465 We would like to thank the ECDC stakeholders that comment- ed upon an earlier version of this document. In addition, we would like to thank Mike Catchpole, Thomas Hoffmann, and Vicky Lefevre for their contributions to this work. 15. European Centre for Disease Prevention and Control (ECDC). Facilitating COVID-19 vaccination acceptance and uptake in the EU/EEA. Stockholm: ECDC; 2021. Available from: https://www.ecdc.europa.eu/en/publications-data/ facilitating-covid-19-vaccination-acceptance-and-uptake 15. European Centre for Disease Prevention and Control (ECDC). Facilitating COVID-19 vaccination acceptance and uptake in the EU/EEA. Stockholm: ECDC; 2021. Available from: https://www.ecdc.europa.eu/en/publications-data/ facilitating-covid-19-vaccination-acceptance-and-uptake 4 www.eurosurveillance.org www.eurosurveillance.org 16. World Health Organization Regional Office for Europe (WHO Regional Office for Europe). Pandemic fatigue: reinvigorating the public to prevent COVID-19: policy framework for supporting pandemic prevention and management: revised version November 2020. Copenhagen: WHO Regional Office for Europe. 2020; Available from: https://apps.who.int/iris/ handle/10665/337574 17. European Centre for Disease Prevention and Control (ECDC). Countering online vaccine misinformation in the EU/EEA. Stockholm: ECDC; 2021. Available from: https://www.ecdc.europa.eu/en/publications-data/ countering-online-vaccine-misinformation-eu-eea 18. European Centre for Disease Prevention and Control (ECDC). Behavioural Insights research to support the response to COVID-19: a survey of implementation in the EU/EEA. Stockholm: ECDC; 17 Feb 2021. Available from: https://www.ecdc.europa.eu/en/publications-data/ behavioural-insights-research-support-response-covid-19 19. Moss P. The T cell immune response against SARS-CoV-2. Nat Immunol. 2022;23(2):186-93. https://doi.org/10.1038/s41590- 021-01122-w PMID: 35105982 20. Lapuente D, Fuchs J, Willar J, Vieira Antão A, Eberlein V, Uhlig N, et al. Protective mucosal immunity against SARS-CoV-2 after heterologous systemic prime-mucosal boost immunization. Nat Commun. 2021;12(1):6871. https://doi.org/10.1038/s41467- 021-27063-4 PMID: 34836955 21. Tan C-W, Chia W-N, Young BE, Zhu F, Lim B-L, Sia W-R, et al. Pan-Sarbecovirus Neutralizing Antibodies in BNT162b2-Immunized SARS-CoV-1 Survivors. N Engl J Med. 2021;385(15):1401-6. https://doi.org/10.1056/NEJMoa2108453 PMID: 34407341 22. European Centre for Disease Prevention and Control (ECDC). Objectives of vaccination strategies against COVID-19. Stockholm: ECDC; 2021. Available from: https://www.ecdc.europa.eu/en/publications-data/ objectives-vaccination-strategies-against-covid-19 23. Reid M, Abdool-Karim Q, Geng E, Goosby E. How will COVID-19 transform global health post-pandemic? Defining research and investment opportunities and priorities. PLoS Med. 2021;18(3):e1003564. https://doi.org/10.1371/journal. pmed.1003564 PMID: 33705396 24. Mallapaty S. Where did Omicron come from? Three key theories. Nature. 2022;602(7895):26-8. https://doi. org/10.1038/d41586-022-00215-2 PMID: 35091701 www.eurosurveillance.org License, supplementary material and copyright This is an open-access article distributed under the terms of the Creative Commons Attribution (CC BY 4.0) Licence. You may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence and indicate if changes were made. Any supplementary material referenced in the article can be found in the online version. This article is copyright of the authors or their affiliated in- stitutions, 2022. www.eurosurveillance.org 5
https://openalex.org/W1968325234	https://europepmc.org/articles/pmc3017552?pdf=render	English	null	Interactions Between Laminin Receptor and the Cytoskeleton During Translation and Cell Motility	PloS one	2,011	cc-by	10,237	Abstract Human laminin receptor acts as both a component of the 40S ribosomal subunit to mediate cellular translation and as a cell surface receptor that interacts with components of the extracellular matrix. Due to its role as the cell surface receptor for several viruses and its overexpression in several types of cancer, laminin receptor is a pathologically significant protein. Previous studies have determined that ribosomes are associated with components of the cytoskeleton, however the specific ribosomal component(s) responsible has not been determined. Our studies show that laminin receptor binds directly to tubulin. Through the use of siRNA and cytoskeletal inhibitors we demonstrate that laminin receptor acts as a tethering protein, holding the ribosome to tubulin, which is integral to cellular translation. Our studies also show that laminin receptor is capable of binding directly to actin. Through the use of siRNA and cytoskeletal inhibitors we have shown that this laminin receptor-actin interaction is critical for cell migration. These data indicate that interactions between laminin receptor and the cytoskeleton are vital in mediating two processes that are intimately linked to cancer, cellular translation and migration. Citation: Venticinque L, Jamieson KV, Meruelo D (2011) Interactions Between Laminin Receptor and the Cytoskeleton During Translation and Cell Motility. PLoS ONE 6(1): e15895. doi:10.1371/journal.pone.0015895 Editor: Maddy Parsons, Kings College London, United Kingdom Received September 10, 2010; Accepted November 30, 2010; Published January 7, 2011 Received September 10, 2010; Accepted November 30, 2010; Published January 7, 2011 Copyright: 2011 Venticinque 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 funded by a generous donation from the Litwin foundation and the U.S. Public Health grant CA100687 from the National Cancer Institute, National Institutes of Health, and Department of Health and Human Services. 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 read the journal’s policy and have the following conflicts: the contents of this paper are being utilized for a patent. According to the rules and regulations of New York University School of Medicine, if this patent is licensed by a third party, the authors (DM, LV, KVJ) may receive benefits in the form of royalties or equity participation. Abstract This does not alter their adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: DM01@mac.com associated [22] and involved in maturation of the 40S ribosome, specifically processing of the 20S to 18S rRNA [23]. In addition, LamR plays a role in maintaining cell viability in yeast [23] and in a number of human cells [20,23,24,25,26]. Lisa Venticinque, Kelly V. Jamieson, Daniel Meruelo* Gene Therapy Center, Cancer Institute and Department of Pathology, New York University School of Medicine, New er Institute and Department of Pathology, New York University School of Medicine, New York, New York, United States of America Interactions Between Laminin Receptor and the Cytoskeleton During Translation and Cell Motility Lisa Venticinque, Kelly V. Jamieson, Daniel Meruelo* Introduction The 37/67 kDa laminin receptor (LamR), originally discovered as a 67 kDa cell surface receptor for laminin-1 in the extracellular matrix (ECM) [1,2,3], has a dual function as a component of the translational machinery and a cell surface receptor. The relationship between the 37 kDa and 67 kDa forms of LamR is not completely understood. The 67 kDa form of LamR is predicted to be a dimer, but whether LamR forms a homo-dimer [4] or hetero-dimer [5,6] has yet to be resolved. Amino acid composition analysis indicates that LamR exists as a homo-dimer at the cell surface [4]. Immunoblotting of detergent extracts indicates that LamR forms a hetero-dimer with galectin3 [5]. Additionally, post translational modifications have been suggested to stabilize the 67 kDa form and may be required for LamR association with the cell membrane [4,5]. At the cell surface, LamR also acts as the receptor for several viruses including Sindbis virus [7], Venezuelan equine encephalitis virus [8] and Dengue virus [9,10] as well as for prion proteins [11]. LamR, which is upregulated on a number of human cancers [12,13,14,15,16], plays a role in migration, tumor invasion and metastasis [17,18] through interactions with laminin-1. Intracellularly, LamR, also known as p40 ribosomal protein and RPSA, acts as an integral component of the 40S ribosomal subunit [19] and is involved in cellular translation and proliferation [20]. LamR is highly conserved across species from bacteria to humans [21]. The LamR orthologs in yeast have been shown to be polysome- [ ] Previous studies have also implicated 67 kDa LamR in binding interactions with actin at the cell membrane. A 70 kDa cell-surface protein, originally called connectin, was found to bind both laminin and actin in vitro [27]. It was also found that clustering of laminin in the ECM results in LamR clustering and subsequent actin remodeling [28]. Further, a 69 kDa laminin-binding protein was found to interact with microfilaments to mediate cell attachment and migration [29]. These data indicate that LamR interactions with the cytoskeleton might play a role in cell motility. The cytoskeleton, an elaborate network of proteins, is responsible for providing structure and shape to the cell and manipulating the cell membrane to induce cell motility. Laminin Receptor and the Cytoskeleton study the effect knockdown had on association with a-tubulin. In cells where expression of LamR had been ablated, cytoplasmic S6 staining became diffuse, indicating that S6 was no longer associated with a-tubulin (Figure 2A, middle panels). Additionally, siLamR induced accumulation of S6 in the nucleus. LamR plays a role in ribosome maturation [23], which could be inhibited in siLamR treated cells. Since some ribosomal proteins have been shown to bind to pre-ribosomes in the nucleus [51], loss of ribosome maturation may cause these ribosomal proteins to accumulate in the nucleus. This may account for the accumulation of S6 in the nucleus in siLamR treated cells. In cells where expression of S6 had been ablated, no change in LamR’s cytoplasmic localization was observed, indicating that S6 is not required for LamR association with a-tubulin (Figure 2A, bottom panels). Treatment with siS6 did induce the accumulation of LamR in the perinuclear region possibly near the microtubule organizing center, which could be the result of S6-induced cell cycle arrest. Knockdown of either LamR or S6 had no effect on cytoplasmic a-tubulin localization (Figure 2B), however siLamR treatment did result in the accumulation of a-tubulin in the nucleus. Tubulin shuttles between the nucleus and cytoplasm [52] and the cell cycle arrest induced by siRNA treatment could induce its accumulation in the nucleus. These data indicate that LamR is required for components of the 40S ribosomal subunit to co- localize with a-tubulin. repolymerizes, polarizing the cell and enabling the formation of lamellipodia and filapodia protrusions [37]. These protrusions, which are stabilized by transmembrane receptors interacting with the ECM, enable the cell to crawl by the use of these adhesions at the leading edge [38]. In addition, the cytoskeleton plays a role in cellular translation. It was originally thought that translation of select transcripts occurred at the cytoskeleton [39], however new evidence indicates that a significant portion of translation may occur bound to the cytoskeleton [40]. Immunofluorescence staining and electron microscopy indicates that polysomes co- localize with cytoskeletal components [41,42,43,44,45]. Detergent treatment of cells, which removes polysomes bound to the endoplasmic reticulum, indicates that polyribosomes bind to the cytoskeleton [42,46,47]. Treatment with agents that depolymerize actin or induce improper organization causes the release of polysomes from the cytoskeleton and inhibits protein synthesis [42,48,49,50]. This study examines LamR interactions with the cytoskeleton. Translation and the Cytoskeleton Our data demonstrating the co-localization of components of the 40S ribosome with a-tubulin indicates that some cellular translation occurs at the cytoskeleton (Figure 1). To assess the requirement of different components of the cytoskeleton for cellular translation cells were treated with either cytochalasin B (CB), which blocks monomer addition to actin filaments, or taxol, which stabilizes microtubules and inhibits tubulin dynamics. Treatment with CB resulted in loss of actin filament structure, but had no effect on LamR or S6 localization (Figure 3A, middle panels). While CB treatment had no effect on the a-tubulin localization, taxol treatment resulted in an alteration of tubulin structure (Figure 3B). The alteration in tubulin structure was likely the result of a long incubation with taxol and not a loss of cell viability, which was monitored in parallel. Treatment with taxol also had a dramatic effect on both LamR and S6 localization, causing both proteins to adopt a diffuse staining pattern (Figure 3A, bottom panels). Through microscopy we have found that LamR and S6, another 40S ribosomal component co-localize with a-tubulin. Co- localization is lost with treatment that disrupts microtubule dynamics. The direct interaction of LamR with tubulin, coupled with the dissociation of S6 from the cytoskeleton following treatment with taxol or siLamR, suggests that LamR may act as a tether to bind the translation complex to the cytoskeleton. This study demonstrates that interactions between LamR and compo- nents of the cytoskeleton, actin and tubulin, play a role in mediating cell motility and translation. Laminin Receptor and the Cytoskeleton Utilizing microscopy we have shown that the interaction between LamR and F-actin is related to LamR functions at the cell membrane. Microscopy revealed actin reorganization, formation of lamellipodia, and LamR localization at the leading edge following cell plating on laminin. Ablation of LamR expression inhibited cell migration comparable to the effects of inhibiting actin filament formation. Further, 37 kDa LamR was found to directly bind actin in an in vitro binding assay. These data indicate that the interaction between actin and LamR mediates cell motility. LamR Localization LamR is an integral ribosomal component, which is required for protein translation [19,20]. Through the use of immunofluores- cence, we confirmed co-localization of LamR with S6, used throughout this study as a marker for the 40S ribosomal subunit (Figure 1A). In order to study LamR interactions with cytoskeletal components, we examined the cellular localization of a-tubulin in relation to LamR. These data revealed that LamR also co- localizes with a-tubulin (Figure 1B), which indicates that LamR in complex with the ribosome may be associated with tubulin. The association between a-tubulin and 40S ribosomal compo- nents S6 and LamR indicates that this interaction is related to cellular translation. To further characterize the relationship between tubulin and protein synthesis, cells were treated with either CB or taxol and subjected to 35S labeling. Interestingly, there was no change in protein synthesis in cells treated with CB (Figure 3C). However, in cells treated with taxol, new protein synthesis was significantly decreased (Figure 3C). These data indicate that tubulin plays a critical role in mediating cellular translation. Characterization of the LamR-a-Tubulin Interaction To study interactions between the ribosome and a-tubulin, siRNA was employed to ablate expression of either LamR or S6 ribosomal protein. LamR-specific siRNA successfully ablates expression of both the 37 and 67 kDa forms of the protein. Successful knockdown of LamR and S6 was confirmed by western blot analysis (Figure S1A and B). Although knockdown of S6 expression was less efficient than LamR, 35S labeling confirmed inhibition of protein synthesis (Figure S1C). siGLO, a RISC free fluorescently conjugated control oligo, coupled with FACS analysis was utilized to assess transfection efficiency (Figure S1D). siLamR and siS6 treated samples were subjected to immunofluorescence to Introduction This network is comprised of three main types of proteins: microfila- ments comprised of helical assemblies of actin, microtubules comprised of alpha and beta tubulin dimers and intermediate filaments comprised of a number of different proteins, depending on cell type. The cytoskeleton is also associated with many cellular components such as the nucleus, the cell membrane, vesicles and other macromolecules [30,31,32,33]. This protein meshwork acts as a highway connecting different points of the cell and utilizing molecular motors powered by filament assembly forces to transport proteins and organelles across the cell’s span [34,35,36]. In response to migration-inducing stimuli, actin Previous studies have also implicated 67 kDa LamR in binding interactions with actin at the cell membrane. A 70 kDa cell-surface protein, originally called connectin, was found to bind both laminin and actin in vitro [27]. It was also found that clustering of laminin in the ECM results in LamR clustering and subsequent actin remodeling [28]. Further, a 69 kDa laminin-binding protein was found to interact with microfilaments to mediate cell attachment and migration [29]. These data indicate that LamR interactions with the cytoskeleton might play a role in cell motility. The cytoskeleton, an elaborate network of proteins, is responsible for providing structure and shape to the cell and manipulating the cell membrane to induce cell motility. This network is comprised of three main types of proteins: microfila- ments comprised of helical assemblies of actin, microtubules comprised of alpha and beta tubulin dimers and intermediate filaments comprised of a number of different proteins, depending on cell type. The cytoskeleton is also associated with many cellular components such as the nucleus, the cell membrane, vesicles and other macromolecules [30,31,32,33]. This protein meshwork acts as a highway connecting different points of the cell and utilizing molecular motors powered by filament assembly forces to transport proteins and organelles across the cell’s span [34,35,36]. In response to migration-inducing stimuli, actin PLoS ONE \| www.plosone.org 1 January 2011 \| Volume 6 \| Issue 1 \| e15895 January 2011 \| Volume 6 \| Issue 1 \| e15895 Laminin Receptor and the Cytoskeleton Characterization of the LamR-Actin Interaction dish induced the reorganization of actin filaments and the formation of lamellipodia with LamR concentrated at the terminal ends (Figure 4B, top panels). Cellular fractionation coupled with western blotting illustrated that there was no change in LamR localization or concentration when cells were cultured on laminin (Figure 4C), suggesting that the altered immunofluorescence pattern resulted from a redistribution of LamR within the cellular compartments. The additional band in the cytoplasmic fraction likely represents LamR prior to post-translational modification, which is important for membrane localization. effect on cell migration indicating that the reduction in migration was specific to the loss of LamR expression. These data indicate that both LamR and actin play an important role in cell migration. LamR Binds to Components of the Cytoskeleton Immunofluorescence data indicate that LamR interacts with tubulin (Figures 1–3) and both immunofluorescence and migration data (Figure 4) demonstrate that LamR interacts with actin. To determine whether LamR- cytoskeleton interactions are direct binding events, we utilized purified recombinant LamR in a binding enzyme-linked immunosorbent assay (ELISA) to measure binding activity in vitro. Either tubulin or actin was immobilized on an ELISA plate and LamR binding activity was assessed (Figure 5A and B). A bacterial ortholog of LamR, A. fulgidus S2p ribosomal protein (RPS2), was used as a negative control. LamR exhibits specific binding to both tubulin and actin compared with A. fulgidus S2p. LamR binding affinity was in the low micromolar range, which suggests that interactions between LamR and both tubulin and actin are of high affinity. To study the formation of lamellipodia structures, cells were treated with CB or LamR-specific siRNA to prevent formation of actin filaments and translation of LamR, respectively. Treatment with CB resulted in a loss of lamellipodia (Figure 4B, middle panels). Ablation of LamR expression did not inhibit lamellipodia formation (Figure 4B, bottom panels). Treatment with siLamR or CB indicates that lamellipodia formation is dependent on a functional actin structure rather than LamR. The formation of lamellipodia is indicative of cell migration [53]. To determine if both of these proteins are required for cell migration, cells were either treated with CB or transfected with siLamR and their ability to migrate to either purified laminin or 10% fetal calf serum (FCS) was assessed. Following either treatment migration was reduced by 80% (Figure 4D). To confirm that LamR expression and not inhibition of translation or cell cycle arrest was responsible for the migration inhibition, cells were treated with cycloheximide (CHX) or serum starved, respectively. The efficacy of CHX treatment or serum starvation was confirmed by 35S labeling and propidium iodide staining respectively (Figure S2). Both CHX treatment and G1 phase cell cycle arrest had no This study demonstrates that interactions between LamR and tubulin or actin mediate both intracellular and extracellular functions. Together these data indicate that LamR directly interacts with components of the cytoskeleton and that these interactions are important for mediating cellular translation and migration. Characterization of the LamR-Actin Interaction LamR interactions with tubulin appear to mediate translation and are concentrated within the intracellular environment (Figures 1–3). Interactions between LamR and actin appear to affect extracellular functions. Under normal cell culture condi- tions, LamR did not co-localize with F-actin at the cell surface (Figure 4A). Plating cells on laminin coated chamber slides induced a change in both cell morphology and LamR localization (Figure 4B, top panels). The presence of laminin on the culture January 2011 \| Volume 6 \| Issue 1 \| e15895 PLoS ONE \| www.plosone.org 2 Laminin Receptor and the Cytoskeleton Figure 1. The S6 ribosomal protein and a-tubulin co-localize with LamR. (A) Immunofluorescence of fixed NIH 3T3 cells stained for LamR (H141) (left), S6 (middle) and a merged image (right) illustrate co-localization of S6 with LamR. (B) Immunofluorescence of a fixed specimen stained for LamR (H141) (left), a-tubulin (middle) and the merged image (right) indicate the co-localization of LamR with a-tubulin. Scale bars in A and B represent 10 mm. doi:10 1371/journal pone 0015895 g001 Figure 1. The S6 ribosomal protein and a-tubulin co-localize with LamR. (A) Immunofluorescence of fixed NIH 3T3 cells stained for LamR (H141) (left), S6 (middle) and a merged image (right) illustrate co-localization of S6 with LamR. (B) Immunofluorescence of a fixed specimen stained for LamR (H141) (left), a-tubulin (middle) and the merged image (right) indicate the co-localization of LamR with a-tubulin. Scale bars in A and B represent 10 mm. doi:10.1371/journal.pone.0015895.g001 Figure 1. The S6 ribosomal protein and a-tubulin co-localize with LamR. (A) Immunofluorescence of fixed NIH 3T3 cells stained for LamR (H141) (left), S6 (middle) and a merged image (right) illustrate co-localization of S6 with LamR. (B) Immunofluorescence of a fixed specimen stained for LamR (H141) (left), a-tubulin (middle) and the merged image (right) indicate the co-localization of LamR with a-tubulin. Scale bars in A and B represent 10 mm. doi:10 1371/journal pone 0015895 g001 dish induced the reorganization of actin filaments and the formation of lamellipodia with LamR concentrated at the terminal ends (Figure 4B, top panels). Cellular fractionation coupled with western blotting illustrated that there was no change in LamR localization or concentration when cells were cultured on laminin (Figure 4C), suggesting that the altered immunofluorescence pattern resulted from a redistribution of LamR within the cellular compartments. The additional band in the cytoplasmic fraction likely represents LamR prior to post-translational modification, which is important for membrane localization. PLoS ONE \| www.plosone.org Discussion LamR plays a role in intracellular functions, such as translation, through its role as a component of the 40S ribosome [19,20] and extracellular functions, such as cell migration and adhesion, PLoS ONE \| www.plosone.org January 2011 \| Volume 6 \| Issue 1 \| e15895 3 Laminin Receptor and the Cytoskeleton Figure 2. LamR tethers the ribosome to the cytoskeleton. (A and B) Immunofluorescence following knockdown of either LamR or S6. (A) Microscopy image of samples stained for LamR (H141) (left), S6 (middle) or a merged overlay (right). Knockdown of LamR results in a change of the S6 localization from cytoskeleton-associated to diffuse. Knockdown of S6 has minimal effect on LamR localization, indicating that LamR tethers S6 to the cytoskeleton. (B) Immunofluorescence of samples stained for LamR (H141) (left), a-tubulin (middle) or merged images (right). Microscopy indicates that a-tubulin localization remains unaffected by knockdown of either protein. Scale bars in A and B represent 10 mm. doi:10.1371/journal.pone.0015895.g002 Figure 2. LamR tethers the ribosome to the cytoskeleton. (A and B) Immunofluorescence fol Figure 2. LamR tethers the ribosome to the cytoskeleton. (A and B) Immunofluorescence following knockdown of either LamR or S6. (A) Microscopy image of samples stained for LamR (H141) (left), S6 (middle) or a merged overlay (right). Knockdown of LamR results in a change of the S6 localization from cytoskeleton-associated to diffuse. Knockdown of S6 has minimal effect on LamR localization, indicating that LamR tethers S6 to the cytoskeleton. (B) Immunofluorescence of samples stained for LamR (H141) (left), a-tubulin (middle) or merged images (right). Microscopy indicates that a-tubulin localization remains unaffected by knockdown of either protein. Scale bars in A and B represent 10 mm. doi:10.1371/journal.pone.0015895.g002 through its role as a cell surface receptor [2,17]. Previously, interactions between LamR and the cytoskeleton, specifically actin, were thought to be exclusively related to cell motility and attachment [29]. In addition, cytoskeletal interactions with the ribosome have been shown, although the role of LamR has not been previously elucidated. The studies presented here demon- strate the direct, high affinity interaction between LamR and both actin and tubulin. These data also suggest that LamR interactions with tubulin are vital to LamR ribosomal functions, specifically protein translation. Co-localization of ribosomes with cytoskeletal components has been verified by electron microscopy [54] and cell fractionation studies [42]. Discussion Previous studies have implicated actin [41], intermediate filaments [43] and tubulin [45,55] as the cytoskeletal January 2011 \| Volume 6 \| Issue 1 \| e15895 PLoS ONE \| www.plosone.org 4 Laminin Receptor and the Cytoskeleton t ibl f ib l i t ti A l l i t di h d th t L R d t b li l l Figure 3. The cytoskeleton is important for translation. (A) Treatment of cells with agents to disrupt normal dynamics of the cytoskele induces changes in LamR localization. NIH 3T3 cells treated with either CB to disrupt the actin filaments or, taxol to block tubulin dynamics w subjected to immunofluorescence. Samples were stained for LamR (H141) (left) or S6 (middle) with the merged image. While treatment with CB no effect, taxol treatment produced a diffuse staining pattern for both LamR and S6, indicating that these proteins dissociated from the cytoskele (B) Samples treated similarly to panel A, were stained with LamR (H141) (left) or a-tubulin (middle) and overlaid image. In A and B panels showin actin staining (right) were included as confirmation that the CB treatment was successful. Scale bars in A and B represent 10 mm. (C) 35S labeling cells following treatment with either CB or taxol. Protein synthesis was inhibited in taxol treated samples, however CB treatment had no effec doi:10.1371/journal.pone.0015895.g003 Figure 3. The cytoskeleton is important for translation. (A) Treatment of cells with agents to disrupt normal dynamics of the cytoskeleto induces changes in LamR localization. NIH 3T3 cells treated with either CB to disrupt the actin filaments or, taxol to block tubulin dynamics wer subjected to immunofluorescence. Samples were stained for LamR (H141) (left) or S6 (middle) with the merged image. While treatment with CB ha no effect, taxol treatment produced a diffuse staining pattern for both LamR and S6, indicating that these proteins dissociated from the cytoskeleton (B) Samples treated similarly to panel A, were stained with LamR (H141) (left) or a-tubulin (middle) and overlaid image. In A and B panels showing F actin staining (right) were included as confirmation that the CB treatment was successful. Scale bars in A and B represent 10 mm. (C) 35S labeling o cells following treatment with either CB or taxol. Protein synthesis was inhibited in taxol treated samples, however CB treatment had no effect. doi:10.1371/journal.pone.0015895.g003 Figure 3. The cytoskeleton is important for translation. January 2011 \| Volume 6 \| Issue 1 \| e15895 Discussion (A) Treatment of cells with agents to disrupt normal dynamics of the cytoskeleton induces changes in LamR localization. NIH 3T3 cells treated with either CB to disrupt the actin filaments or, taxol to block tubulin dynamics were subjected to immunofluorescence. Samples were stained for LamR (H141) (left) or S6 (middle) with the merged image. While treatment with CB had no effect, taxol treatment produced a diffuse staining pattern for both LamR and S6, indicating that these proteins dissociated from the cytoskeleton. (B) Samples treated similarly to panel A, were stained with LamR (H141) (left) or a-tubulin (middle) and overlaid image. In A and B panels showing F- actin staining (right) were included as confirmation that the CB treatment was successful. Scale bars in A and B represent 10 mm. (C) 35S labeling of cells following treatment with either CB or taxol. Protein synthesis was inhibited in taxol treated samples, however CB treatment had no effect. doi:10.1371/journal.pone.0015895.g003 microscopy studies, showed that LamR and a-tubulin co-localize. Ablation of LamR and S6 expression demonstrated that LamR, but not S6 was critical for interactions between a-tubulin and the ribosome. In vitro binding assays verified that LamR specifically binds tubulin with micromolar affinity. Together, these data components responsible for ribosomal interactions. A large-scale proteomics study aimed at finding microtubule binding proteins in Arabidopsis identified LamR (ribosomal protein S2) [55]. Our study indicates an interaction between LamR and tubulin in mammalian cells, and also provides functional insight into this interaction. Our January 2011 \| Volume 6 \| Issue 1 \| e15895 PLoS ONE \| www.plosone.org January 2011 \| Volume 6 \| Issue 1 \| e15895 5 Laminin Receptor and the Cytoskeleton Figure 4. Characterization of the interaction between LamR and F-actin. (A) Immunofluorescence of NIH 3T3 cells stained for LamR (F (left) or F-Actin (middle) and merged (right). (B) Plating cells on laminin-coated plates induces lamellipodia formation and LamR localizes to terminal ends of the actin fibers. Mock treated NIH 3T3 cells (top), cells treated with CB (middle) or cells transfected with siLamR (bottom) were pla on laminin coated chamber slides. Cells were processed for immunofluorescence, staining for LamR (F18) (left) or F-actin (middle) and merged (rig PLoS ONE \| www.plosone.org 6 January 2011 \| Volume 6 \| Issue 1 \| e15 Figure 4. Characterization of the interaction between LamR and F-actin. Laminin Receptor and the Cytoskeleton Translation at the cytoskeleton was originally believed to be responsible for the targeted protein synthesis of a small number of mRNAs [39]. Evidence is now supporting the concept that a large proportion of cellular translation takes place at the cytoskeleton [40]. Through the use of CB and taxol, under conditions where greater than 80% of cells are viable, we were able to inhibit the dynamics of either actin filaments or microtubules, respectively. These manipulations enabled the study of these two cytoskeletal Figure 5. LamR binds directly to components of the cytoskel- eton. (A and B) LamR binds directly to the cytoskeleton as shown by an in vitro ELISA assay whereby purified microtubules (A) or filamentous actin (B) have been coated onto plates and the ability of purified recombinant LamR to bind was assessed. Graphical representations of the binding of either LamR (solid line) or ortholog, A. fulgidus S2p (RPS2) (dashed line). LamR is able to bind directly to both tubulin and actin at a significantly higher affinity than RPS2. Data in A and B represents the SEM (error bars) of three experiments. doi:10.1371/journal.pone.0015895.g005 LamR plays an important role in attachment to the ECM. As a cell surface receptor, LamR also plays a role in tumor invasion and metastasis. Previous studies have shown co-localization of F-actin with LamR within the cytoplasm of bovine vascular smooth muscle cells [29]. Our in vitro binding studies showed a direct and high affinity interaction between LamR and actin. Additionally, our microscopy studies showed the reorganization of actin filaments when cells are plated on laminin. We have also observed the formation of lamellipodia in which LamR localized at the termini of the actin fibers. Treatment with CB or transfection with siLamR reveals that lamellipodia formation is induced by actin filament reorganization, rather than LamR, since lamellipodia still formed in siLamR transfected samples. Lamellipodia formation is an important step in cell migration during which actin filament reorganization is coupled with interactions with the ECM [53]. Our microscopy data examined the role of both LamR and actin for efficient cell migration. We showed that CB treatment and siLamR transfection independently reduced cell migration to either 10% FCS or purified laminin by 80%, indicating that both proteins are important for cell migration. Laminin Receptor and the Cytoskeleton Laminin Receptor and the Cytoskeleton The additional panel shown with the mock-treated sample represents a zoomed image of the area indicated by the white square. Following treatment with CB, lamellipodia formation is inhibited, however, after knockdown of LamR these structures still form but LamR is not available to localize to the actin fibers. Scale bars in A and B represent 10 mm. (C) Culturing cells on laminin-coated plates had no effect on localization or concentration of LamR within the samples. Fractionation of cells plated on tissue culture coated or laminin coated plates. Samples were separated into cytosolic (c), membrane (m) and nuclear (n) fractions. (D) Treatment with CB or ablation of LamR expression reduces cell migration. A graphical representation of the ability of NIH 3T3 cells treated with CB or transfected with siLamR to migrate toward 10% FCS or laminin. CHX and serum starved samples serve as controls for translation inhibition and cell cycle arrest respectively. Data in D represents the SEM (error bars) of three experiments. Each sample was corrected for percent viability and was compared to the mock treated control. Statistical significance was calculated by a two-tailed student t-test (* P,0.005). doi:10.1371/journal.pone.0015895.g004 components to identify their specific interactions with LamR. Taxol treatment inhibits tubulin dynamics through the hyperst- abilization of microtubules. However, in our studies taxol disrupted the microtubule structure, similarly to treatment with nocodazole. The alteration in tubulin staining following taxol treatment could have resulted from the extended incubation time and was not the result of a loss in cell viability. In addition to the disruption of the tubulin structure, taxol treatment dramatically altered the localization of both S6 and LamR indicating that they are bound to microtubules. Conversely, treatment of cells with CB had no effect on LamR or S6 localization, indicating that neither protein was associated with actin. These treatments were also used in conjunction with 35S labeling to study the role of microfilaments and microtubules in translation. These studies revealed that treatment with taxol dramatically inhibits new protein synthesis. Previous studies have indicated that taxol induces translational arrest [56,57]. These studies confirm taxol-induced translational arrest and indicate that not only is ribosomal association with tubulin vital to translation, but also that a significant amount of translation occurs at the cytoskeleton. strongly implicate LamR as the protein responsible for tethering the ribosome to the cytoskeleton. January 2011 \| Volume 6 \| Issue 1 \| e15895 Discussion (A) Immunofluorescence of NIH 3T3 cells stained for LamR (F18) (left) or F-Actin (middle) and merged (right). (B) Plating cells on laminin-coated plates induces lamellipodia formation and LamR localizes to the terminal ends of the actin fibers. Mock treated NIH 3T3 cells (top), cells treated with CB (middle) or cells transfected with siLamR (bottom) were plated on laminin coated chamber slides. Cells were processed for immunofluorescence, staining for LamR (F18) (left) or F-actin (middle) and merged (right). Figure 4 Characterization of the interaction between LamR and F actin (A) Immunofluorescence of NIH 3T Figure 4. Characterization of the interaction between LamR and F-actin. (A) Immunofluorescence of NIH 3T3 cells stained for LamR (F18) (left) or F-Actin (middle) and merged (right). (B) Plating cells on laminin-coated plates induces lamellipodia formation and LamR localizes to the terminal ends of the actin fibers. Mock treated NIH 3T3 cells (top), cells treated with CB (middle) or cells transfected with siLamR (bottom) were plated on laminin coated chamber slides. Cells were processed for immunofluorescence, staining for LamR (F18) (left) or F-actin (middle) and merged (right). PLoS ONE \| www.plosone.org January 2011 \| Volume 6 \| Issue 1 \| e15895 6 Laminin Receptor and the Cytoskeleton LamR tethers the ribosomal components to tubulin and translation can occur. When cells are plated on poly D-lysine, actin is localized to the membrane and does not co-localize with LamR. (B) When cells are cultured on laminin-coated plates, actin reorganization is induced, resulting in the formation of lamellipodia with LamR concentrated at the terminal ends. The LamR- actin interaction is most likely important for mediating the cell surface functions of LamR, whereas the interaction with tubulin is integral to cellular translation. doi:10.1371/journal.pone.0015895.g006 NIH 3T3 cells were treated with CB (Sigma) at a final concentration of 5 mg/ml for 30 minutes at 37uC to disrupt the actin filament structure. Cells were washed with phosphate buffered saline (PBS) and used for downstream experiments. Cell viability was assessed in parallel to each experiment (as described below) following similar treatment and only samples greater than 80% viable were used. plated on tissue culture coated or poly D-lysine coated plates, LamR does not co-localize with F-actin at the membrane (Figure 6A). However, when cells are cultured on laminin-coated plates, lamellipodia form and LamR is reorganized to the terminal ends of the actin fibers (Figure 6B). The interaction between LamR and actin is most likely important for the functions of LamR as a cell surface receptor, specifically cell attachment and motility. NIH 3T3 cells were treated with Paclitaxel (taxol) (NovaPlus) at a final concentration of 5 mM for 5 hours at 37uC to inhibit microtubule dynamics. Cells were washed with PBS and used for downstream experiments. Cell viability was assessed in parallel to each experiment (as described below) following similar treatment and only samples greater than 80% viable were used. LamR is involved in numerous pathologies due to its role in binding virus [8,9,10,59], prion proteins [11] and its overexpression in cancer [12,13,14,15,16]. The expression of several other ribosomal proteins is elevated in human cancers [60,61,62] supporting the link between overexpression of LamR and cellular transformation. LamR is involved in many processes that mediate tumor aggressiveness including, cell migration [17], invasion [63] and ECM remodeling [18]. The correlation between LamR expression and tumor aggressiveness underscores the importance of understanding LamR’s diverse functions. This study demon- strates LamR’s ability to directly bind tubulin and actin. Through its interaction with tubulin, LamR acts as a molecular tether linking the ribosomal components to tubulin and mediating cellular translation. Laminin Receptor and the Cytoskeleton Through other studies in our lab aimed at characterizing the functions of LamR and its interactions with laminin, it was determined that incubation of cells with purified recombinant LamR can block their ability to migrate toward laminin. Additionally, mutational analysis revealed that the binding sites for actin and tubulin are separate from the binding site of laminin [58]. These data underscore the importance of LamR in cell migration. Figure 5. LamR binds directly to components of the cytoskel- eton. (A and B) LamR binds directly to the cytoskeleton as shown by an in vitro ELISA assay whereby purified microtubules (A) or filamentous actin (B) have been coated onto plates and the ability of purified recombinant LamR to bind was assessed. Graphical representations of the binding of either LamR (solid line) or ortholog, A. fulgidus S2p (RPS2) (dashed line). LamR is able to bind directly to both tubulin and actin at a significantly higher affinity than RPS2. Data in A and B represents the SEM (error bars) of three experiments. doi:10.1371/journal.pone.0015895.g005 We propose a model of interactions between LamR and the cytoskeletal components actin and tubulin (Figure 6). LamR directly interacts with both actin and tubulin, however, each interaction appears to serve a different purpose. LamR interactions with tubulin are critical for cellular translation, where LamR acts as a molecular tether linking the ribosomal components to tubulin (Figure 6A). LamR interactions with actin are more complex. When cells are January 2011 \| Volume 6 \| Issue 1 \| e15895 January 2011 \| Volume 6 \| Issue 1 \| e15895 7 PLoS ONE \| www.plosone.org Laminin Receptor and the Cytoskeleton Figure 6. Model depicting the interactions of LamR with the cytoskeleton. (A) LamR’s interaction with tubulin is associated with cellular translation. LamR tethers the ribosomal components to tubulin and translation can occur. When cells are plated on poly D-lysine, actin is localized to the membrane and does not co-localize with LamR. (B) When cells are cultured on laminin-coated plates, actin reorganization is induced, resulting in the formation of lamellipodia with LamR concentrated at the terminal ends. The LamR- actin interaction is most likely important for mediating the cell surface functions of LamR, whereas the interaction with tubulin is integral to cellular translation. doi:10.1371/journal.pone.0015895.g006 Figure 6. Model depicting the interactions of LamR with the cytoskeleton. (A) LamR’s interaction with tubulin is associated with cellular translation. Laminin Receptor and the Cytoskeleton The LamR-actin interaction is critical for cell migration, indicating that this complex may be important to metastasis. These data indicate that interactions between LamR and the cytoskeleton are vital in mediating two processes that are intimately linked to cancer. Study of the mechanisms of LamR and cytoskeletal interaction may lead to the development of novel anti-cancer therapeutics. NIH 3T3 cells were treated with CHX (Sigma) at a final concentration of 50 mg/ml for 5 hours at 37uC to inhibit cellular translation. For downstream experiments cells were maintained in CHX throughout. Cell viability was assessed in parallel (as described below) following similar treatment and only samples greater than 80% viable were used. For each experiment inhibition of translation was confirmed using 35S labeling. G1 phase cell cycle arrest was induced by incubating NIH 3T3 cells in serum free media for 24 hours at 37uC. Cell cycle arrest was assessed through propidium iodide staining followed by fluorescence activated cell sorting (FACS) analysis. Cell viability was also assessed in parallel (as described below). Materials and Methods NIH 3T3 cells were cultured on poly-D Lysine coated chamber slides. Poly-D Lysine/laminin coated chamber slides were also used as indicated (BD Bioscience). After CB or taxol treatment described above or transfection with siRNA (as described below) samples were processed for immunofluorescence. Briefly, cells were washed with PBS, fixed with 4% paraformaldehyde and permeabilized with 1% Triton X100. Cells were blocked at room temperature with blocking buffer (0.1% Triton X100 containing Cell Lines and Treatments Short Interfering RNA Studies NIH 3T3 cells were cultured on tissue culture plates or laminin coated plates (BD Bioscience). Cells were trypsinized, washed with PBS and resuspended at 56106 cells/ml. Cells were subjected to fractionation to separate cytosolic, membrane and nuclear fractions using the Qproteome Cell Compartment Kit (Qiagen) according to the manufacturer’s instructions. Successful fraction- ation was confirmed by western blotting with markers specific to each fraction. To ablate expression of LamR or S6, cells were transfected with siGENOME SMARTpool siRNA (Dharmacon). As a control, as well as a measure of transfection efficiency, siGLO, a fluorescently labeled RISC-free siRNA was used (Dharmacon). Briefly, transfections were performed in 12 well plates with cells at 70% confluency. Each oligo was used at a final concentration of 100 nmol/l. Oligos were incubated with Dharmafect reagent IV (Dharmacon) for 15 minutes at room temperature. Then, 0.8 ml of media was added and the mixture was added to the cells. After 24 hours cells were plated according to downstream experiments. siGLO was used to calculate transfection efficiency through FACS analysis. Only samples with greater than 80% transfection efficiency were used. Efficient knockdown of LamR or S6 was assessed through western blotting. Cell Migration Assay Cells transfected with siRNA or untreated NIH 3T3 cells were used for these assays. Transfected cells were harvested in the optimal knockdown period, as confirmed by western blotting done in parallel. Cell viability was also tested in parallel (as described above). siRNA transfected, CHX, serum starved, CB or mock treated cells were tested for their ability to migrate to either 10% FCS or 15 mg/ml purified laminin (Invitrogen) using the CytoSelectTM 24-Well Cell Migration Assay (8 mm, Colorimetric Format) (Cell Biolabs, Inc.). Briefly, 1.56105 cells in unsupple- Western Blotting Cell lysates were collected using Mammalian Protein Extraction Reagent (Pierce) supplemented with protease inhibitors (Roche) according to the manufacturer’s instructions. Protein content was measured using BioRad Dc Protein Reagent according to the manufacturer’s instructions (BioRad). Lysates containing 25 mg total protein were run on polyacrylamide gels (BioRad) under reducing conditions. Protein was transferred to polyvinylidine fluoride membrane (Millipore). Membranes were blocked with non-fat dry milk and probed with anti-LamR H141 (1:2000) (Santa Cruz), anti-S6 (1:1000) (Cell Signaling Technologies) or b- actin (1:10,000) (Sigma) antibodies diluted in tris buffered saline, 0.05%Tween, 5% BSA. Proteins were detected using horseradish peroxidase (HRP) conjugated secondary antibodies (Santa Cruz) and then exposed by chemiluminescence (Pierce). 35S Labeling Following CB or taxol treatment, as described above, cells were incubated with 35S methionine/cysteine (20 mCi/ml) (Perkin Elmer) in methionine-free media for 2 hours at 37uC. Cells were washed to remove unbound label and then incubated in DMEM supplemented with 10% FCS for 30 minutes. Lysates were collected with Mammalian Protein Extraction Reagent (Pierce) and samples containing 20 mg of total protein were run on a 4– 15% gradient gel (BioRad). The gel was fixed in a solution of 50% methanol and 10% acetic acid for 30 minutes with agitation. The gel was then incubated with enhancer solution (GE Healthcare) for 10 minutes, dried for 2 hours at 80uC under vacuum and exposed to film overnight at 280uC. Cell Lines and Treatments Slides were washed with PBS and incubated with Alexafluor 488 conjugated goat anti-mouse (1:500) (Molec- ular Probes), Alexafluor 594 conjugated donkey anti-rabbit (1:500) (Molecular Probes) secondary antibodies and Alexafluor 647 conjugated phalloidin (1:40), used for F-actin staining, (Molecular Probes). Coverslips were mounted with Prolong Gold Antifade Reagent (Invitrogen). For each experiment, samples stained with secondary antibody alone were processed in parallel to control for non-specific staining. Additionally, for all co- localization studies, samples stained for localization of each protein individually were processed in parallel and the fluores- cence of adjacent channels was monitored for bleed through. Samples were visualized with a confocal microscope (Axiovert 100m; Carl Zeiss MicroImaging, Inc.) fitted with a plan- Apochroma 100/1.40 oil DIC objective lens. Images were captured with a DKC-5000 digital camera (Sony) using the LSM510 version 3.2 SP2 software (Carl Zeiss MicroImaging, Inc.). Images were divided into individual channels for single color visualization using Adobe Photoshop CS4. at 3006 g for 5 minutes at 4uC. Samples were washed with PBS and then resuspended in PBS: 4% paraformaldehyde (1:1) for fixation. Samples were run on a FACSCaliber instrument (Becton Dickinson). Data analysis was performed with FlowJo version 8.2 software (Tree Star, Inc.). For cell cycle analysis following serum starvation cells were trypsinized at 37uC and centrifuged at 3006 g for 5 minutes at 4uC. Samples were washed with PBS and resuspended in PBS. Ice cold 100% ethanol was added dropwise and RNase was added at a final concentration of 100 mg/ml. Samples were incubated overnight at 4uC. Samples were then centrifuged at 3006 g for 5 minutes at 4uC and washed with PBS. Samples were resuspended in PBS with the addition of propidium iodide at a final concentration of 50 mg/ml. Samples were run on a FACScan instrument (Becton Dickinson). Data analysis was performed with ModFit LT v3.0 software (Verity Software House, Inc.). Assessment of Cell Viability Cells were cultured on 96 well luminescence plates (BD Bioscience). After initial treatment with CB, taxol, CHX, serum starvation or siRNA transfection, cell viability was assayed with the Cell Titer Glo assay (Promega). Briefly, Cell Titer Glo reagent was added 1:1 directly to the cell culture media. Samples were incubated at room temperature for 2 minutes with agitation and then for an additional 10 minutes. After incubation luminescence was read with a multiwell plate reader, Wallac EnVision (Perkin Elmer). To calculate cell viability each sample was compared with a similarly treated mock control. Cell Lines and Treatments Cell Lines and Treatments NIH 3T3 cells were obtained from the American Type Culture Collection. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FCS supplemented with 100 mg/ml of penicillin-streptomycin and 0.5 mg/ml amphotericin B (all from Mediatech). PLoS ONE \| www.plosone.org January 2011 \| Volume 6 \| Issue 1 \| e15895 8 Laminin Receptor and the Cytoskeleton 3% bovine serum albumin (BSA)). Slides were incubated with anti-LamR H141 (1:200) (Santa Cruz), which recognizes amino acids 110-250 of human LamR, to monitor intracellular functions, used for co-staining in all a-tubulin experiments, anti-LamR F18 (1:200) (Santa Cruz), which recognizes amino acids 245-295 of human LamR, to study cell surface functions, used for co-staining in all F-actin experiments, anti-a-Tubulin (1:1000) (Cell Signaling Technologies) or anti-S6 (1:50) (Cell Signaling Technologies) antibodies, all diluted in blocking buffer, overnight at 4uC. Slides were washed with PBS and incubated with Alexafluor 488 conjugated goat anti-mouse (1:500) (Molec- ular Probes), Alexafluor 594 conjugated donkey anti-rabbit (1:500) (Molecular Probes) secondary antibodies and Alexafluor 647 conjugated phalloidin (1:40), used for F-actin staining, (Molecular Probes). Coverslips were mounted with Prolong Gold Antifade Reagent (Invitrogen). For each experiment, samples stained with secondary antibody alone were processed in parallel to control for non-specific staining. Additionally, for all co- localization studies, samples stained for localization of each protein individually were processed in parallel and the fluores- cence of adjacent channels was monitored for bleed through. Samples were visualized with a confocal microscope (Axiovert 100m; Carl Zeiss MicroImaging, Inc.) fitted with a plan- Apochroma 100/1.40 oil DIC objective lens. Images were captured with a DKC-5000 digital camera (Sony) using the LSM510 version 3.2 SP2 software (Carl Zeiss MicroImaging, Inc.). Images were divided into individual channels for single color visualization using Adobe Photoshop CS4. 3% bovine serum albumin (BSA)). Slides were incubated with anti-LamR H141 (1:200) (Santa Cruz), which recognizes amino acids 110-250 of human LamR, to monitor intracellular functions, used for co-staining in all a-tubulin experiments, anti-LamR F18 (1:200) (Santa Cruz), which recognizes amino acids 245-295 of human LamR, to study cell surface functions, used for co-staining in all F-actin experiments, anti-a-Tubulin (1:1000) (Cell Signaling Technologies) or anti-S6 (1:50) (Cell Signaling Technologies) antibodies, all diluted in blocking buffer, overnight at 4uC. References 12. Basolo F, Pollina L, Pacini F, Fontanini G, Menard S, et al. (1996) Expression of the Mr 67,000 laminin receptor is an adverse prognostic indicator in human thyroid cancer: an immunohistochemical study. Clin Cancer Res 2: 1777–1780. 1. Rao NC, Barsky SH, Terranova VP, Liotta LA (1983) Isolation of a tumor cell laminin receptor. Biochem Biophys Res Commun 111: 804–808. 2. Malinoff HL, Wicha MS (1983) Isolation of a cell surface receptor protein for laminin from murine fibrosarcoma cells. J Cell Biol 96: 1475–1479. thyroid cancer: an immunohistochemical study. Clin Cancer Res 2: 1777–1780. 13. Menard S, Tagliabue E, Colnaghi MI (1998) The 67 kDa laminin receptor as a prognostic factor in human cancer. Breast Cancer Res Treat 52: 137–145. 3. Lesot H, Kuhl U, Mark KV (1983) Isolation of a laminin-binding protein from muscle cell membranes. Embo J 2: 861–865. 4. Landowski TH, Dratz EA, Starkey JR (1995) Studies of the structure of the metastasis-associated 67 kDa laminin binding protein: fatty acid acylation and evidence supporting dimerization of the 32 kDa gene product to form the mature protein. Biochemistry 34: 11276–11287. 14. Pelosi G, Pasini F, Bresaola E, Bogina G, Pederzoli P, et al. (1997) High-affinity monomeric 67-kD laminin receptors and prognosis in pancreatic endocrine tumours. J Pathol 183: 62–69. 15. Sanjuan X, Fernandez PL, Miquel R, Munoz J, Castronovo V, et al. (1996) Overexpression of the 67-kD laminin receptor correlates with tumour progression in human colorectal carcinoma. J Pathol 179: 376–380. 5. Buto S, Tagliabue E, Ardini E, Magnifico A, Ghirelli C, et al. (1998) Formation of the 67-kDa laminin receptor by acylation of the precursor. J Cell Biochem 69: 244–251. 16. van den Brule FA, Castronovo V, Menard S, Giavazzi R, Marzola M, et al. (1996) Expression of the 67 kD laminin receptor in human ovarian carcinomas as defined by a monoclonal antibody, MLuC5. Eur J Cancer 32A: 1598–1602. 6. Hundt C, Peyrin JM, Haik S, Gauczynski S, Leucht C, et al. (2001) Identification of interaction domains of the prion protein with its 37-kDa/67- kDa laminin receptor. EMBO J 20: 5876–5886. 17. Wewer UM, Taraboletti G, Sobel ME, Albrechtsen R, Liotta LA (1987) Role of laminin receptor in tumor cell migration. Cancer Res 47: 5691–5698. 7. Wang KS, Kuhn RJ, Strauss EG, Ou S, Strauss JH (1992) High-affinity laminin receptor is a receptor for Sindbis virus in mammalian cells. J Virol 66: 4992–5001. 18. Protein Binding ELISA We thank Dr. Christine Pampeno for the critical reading of this manuscript. ELISA plates (Costar) were coated with 1.5 mg per well of either filamentous actin or preformed microtubules (Cytoskeleton Inc.) dissolved in coating buffer (0.1 M sodium bicarbonate pH 9.2) overnight at 4uC. Plates were washed with wash buffer (PBS containing 0.5% Tween). Plates were then incubated in blocking buffer (2% FCS, 1 mg/ml BSA, 0.1% sodium azide in PBS) for 1 hour at 37uC. After incubation, plates were washed with wash buffer, 3 times for 5 minutes each. Triplicate wells were incubated Protein Purification Recombinant LamR was purified as described previously [64]. Briefly, a construct generated from human full length LamR was transformed into E.coli strain BL21 (DE3*) and grown in Luria broth to OD600 of 0.6 at 37uC. Protein expression was induced by the addition of isopropyl-thiogalactopyranoside, 0.1 mM, at 20uC. Cells were harvested and lysed by French press. The lysate was cleared by centrifugation at 16,000 RPM for 30 minutes. The supernatant was purified in two steps: by Ni-NTA chromatogra- phy (Qiagen) followed by gel filtration chromatography (Superdex 75) (Amersham). A. fulgidus S2p (RPS2) (residues 1–208) was expressed and purified under the same conditions. Figure S2 CHX and serum starvation treatment con- trols. (A) 35S labeling of CHX treated NIH 3T3 cells. CHX inhibited protein synthesis relative to the mock treated control. (B) Cell cycle profile of mock and serum starved samples. Serum starvation efficiently induced G1 phase cell cycle arrest. (TIF) FACS Analysis To assess transfection efficiency with siGLO, FACS analysis was employed. Briefly, cells were trypsinized at 37uC and centrifuged PLoS ONE \| www.plosone.org January 2011 \| Volume 6 \| Issue 1 \| e15895 9 Laminin Receptor and the Cytoskeleton mented media were added to the upper insert chamber of each well with the reservoir below containing 500 ml of either 10% FCS, purified laminin or unsupplemented media (used to calculate assay background). The plate was incubated at 37uC for 5 hours. After the incubation was completed the upper membrane of each insert was thoroughly washed with dH2O to remove non- migratory cells. Inserts were then incubated in cell stain solution for 10 minutes at room temperature, washed again in dH2O, and allowed to dry. Inserts were then incubated with extraction solution at room temperature for 10 minutes with gentle agitation. 100 ml of each sample was transferred to an ELISA plate and read at 630 nm with the ELx800 plate reader (Biotek Instruments, Inc.). To calculate migration ability, background was subtracted and then each sample was corrected for cell viability and compared to the similarly treated mock sample. with indicated concentrations of either purified LamR or A. fulgidus S2p (RPS2) for 1 hour at 37uC. Plates were washed with wash buffer. Wells were incubated with Penta-His HRP conjugate (Qiagen) for 2 hours at room temperature. Plates were washed and substrate solution was added. Absorbance at 490 nm was measured on the ELx800 ELISA plate reader (Biotek Instruments, Inc.) Each sample was normalized to background absorbance and a binding curve was generated with Prism4. Supporting Information Figure S1 siRNA treatment controls. (A and B) Western blot analysis of lysates collected from siLamR (A) or siS6 (B) transfected NIH 3T3 cells illustrates efficient ablation of protein expression. In A and B bactin was used as a loading control. (C) 35S labeling of siRNA transfected samples. Protein synthesis remains unaffected in the siGLO-transfected, control sample. (D) Transfection efficiency was monitored through transfection of siGLO, a fluorescently labeled, RISC-free control oligo and quantified by FACS analysis. Figure S1 siRNA treatment controls. (A and B) Western blot analysis of lysates collected from siLamR (A) or siS6 (B) transfected NIH 3T3 cells illustrates efficient ablation of protein expression. In A and B bactin was used as a loading control. (C) 35S labeling of siRNA transfected samples. Protein synthesis remains unaffected in the siGLO-transfected, control sample. (D) Transfection efficiency was monitored through transfection of siGLO, a fluorescently labeled, RISC-free control oligo and quantified by FACS analysis. Author Contributions Conceived and designed the experiments: LV KVJ DM. Performed the experiments: LV. Analyzed the data: LV. Contributed reagents/materials/ analysis tools: LV KVJ DM. Wrote the paper: LV KVJ DM. Conceived and designed the experiments: LV KVJ DM. Performed the experiments: LV. Analyzed the data: LV. Contributed reagents/materials/ analysis tools: LV KVJ DM. Wrote the paper: LV KVJ DM. Laminin Receptor and the Cytoskeleton 22. Demianova M, Formosa TG, Ellis SR (1996) Yeast proteins related to the p40/ laminin receptor precursor are essential components of the 40 S ribosomal subunit. J Biol Chem 271: 11383–11391. 45. Hamill D, Davis J, Drawbridge J, Suprenant KA (1994) Polyribosome targeting to microtubules: enrichment of specific mRNAs in a reconstituted microtubule preparation from sea urchin embryos. J Cell Biol 127: 973–984. J 23. Ford CL, Randal-Whitis L, Ellis SR (1999) Yeast proteins related to the p40/ laminin receptor precursor are required for 20S ribosomal RNA processing and the maturation of 40S ribosomal subunits. Cancer Res 59: 704–710. 46. Cervera M, Dreyfuss G, Penman S (1981) Messenger RNA is translated when associated with the cytoskeletal framework in normal and VSV-infected HeLa cells. Cell 23: 113–120. 24. Susantad T, Smith DR (2008) siRNA-mediated silencing of the 37/67-kDa high affinity laminin receptor in Hep3B cells induces apoptosis. Cell Mol Biol Lett 13: 452–464. 47. van Venrooij WJ, Sillekens PT, van Eekelen CA, Reinders RJ (1981) On the association of mRNA with the cytoskeleton in uninfected and adenovirus- infected human KB cells. Exp Cell Res 135: 79–91. 25. Kaneda Y, Kinoshita K, Sato M, Saeki Y, Yamada R, et al. (1998) The induction of apoptosis in HeLa cells by the loss of LBP-p40. Cell Death Differ 5: 20–28. 48. Gross SR, Kinzy TG (2007) Improper organization of the actin cytoskeleton affects protein synthesis at initiation. Mol Cell Biol 27: 1974–1989. 49. Vedeler A, Pryme IF, Hesketh JE (1991) The characterization of free, cytoskeletal and membrane-bound polysomes in Krebs II ascites and 3T3 cells. Mol Cell Biochem 100: 183–193. 26. Scheiman J, Jamieson KV, Ziello J, Tseng JC, and Meruelo D (2010) Extraribosomal functions associated with the c terminus of the 37/67 kDa laminin receptor are required for maintaining cell viability. Cell Death and Disease 1: 1–9. 50. Ornelles DA, Fey EG, Penman S (1986) Cytochalasin releases mRNA from the cytoskeletal framework and inhibits protein synthesis. Mol Cell Biol 6: 1650–1662. 27. Brown SS, Malinoff HL, Wicha MS (1983) Connectin: cell surface protein that binds both laminin and actin. Proc Natl Acad Sci U S A 80: 5927–5930. 51. Hugle B, Hazan R, Scheer U, Franke WW (1985) Localization of ribosomal protein S1 in the granular component of the interphase nucleolus and its distribution during mitosis. J Cell Biol 100: 873–886. 28. Laminin Receptor and the Cytoskeleton Cody RL, Wicha MS (1986) Clustering of cell surface laminin enhances its association with the cytoskeleton. Exp Cell Res 165: 107–116. y p 29. Yannariello-Brown J, Wewer U, Liotta L, Madri JA (1988) Distribution of a 69- kD laminin-binding protein in aortic and microvascular endothelial cells: modulation during cell attachment, spreading, and migration. J Cell Biol 106: 1773–1786. 52. Akoumianaki T, Kardassis D, Polioudaki H, Georgatos SD, Theodoropoulos PA (2009) Nucleocytoplasmic shuttling of soluble tubulin in mammalian cells. J Cell Sci 122: 1111–1118. 53. Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, et al. (2003) Cell migration: integrating signals from front to back. Science 302: 1704–1709. 30. Brown S, Levinson W, Spudich JA (1976) Cytoskeletal elements of chick embryo fibroblasts revealed by detergent extraction. J Supramol Struct 5: 119–130. 54. Wolosewick JJ, Porter KR (1976) Stereo high-voltage electron microscopy of whole cells of the human diploid line, WI-38. Am J Anat 147: 303–323. 31. Goldman RD, Lazarides E, Pollack R, Weber K (1975) The distribution of actin in non-muscle cells. The use of actin antibody in the localization of actin within the microfilament bundles of mouse 3T3 cells. Exp Cell Res 90: 333–344. 55. Chuong SD, Good AG, Taylor GJ, Freeman MC, Moorhead GB, et al. (2004) Large-scale identification of tubulin-binding proteins provides insight on subcellular trafficking, metabolic channeling, and signaling in plant cells. Mol Cell Proteomics 3: 970–983. p 32. Lazarides E (1980) Intermediate filaments as mechanical integrators of cellular space. Nature 283: 249–256. p 33. Osborn M, Weber K (1977) The detertent-resistant cytoskeleton of tissue culture cells includes the nucleus and the microfilament bundles. Exp Cell Res 106: 339–349. 56. Pineiro D, Gonzalez VM, Hernandez-Jimenez M, Salinas M, Martin ME (2007) Translation regulation after taxol treatment in NIH3T3 cells involves the elongation factor (eEF)2. Exp Cell Res 313: 3694–3706. 34. Goldberg MB (2001) Actin-based motility of intracellular microbial pathogens. Microbiol Mol Biol Rev 65: 595–626, table of contents. 57. Pineiro D, Gonzalez VM, Salinas M, Elena Martin M (2010) Analysis of the protein expression changes during taxol-induced apoptosis under translation inhibition conditions. Mol Cell Biochem. 35. Pollard TD, Borisy GG (2003) Cellular motility driven by assembly and disassembly of actin filaments. Cell 112: 453–465. y 36. Mitchison TJ (1995) Evolution of a dynamic cytoskeleton. Philos Trans R Soc Lond B Biol Sci 349: 299–304. 58. References Berno V, Porrini D, Castiglioni F, Campiglio M, Casalini P, et al. (2005) The 67 kDa laminin receptor increases tumor aggressiveness by remodeling laminin- 1. Endocr Relat Cancer 12: 393–406. 8. Ludwig GV, Kondig JP, Smith JF (1996) A putative receptor for Venezuelan equine encephalitis virus from mosquito cells. J Virol 70: 5592–5599. 19. Auth D, Brawerman G (1992) A 33-kDa polypeptide with homology to the laminin receptor: component of translation machinery. Proc Natl Acad Sci U S A 89: 4368–4372. 9. Thepparit C, Smith DR (2004) Serotype-specific entry of dengue virus into liver cells: identification of the 37-kilodalton/67-kilodalton high-affinity laminin receptor as a dengue virus serotype 1 receptor. J Virol 78: 12647–12656. 20. Scheiman J, Tseng JC, Zheng Y, Meruelo D Multiple functions of the 37/67-kd laminin receptor make it a suitable target for novel cancer gene therapy. Mol Ther 18: 63–74. 10. Tio PH, Jong WW, Cardosa MJ (2005) Two dimensional VOPBA reveals laminin receptor (LAMR1) interaction with dengue virus serotypes 1, 2 and 3. Virol J 2: 25. 21. Ardini E, Pesole G, Tagliabue E, Magnifico A, Castronovo V, et al. (1998) The 67-kDa laminin receptor originated from a ribosomal protein that acquired a dual function during evolution. Mol Biol Evol 15: 1017–1025. 11. Gauczynski S, Peyrin JM, Haik S, Leucht C, Hundt C, et al. (2001) The 37- kDa/67-kDa laminin receptor acts as the cell-surface receptor for the cellular prion protein. Embo J 20: 5863–5875. PLoS ONE \| www.plosone.org January 2011 \| Volume 6 \| Issue 1 \| e15895 January 2011 \| Volume 6 \| Issue 1 \| e15895 10 Laminin Receptor and the Cytoskeleton Jamieson KV, Hubbard SR, Meruelo D (2010) Structure-guided identification of a laminin binding site on the laminin receptor precursor. J Mol Biol. 37. Small JV, Stradal T, Vignal E, Rottner K (2002) The lamellipodium: where motility begins. Trends Cell Biol 12: 112–120. 59. Wang KS, Kuhn RJ, Strauss EG, Ou S, Strauss JH (1992) High-affinity laminin receptor is a receptor for Sindbis virus in mammalian cells. Journal of virology 66: 4992–5001. y g 38. Lauffenburger DA, Horwitz AF (1996) Cell migration: a physically integrated molecular process. Cell 84: 359–369. 60. Ferrari S, Manfredini R, Tagliafico E, Rossi E, Donelli A, et al. (1990) Noncoordinated expression of S6, S11, and S14 ribosomal protein genes in leukemic blast cells. Cancer Res 50: 5825–5828. 39. Hesketh J (1994) Translation and the cytoskeleton: a mechanism for targeted protein synthesis. Mol Biol Rep 19: 233–243. 40. Kim S, Coulombe PA (2010) Emerging role for the cytoskeleton as an organizer and regulator of translation. Nat Rev Mol Cell Biol 11: 75–81. 61. Loging WT, Reisman D (1999) Elevated expression of ribosomal protein genes L37, RPP-1, and S2 in the presence of mutant p53. Cancer Epidemiol Biomarkers Prev 8: 1011–1016. 41. Toh BH, Lolait SJ, Mathy JP, Baum R (1980) Association of mitochondria with intermediate filaments and of polyribosomes with cytoplasmic actin. Cell Tissue Res 211: 163–169. 62. Kondoh N, Shuda M, Tanaka K, Wakatsuki T, Hada A, et al. (2001) Enhanced expression of S8, L12, L23a, L27 and L30 ribosomal protein mRNAs in human hepatocellular carcinoma. Anticancer Res 21: 2429–2433. 42. Lenk R, Ransom L, Kaufmann Y, Penman S (1977) A cytoskeletal structure with associated polyribosomes obtained from HeLa cells. Cell 10: 67–78. hepatocellular carcinoma. Anticancer Res 21: 2429–2433. 63. Mafune K, Ravikumar TS (1992) Anti-sense RNA of 32-kDa laminin-binding protein inhibits attachment and invasion of a human colon carcinoma cell line. J Surg Res 52: 340–346. 43. Traub P, Bauer C, Hartig R, Grub S, Stahl J (1998) Colocalization of single ribosomes with intermediate filaments in puromycin-treated and serum-starved mouse embryo fibroblasts. Biol Cell 90: 319–337. 64. Jamieson KV, Wu J, Hubbard SR, Meruelo D (2008) Crystal structure of the human laminin receptor precursor. J Biol Chem 283: 3002–3005. y 44. Hesketh JE, Horne Z, Campbell GP (1991) Immunohistochemical evidence for an association of ribosomes with microfilaments in 3T3 fibroblasts. Cell Biol Int Rep 15: 141–150. Laminin Receptor and the Cytoskeleton PLoS ONE \| www.plosone.org January 2011 \| Volume 6 \| Issue 1 \| e15895 11
https://openalex.org/W2803533463	https://www.frontiersin.org/articles/10.3389/fnagi.2018.00164/pdf	English	null	The Role of Fluoxetine in Activating Wnt/β-Catenin Signaling and Repressing β-Amyloid Production in an Alzheimer Mouse Model	Frontiers in aging neuroscience	2,018	cc-by	10,207	ORIGINAL RESEARCH published: 01 June 2018 doi: 10.3389/fnagi.2018.00164 The Role of Fluoxetine in Activating Wnt/β-Catenin Signaling and Repressing β-Amyloid Production in an Alzheimer Mouse Model Min Huang1†, Yubin Liang2†, Hongda Chen3†, Binchu Xu1, Cuicui Chai1 and Pengfei Xing1 1 Department of Neurology, The Seventh Afﬁliated Hospital, Sun Yat-sen University, Shenzhen, China, 2 Department of Neurology, The First Afﬁliated Hospital of Jinan University, Guangzhou, China, 3 Department of Traditional Chinese Medicine, The Seventh Afﬁliated Hospital, Sun Yat-sen University, Shenzhen, China Fluoxetine (FLX) is one of the selective serotonin reuptake inhibitors (SSRIs) antidepressants, which could be used to relieve depression and anxiety among AD patients. This study was designed to search for new mechanisms by which ﬂuoxetine could activate Wnt/β-catenin signaling pathway and reduce amyloidosis in AD brain. Fluoxetine was administered via intragastric injection to APP/tau/PS1 mouse model of Alzheimer’s disease (3×Tg-AD) mice for 4 months. In the hippocampus of AD mouse model, there could be observed neuronal apoptosis, as well as an increase in Aβ (amyloid-β) production. Moreover, there is a strong association between down- regulation of Wnt/β-catenin signaling and the alteration of AD pathology. The activity of protein phosphatases of type 2A (PP2A) could be signiﬁcantly enhanced by the treatment of ﬂuoxetine. The activation of PP2A, caused by ﬂuoxetine, could then play a positive role in raising the level of active β-catenin, and deliver a negative impact in GSK3β activity in the hippocampal tissue. Both the changes mentioned above would lead to the activation of Wnt/β-catenin signaling. Meanwhile, ﬂuoxetine treatment would reduce APP cleavage and Aβ generation. It could also prevent apoptosis in 3×Tg-AD primary neuronal cell, and have protective effects on neuron synapse. These ﬁndings imply that Wnt/β-catenin signaling could be a potential target outcome for AD prevention, and ﬂuoxetine has the potential to be a promising drug in both AD prevention and treatment. Keywords: ﬂuoxetine, Alzheimer’s disease, Wnt/β-catenin signaling, protein phosphatases of type 2A (PP2A), amyloid-β Edited by: Ghulam Md Ashraf, King Abdulaziz University, Saudi Arabia Reviewed by: Gloria Patricia Cardona Gomez, Universidad de Antioquía, Colombia Mahmood Ahmad Khan, University College of Medical Sciences, India Correspondence: Min Huang minhuanghm@163.com †These authors have contributed Edited by: Ghulam Md Ashraf, King Abdulaziz University, Saudi Arabia Reviewed by: Gloria Patricia Cardona Gomez, Universidad de Antioquía, Colombia Mahmood Ahmad Khan, University College of Medical Sciences, India *Correspondence: Min Huang minhuanghm@163.com †These authors have contributed equally to this work. Received: 10 January 2018 Accepted: 15 May 2018 Published: 01 June 2018 INTRODUCTION Huang M, Liang Y, Chen H, Xu B, Chai C and Xing P (2018) The Role of Fluoxetine in Activating Wnt/β-Catenin Signaling and Repressing β-Amyloid Production in an Alzheimer Mouse Model. Front. Aging Neurosci. 10:164. doi: 10.3389/fnagi.2018.00164 Alzheimer’s disease (AD) is a chronic neurodegenerative disease characterized by progressive memory decline and cognitive impairment (Scharre and Chang, 2002). AD has various histopathological hallmarks, including neuroﬁbrillary tangles, cerebral amyloid senile plaques, synaptic and neuronal loss in the brain (Suh and Checler, 2002). Senile plaques, one of the important histopathological hallmarks, consist of a dense core of amyloid-β peptide (Aβ) and the June 2018 \| Volume 10 \| Article 164 Frontiers in Aging Neuroscience \| www.frontiersin.org The Therapeutic Value of Fluoxetine in AD Huang et al. dystrophic neuritis surrounded (Selkoe, 2004). The sequential proteolytic process of β-amyloid precursor protein (APP) through β- and γ-secretases could generate Aβ (Oulès et al., 2012). Previous study shows that Aβ could disrupt synapses and initiate a cascade of toxic events, which might result in neuronal loss (Liu et al., 2014). Another early feature of AD, besides amyloid pathogenesis, is synaptic dysfunction, which might be even prior to Aβ deposition (Miller-Thomas et al., 2016; Zuroﬀ et al., 2017). PP2A plays a core part in dephosphorylation of inactive β-catenin and phosphorylated APP (Seeling et al., 1999; Sontag et al., 2007). Additionally, Aβ generation could also be inhabited simultaneously by drugs that target inactive PP2A (Liu and Wang, 2009; Triaca et al., 2016). dystrophic neuritis surrounded (Selkoe, 2004). The sequential proteolytic process of β-amyloid precursor protein (APP) through β- and γ-secretases could generate Aβ (Oulès et al., 2012). Previous study shows that Aβ could disrupt synapses and initiate a cascade of toxic events, which might result in neuronal loss (Liu et al., 2014). Another early feature of AD, besides amyloid pathogenesis, is synaptic dysfunction, which might be even prior to Aβ deposition (Miller-Thomas et al., 2016; Zuroﬀ et al., 2017). PP2A plays a core part in dephosphorylation of inactive β-catenin and phosphorylated APP (Seeling et al., 1999; Sontag et al., 2007). Additionally, Aβ generation could also be inhabited simultaneously by drugs that target inactive PP2A (Liu and Wang, 2009; Triaca et al., 2016). mouse. Moreover, during the process where ﬂuoxetine positively inﬂuences the activation of Wnt/β-catenin signaling, promotion of PP2A activity is found to play a signiﬁcant role. INTRODUCTION Ultimately, the mechanisms behind the regulation of ﬂuoxetine in the Wnt/β-catenin signaling pathway were explored. Drugs and Reagents Fluoxetine was manufactured by Sigma-Aldrich (St. Louis, MO, United States). All cell culture reagents were produced from Invitrogen. Penicillin–streptomycin and poly-D-lysine, were manufactured by Sigma-Aldrich (St. Louis, MO, United States). Papain was obtained from Worthington. The Aβ1–42 ELISA kits and Aβ40 ELISA kits were purchased from Nanjing SenBeiJia Biological Technology Co., Ltd. (Nanjing, China). Antibodies information was in the Table 1. All other reagents were reagent grade. g The Wnt/β-catenin signaling pathway has been found to be critical for both neuronal development and maintenance of the nervous system (Patapoutian and Reichardt, 2000). Research shows that Wnt/β-catenin signaling pathway would inﬂuence various neuronal processes, such as synaptic diﬀerentiation, synaptic function, the function of neuronal circuits, dendrite development, and neuronal plasticity (Rosso and Inestrosa, 2013). Without the activation of the Wnt/β-catenin pathway, β-catenin in the cytoplasm could be phosphorylated by a complex set of proteins, such as glycogen synthase kinase- 3β (GSK3β), for ubiquitylation and degradation. However, the GSK3β activity could be inhibited by the activation of the Wnt/β-catenin pathway, which would in term lead to the repression of β-catenin phosphorylation, and, ultimately, result in the degradation of proteasome (Davidson et al., 2005; Zeng et al., 2008). Previous research shows that, in both sporadic and familial AD patients, there could be observed a decreased level of active β-catenin, inactive PP2A, and hyper active GSK3β (Voronkov et al., 2011; Inestrosa and Varela-Nallar, 2014). According to mentioned studies, it could be implied that it would be constructive and therapeutic for AD patients to maintain and rescue Wnt/β-catenin signaling. Frontiers in Aging Neuroscience \| www.frontiersin.org ELISA for Aβ1–42 and Aβ40 Levels β β Aβ1–42 and Aβ40 levels were assessed by ELISA (enzyme-linked immunosorbent assay). Primary cultured hippocampal neurons and medium from WT, 3×Tg-AD and 3×Tg-AD+ﬂuoxetine groups were collected. The levels of extracellular and intracellular Aβ1–42 and Aβ40 were evaluated through a sandwich ELISA kit, following the manufacturer’s instruction. Animals and Treatment Fluoxetine treatment dramatically slowing down the production of Aβ in the hippocampus of AD June 2018 \| Volume 10 \| Article 164 2 The Therapeutic Value of Fluoxetine in AD Huang et al. at ages between 3 and 6 months, and cognitive impairment was detected at the age of 6 months (Oddo et al., 2003a,b; Billings et al., 2005). In the treatment group (Tg + FLX) (n = 12; 6 males and 6 females), ﬂuoxetine was administrated at 10 mg/kg/day, intragastrical injection for 4 months. The ﬂuoxetine administration began at the 4 months of age, just before they developed cognitive impairment and key pathologic features. The dose of ﬂuoxetine was chosen aligning with previous studies (Li et al., 2009), with no gender diﬀerences. The rest two groups, the 3×Tg-AD mice group (Tg) (n = 12; 6 males, and 6 females) and male non-transgenic wild-type (WT) mice group (n = 12, 12 males), were treated with drinking water instead. All these three groups were kept under the same standard laboratory conditions with the treatment group, including temperature of 22 ± 2◦C, 12-h light/dark cycle, and free access to water and food. Each cage contained 3 or 4 subjects with the same genotype. All experiments were conducted following the Animal Care and Institutional Ethical Guidelines in China to minimize animal suﬀering, for instance, reducing the number of animals used, and utilizing alternatives to in vivo techniques, if available. The experiments and procedures utilized in this study were conducted strictly according to the institutional guidelines regarding experimental animal use in Sun Yat-sen University. The protocol was approved by the Animal Ethical and welfare Committee of Sun Yat-sen University (Permit Number: SYXK 2016-0112). mouse. All trials were recorded using an HVS (human visual system) water maze program for subsequent analyses of escape latency (Water Maze 3, Actimetrics, Evanston, IL, United States). All experimental procedures were performed in a blind method, where investigators were blinded to group assignment of each mouse. Primary Culture of Hippocampal Neurons Primary Culture of Hippocampal Neurons Primary hippocampal neurons were achieved from postnatal (P0–P1) 3×Tg-AD and WT mice pups born within 24 h. After being dissected from the brain, the hippocampi were then digested with 2 mg/mL papain for 30 min at 37◦C. Afterwards, the digested tissue was triturated and suspended in DMEM with 10% FBS. Dissociated cells were cultured in the neurobasal medium with 0.5 mM of L-glutamine, 2% B27 supplement, and 50 U/mL of penicillin–streptomycin. All the cultivation process would be conducted in poly-D-lysine-coated 6-well cell culture plates/culture dishes at a density of 0.5 × 106 cells/per well. The cells were cultured in a 37◦C incubator with 95% O2 and 5% CO2. The medium was completely replaced after 4 h, and half of the medium was then replaced every 3 days. On day 13, the neurons were treated with 1 µM ﬂuoxetine (diluted from 20 mM ﬂuoxetine stock solution dissolved in culture medium) for 24 h. Neurons from 3×Tg-AD mice treated with the culture medium were set up as the control group. Animals and Treatment This study utilized 3×Tg-AD mice expressing APPswe, PS1M146V, and tauP301L human gene mutants, which were purchased from the Jackson Laboratory (Bar Harbor, ME, United States). In these mice, intracellular Aβ was detected TABLE 1 \| Antibody information. Antibody Host Application Source Dilutions Aβ1–42 Rabbit WB/IF Abcam 1:500 APP Mouse WB Abcam 1:3000 CTFβ Mouse WB Santa Cruz 1:500 CTFα Rabbit WB Santa Cruz 1:500 ADAM10 Rabbit WB Abcam 1:1000 BACE1 Rabbit WB Abcam 1:3000 PS1 Mouse WB Abcam 1:1000 sAPPα Mouse WB USBiological 1:1000 sAPPβ Rabbit WB Abcam 1:500 C99 Rabbit WB Pierce 1:2000 C83 Rabbit WB Pierce 1:2000 BDNF Mouse WB Abcam 1:1000 NeuN Rabbit IF Abcam 1:300 Bcl-xL Rabbit WB Abcam 1:1000 Bcl-2 Rabbit WB Abcam 1:1000 Cleave-Caspase3 Rabbit WB Cell signaling 1:1000 Bax Rabbit WB Abcam 1:1000 PP2Ac Rabbit WB Abcam 1:1000 PP2A pY307 Rabbit WB Abcam 1:1000 GSK3β pY216 Rabbit WB Abcam 1:1000 GSK3β Rabbit WB Abcam 1:5000 Active β-catenin Rabbit WB/IF Cell signaling 1:2000 Inactive β-catenin Rabbit WB Cell signaling 1:2000 PSD95 Rabbit WB/IF Abcam 1:500 Synaptophysin Rabbit WB Abcam 1:1000 GAPDH Rabbit WB Abcam 1:2500 WB, western blot blatting; IF, immunoﬂuorescence. g g Fluoxetine (FLX), as a selective serotonin reuptake inhibitors (SSRIs) antidepressant, could be used to relieve depression and anxiety among AD patients (Modrego, 2010). Moreover, studies suggest another potential application of FLX. For patients with mild cognitive impairment (MCI), which is a prodromal state of AD, Fluoxetine could improve the memory and cognitive function (Mowla et al., 2007). Besides, ﬂuoxetine has been shown to be able to inhibit β-amyloid production, and prevent neuronal degeneration in an APP/PS1 mouse model (Wang et al., 2014; Ma et al., 2017; Sun et al., 2017). Furthermore, Li et al. (2004) have showed that ﬂuoxetine could greatly enhance the phosphorylation of GSK3β. And Pilar-Cuellar et al. (2012) have revealed that ﬂuoxetine could increase the β-catenin level. However, more research would be required to clarify if the neuroprotective eﬀect of ﬂuoxetine is related to the action of Wnt/β-catenin. The purpose of our research is to explore the role of ﬂuoxetine and its underlying mechanism in alleviating AD symptom. The research would utilize a triple-transgenic mouse model of AD, and measure the AD symptom by PP2A dependent Wnt/β-catenin signaling. Probe Trial To evaluate short-term and long-term memory consolidation, probe trials were conducted at 24 and 72h, respectively, after the last trial. To perform the memory consolidation evaluation, the platform was removed at ﬁrst. Then, the mice were placed into the quadrant of the pool opposite to the one pre-placed with the platform. The mice were given 60 s to swim in each probe trial. Both the time spent in the quadrant preplaced with the platform and the time spent across the platform position were recorded to evaluate short-term and long-term memory. Frontiers in Aging Neuroscience \| www.frontiersin.org Morris Water Maze Test At 8 months of age, all mice were subjected to the Morris water maze task (Mueller et al., 2008) for 5 days (d), as an evaluation of their learning and memory abilities. During the 5-day evaluation, the treatments in both experiment and control groups remained the same. All the apparatus and the test procedure utilized were described before (Vorhees and Williams, 2006). Brieﬂy, the apparatus included a circular white metal pool, whose diameter was 160 cm and height 50 cm, and the pool was ﬁlled with 26-cm deep water at constant temperature (22 ± 1◦C) throughout the experiment. The water pool was divided into four quadrants by the water maze software, and had a translucent acrylic platform. The translucent acrylic platform was 12 cm in diameter, and 24 cm in height. It was placed in the center of the northwest quadrant, 1∼2.0 cm below the water surface. Immunoﬂuorescence Staining and Histological Analysis The general health conditions of the 3×Tg-AD mice during the ﬂuoxetine treatment were carefully monitored during the trial period, and no signiﬁcant changes were found in the body weight of the mice before and after the trial. Five-micrometer-thick sagittal paraﬃn sections of mouse hippocampus were mounted on glass slides. Before incubation, they were pretreated with 0.01 mol/L citrate buﬀer (pH = 6.0) in hyperthermy for 5 min. 5% goat serum in PBS was used to block the sections for 10 min. After the above pretreatments, there performed two incubations. The ﬁrst one was performed with primary antibodies at 4◦C, overnight. The second incubation was with secondary antibodies (1: 500 in PBS) at 37◦C, 1 h. The primary antibodies used were speciﬁc to Aβ1–42, NeuN, and β-catenin. The Alexa-Fluor ﬂuorescent dye-conjugated secondary antibodies (anti-mouse and anti- rabbit; Alexa Fluor 488 and 695, Multi Sciences Biotech) were used to detect MAP2 and PSD95. Three equidistant sections including the whole hippocampus were assessed per sample. To analyze and quantify immunoreactive areas, these sections were imaged with ﬂuorescence microscopy (Olympus, Japan) and analyzed with Image-Pro Plus 6.0 software (Media Cybernetics). Spatial learning was evaluated by the length of the time to ﬁnd the hidden platform (i.e., escape latency). The results of all mice during the water maze acquisition training could be found in Figure 1A (p < 0.05). From the perspective of daily escape latency, the spatial learning ability of the mice was eﬀectively improved in both groups after the 5-day consecutive training period. Compared with the 3×Tg-AD mice group, a signiﬁcantly shorter escape latency was observed for the ﬂuoxetine-treated 3×Tg-AD mice (p < 0.05). However, in post hoc multiple comparisons, there was no signiﬁcant diﬀerences across all groups regarding swimming speed (Figure 1B, p > 0.05). The data suggests that ﬂuoxetine could have a signiﬁcant inﬂuence in attenuating spatial learning deﬁcits in 8-month-old 3×Tg-AD mice. Following the 5-day training, probe trials were conducted to evaluate short-term (24 h later) and long-term (72 h later) memory on the 6th and 8th day, respectively (Figures 1C,D). Compared with the WT mice group, 3×Tg-AD mice group exhibited a longer path distance (p < 0.01) across all trial sessions (Figures 1C,D). Quantitative RT-PCR Analysis (qRT-PCR) Q y (q ) TRIzol reagent (Invitrogen, Carlsbad, CA, United States) was utilized to exact the total RNA from the cells. And a qSYBR-green-containing PCR kit (Qiagen, Germantown, MD, United States) was utilized to conduct reverse transcription and qRT-PCR reactions. The fold change was determined as 2−11Ct, where Ct standard for the number of fractional cycle where the ﬂuorescence of each sample passed the ﬁxed threshold. All of the real-time PCR assays were performed with the Bio-Rad IQTM5 Multicolor Real-Time PCR Detection System (United States). Fluoxetine Reduced the Production of Aβ in the Brains of 3×Tg-AD Mice g The eﬀect of ﬂuoxetine on Aβ burden in the brain was also investigated in the 3×Tg-AD mice. Figures 2A,B indicated the results of immunoﬂuorescent staining and immunohistochemical staining for β-amyloid in the hippocampus of mice from all three groups. While little aggregation of β-amyloid was found in the WT mice, in the other Western Blot Assay The spatial learning task was conducted for ﬁve training days with four consecutive trials per day. The location of platform was remained constant with the starting position chosen among four quadrants in sequence at the pool rim every day. At the beginning of each trial, the mice would be gently released into the water with their noses against the wall at each starting point (north, south, east, and west). Every mouse was given a maximum of 60 s to ﬁnd the hidden platform. If the mouse failed to ﬁnd the escape platform within 60 s on the training day, it was then manually guided to the platform for 30 s. A camera was mounted in the ceiling directly above the pool to record the escape trace of each In the brain tissue-based assay, lysis buﬀer plus 1 mM PMSF and protease inhibitor cocktail were utilized to homogenize brain tissue samples from diﬀerent group. In the cell-based assay, the utilized cells were harvested after 24 h of 1 µM of ﬂuoxetine treatment. Then, the harvested cells were lysed with lysis buﬀer. BCA protein assay kit was utilized to measure protein concentration. And SDS-PAGE was used to extract the same amount of total protein (20 µg per well) from each sample. The extracted protein would then be transferred to polyvinylidene ﬂuoride (PVDF) membranes. After blocking with 5% fat-free June 2018 \| Volume 10 \| Article 164 Frontiers in Aging Neuroscience \| www.frontiersin.org 3 The Therapeutic Value of Fluoxetine in AD Huang et al. ANOVA was used to analyze the latency of MWM test. And two-way ANOVA and Bonferroni’s post hoc tests were utilized to analyze the rest data. p < 0.05 was considered as statistically signiﬁcant. milk, corresponding primary antibodies (Table 1) were used to probe, which would then be incubated with HRP-conjugated anti-rabbit antibody or HRP-anti-mouse antibody. The blots were developed with ECL detection reagents and visualized with a KODAK Image Station 4000 MM (Carestream Health Inc., New Haven, CT, United States). All band intensities were quantiﬁed using Quantity One software. Immunoﬂuorescence Staining and Histological Analysis Between the ﬂuoxetine -treated group and the 3×Tg-AD mice group, a statistically signiﬁcant diﬀerence was observed in both short-term and long-term memory test in terms of the length of time searching for target quadrant, and searching for non-target quadrants (Figure 1C, p < 0.05). When the 3×Tg-AD mice group was compared with WT group, the former spent signiﬁcantly longer time than the latter group. Based on the assessment of the length of time searching for the pre-placed platform in both short-term and long-term memory test, a statistically signiﬁcant improvement was observed in the ﬂuoxetine treated group, compared with the 3×Tg-AD mice group (Figure 1D, p < 0.05). Mice in the treated groups were gender-matched, and no signiﬁcant gender-speciﬁc diﬀerences in the results were observed. Compared three groups follow tracks, the ﬂuoxetine treated 3×Tg-AD mice group and WT group were more similar (Figure 1E). TdT-Mediated dUTP Nick-End Labeling g Neurons were washed three times with 0.01 MPBS, 5 min each time. 4% paraformaldehyde was used to ﬁx the neurons for 30 min, and 0.1% Triton X-100 in 0.1% sodium citrate was used to permeate them. The TdT-mediated dUTP nick-end labeling (TUNEL) was conducted via In Situ Cell Death Detection Kit, Fluorescein (Roche), following the manufacturer’s protocols. Hoechst 33342 was used to counter stain the neuronal nuclei. Fluorescence images were obtained with an Olympus ﬂuorescent microscope (Olympus), and TUNEL-positive cells were counted under a 20× objective. Statistical Analysis Data were shown as mean ± SD, n = 12 animals/group. #p < 0.05, 3×Tg-AD group vs. WT FIGURE 1 \| Effects of ﬂuoxetine on spatial learning and memory of 3×Tg-AD mice detected by the Morris water maze task. (A) In 5-day training trials, the escaping latencies of mice were measured to evaluate the three groups mouse memory ability. Based on the results, compared with vehicle control 3×Tg-AD mice. (B) Regarding the swimming speed, no signiﬁcant differences were observed in all groups. (C) In the probe trail, the frequency that mice crossed the area where the submerged platform was placed in training trials was recorded. (D) In terms of the time spent searching for the pre-placed platform in the target quadrant, in both the short-term and long-term memory tests. (E) The swimming tracks the mice in the three groups made in the water tank on the last day of the test. WT, wild-type mice; Tg, 3×Tg-AD mice; Tg + FLX, ﬂuoxetine treated 3×Tg-AD mice. Data were shown as mean ± SD, n = 12 animals/group. #p < 0.05, 3×Tg-AD group vs. WT group, ##p < 0.01, 3×Tg-AD group vs. WT group. ∗p < 0.05, ﬂuoxetine treated 3×Tg-AD vs. 3×Tg-AD group. Statistical Analysis All data was in the form of mean ± SEM or mean ± SD. All statistical analysis were performed using SPSS 19.0 software (IBM SPSS Inc., Chicago, IL, United States). Two-way repeated June 2018 \| Volume 10 \| Article 164 Frontiers in Aging Neuroscience \| www.frontiersin.org 4 The Therapeutic Value of Fluoxetine in AD Huang et al. ang et al. The Therapeutic Value of Fluoxetine in AD IGURE 1 \| Effects of ﬂuoxetine on spatial learning and memory of 3×Tg-AD mice detected by the Morris water maze task. (A) In 5-day training trials, the escaping tencies of mice were measured to evaluate the three groups mouse memory ability. Based on the results, compared with vehicle control 3×Tg-AD mice. B) Regarding the swimming speed, no signiﬁcant differences were observed in all groups. (C) In the probe trail, the frequency that mice crossed the area where the ubmerged platform was placed in training trials was recorded. (D) In terms of the time spent searching for the pre-placed platform in the target quadrant, in both e short-term and long-term memory tests. (E) The swimming tracks the mice in the three groups made in the water tank on the last day of the test. WT, wild-type ice; Tg, 3×Tg-AD mice; Tg + FLX, ﬂuoxetine treated 3×Tg-AD mice. Data were shown as mean ± SD, n = 12 animals/group. #p < 0.05, 3×Tg-AD group vs. WT roup, ##p < 0.01, 3×Tg-AD group vs. WT group. ∗p < 0.05, ﬂuoxetine treated 3×Tg-AD vs. 3×Tg-AD group. FIGURE 1 \| Effects of ﬂuoxetine on spatial learning and memory of 3×Tg-AD mice detected by the Morris water maze task. (A) In 5-day training trials, the escaping latencies of mice were measured to evaluate the three groups mouse memory ability. Based on the results, compared with vehicle control 3×Tg-AD mice. (B) Regarding the swimming speed, no signiﬁcant differences were observed in all groups. (C) In the probe trail, the frequency that mice crossed the area where the submerged platform was placed in training trials was recorded. (D) In terms of the time spent searching for the pre-placed platform in the target quadrant, in both the short-term and long-term memory tests. (E) The swimming tracks the mice in the three groups made in the water tank on the last day of the test. WT, wild-type mice; Tg, 3×Tg-AD mice; Tg + FLX, ﬂuoxetine treated 3×Tg-AD mice. Fluoxetine Enhances Non-amyloidogenic Processing of APP in 3×Tg-AD Mice Brain two groups there could observe β-amyloid aggregation in the hippocampus (Figures 2A,B). Moreover, between the 3×Tg AD group and the 3×Tg AD + Flx group, the density of β-amyloid aggregating in the hippocampus was higher in the 3×Tg AD group, and the labeling for β-amyloid was also more intense (Figure 2B, p < 0.05). These ﬁndings were validated by ELISA test. The levels of Aβ42 and Aβ40 increased signiﬁcantly in the hippocampus of 3×Tg AD mice, when compared with the WT mice. Treatment with ﬂuoxetine could inhibit the level of Aβ42 and Aβ40 in the 3×Tg AD mice (Figures 2C,D, p < 0.01). To explore the impacts of ﬂuoxetine on APP processing, the study also measured the levels of APP protein in 3×Tg-AD mouse brain with a 4-month ﬂuoxetine treatment. As shown in Figure 3A, there was a signiﬁcant increase in the levels of APP protein in the hippocampus of 3×Tg AD mice, compared with that of the WT mice (p < 0.01). Treatment with ﬂuoxetine could Frontiers in Aging Neuroscience \| www.frontiersin.org June 2018 \| Volume 10 \| Article 164 5 The Therapeutic Value of Fluoxetine in AD Huang et al. FIGURE 2 \| Fluoxetine reduced the production of Aβ in the brains of 3×Tg-AD mice. Four-month old 3×Tg-AD mouse were treated with vehicle or ﬂuoxetine (10 mg/kg/day) every day for 4 months and sacriﬁced for analysis. (A) Immunohistochemical staining for β-amyloid in the hippocampus of mice from the three groups. Scale bars: 100 µm. (B) Immunoﬂuorescence of Aβ42 (green) in the hippocampi of mice from the three groups. Scale bars: 100 µm. (C,D) The levels Aβ42 and Aβ40 in the brains of three groups were detected by ELISA. WT, wild-type mice; Tg: 3×Tg-AD mice; Tg + FLX: ﬂuoxetine treated 3×Tg-AD mice. Data were shown as mean ± SD, n = 12 animals/group.∗p < 0.05, ∗∗p < 0.01. FIGURE 2 \| Fluoxetine reduced the production of Aβ in the brains of 3×Tg-AD mice. Four-month old 3×Tg-AD mouse were treated with vehicle or ﬂuoxetine (10 mg/kg/day) every day for 4 months and sacriﬁced for analysis. (A) Immunohistochemical staining for β-amyloid in the hippocampus of mice from the three groups. Scale bars: 100 µm. (B) Immunoﬂuorescence of Aβ42 (green) in the hippocampi of mice from the three groups. Scale bars: 100 µm. (C,D) The levels Aβ42 and Aβ40 in the brains of three groups were detected by ELISA. Fluoxetine Enhances Non-amyloidogenic Processing of APP in 3×Tg-AD Mice Brain However, ﬂuoxetine treatment could reduce the levels of protein-CTFs, BACE1, PS1, sAPPβ, and C99 in the hippocampus (Figures 3A,B) of the 3×Tg AD mice. We found that, compared with WT mice, 3×Tg AD mice had lower levels of ADAM10, sAPPα, and C83 in the hippocampus of, while ﬂuoxetine treatment markedly enhanced the expression of ADAM10, sAPPα, and C83 in the hippocampus of 3×Tg AD mice. Fluoxetine Enhances Non-amyloidogenic Processing of APP in 3×Tg-AD Mice Brain WT, wild-type mice; Tg: 3×Tg-AD mice; Tg + FLX: ﬂuoxetine treated 3×Tg-AD mice. Data were shown as mean ± SD, n = 12 animals/group.∗p < 0.05, ∗∗p < 0.01. reduce the level of APP protein in the 3×Tg AD mice (Figure 3A, p < 0.05). Then, it was investigated if ﬂuoxetine was involved in APP cleavage. Western blot analysis was utilized to detect the APP cleavage enzymes and cleavage fragments in ﬂuoxetine- treated 3×Tg-AD mouse brain (Figure 3A). Compared with that of WT mice, the protein levels of CTFs, BACE1, PS1, sAPPβ, and C99 in the hippocampus of 3×Tg AD mice were signiﬁcantly higher. However, ﬂuoxetine treatment could reduce the levels of protein-CTFs, BACE1, PS1, sAPPβ, and C99 in the hippocampus (Figures 3A,B) of the 3×Tg AD mice. We found that, compared with WT mice, 3×Tg AD mice had lower levels of ADAM10, sAPPα, and C83 in the hippocampus of, while ﬂuoxetine treatment markedly enhanced the expression of ADAM10, sAPPα, and C83 in the hippocampus of 3×Tg AD mice. The Promoting Effect of Fluoxetine on BDNF in the Brain of 3×Tg-AD Mice The levels of BDNF in brain of three groups mice were measured. As indicated in Figure 4A, 3×Tg AD mice had remarkably Frontiers in Aging Neuroscience \| www.frontiersin.org lower levels of BDNF protein in the hippocampus than that of the WT mice (p < 0.01). Treatment with ﬂuoxetine could have a positive impact on increasing the levels of BDNF protein in the 3×Tg AD mice (p < 0.01). Figure 4B indicated the results of immunoﬂuorescent staining for NeuN in the hippocampus of mice from three groups, there was a decrease the number of cells stained with NeuN antibody in the 3×Tg AD mice compare with WT mice (Figure 4B, p < 0.05). Treatment with ﬂuoxetine could increase the number of cells stained with NeuN antibody in the 3×Tg AD mice (Figure 4B, p < 0.05). reduce the level of APP protein in the 3×Tg AD mice (Figure 3A, p < 0.05). Then, it was investigated if ﬂuoxetine was involved in APP cleavage. Western blot analysis was utilized to detect the APP cleavage enzymes and cleavage fragments in ﬂuoxetine- treated 3×Tg-AD mouse brain (Figure 3A). Compared with that of WT mice, the protein levels of CTFs, BACE1, PS1, sAPPβ, and C99 in the hippocampus of 3×Tg AD mice were signiﬁcantly higher. Fluoxetine Treatment Inhibited Apoptosis in Hippocampal Primary Neurons As shown in Figure 5A, the 3×Tg AD primary neurons had a larger number of TUNEL-positive cells than the WT primary neurons (p < 0.01). Treatment of ﬂuoxetine at 1 µM signiﬁcantly reduced the number of TUNEL-positive cells (p < 0.05) in the 3×Tg AD primary neurons. As proved in previous research, the ratio between Bcl-2/Bcl-xl and Bax is correlated with The Promoting Effect of Fluoxetine on BDNF in the Brain of 3×Tg-AD Mice g The levels of BDNF in brain of three groups mice were measured. As indicated in Figure 4A, 3×Tg AD mice had remarkably June 2018 \| Volume 10 \| Article 164 Frontiers in Aging Neuroscience \| www.frontiersin.org 6 The Therapeutic Value of Fluoxetine in AD Huang et al. FIGURE 3 \| Fluoxetine enhances the non-amyloidogenic pathway in 3×Tg-AD mice brain. (A) The western blot analysis was used to examine the expression levels of APP and APP cleavage enzymes, including APP, CTFβ, CTFα, ADAM10, BACE1, and PS1. (B) Levels of APP cleavage fragments, including sAPPα, sAPPβ, C83, and C99 generation, in the brain of three groups were assessed and quantiﬁed by immunoblotting. WT, wild-type mice; Tg: 3×Tg-AD mice; Tg + FLX: ﬂuoxetine treated 3×Tg-AD mice. All data were shown as mean ± SD, n = 12 animals/group. ∗p < 0.05, ∗∗p < 0.01. FIGURE 3 \| Fluoxetine enhances the non-amyloidogenic pathway in 3×Tg-AD mice brain. (A) The western blot analysis was used to examine the expression levels of APP and APP cleavage enzymes, including APP, CTFβ, CTFα, ADAM10, BACE1, and PS1. (B) Levels of APP cleavage fragments, including sAPPα, sAPPβ, C83, and C99 generation, in the brain of three groups were assessed and quantiﬁed by immunoblotting. WT, wild-type mice; Tg: 3×Tg-AD mice; Tg + FLX: ﬂuoxetine treated 3×Tg-AD mice. All data were shown as mean ± SD, n = 12 animals/group. ∗p < 0.05, ∗∗p < 0.01. FIGURE 4 \| Fluoxetine promotes BDNF in the brain of 3×Tg-AD mice. (A) The expression levels of BDNF were examined by western blot analysis. (B) Immunoﬂuorescence of NeuN (red) in the hippocampi of mice from the three groups. Scale bars: 100 µm. WT, wild-type mice; Tg, 3×Tg-AD mice; Tg + FLX, ﬂuoxetine treated 3×Tg-AD mice. Data were shown as mean ± SD, n = 12 animals/group. ∗p < 0.05, ∗∗p < 0.01. FIGURE 3 \| Fluoxetine enhances the non-amyloidogenic pathway in 3×Tg-AD mice brain. (A) The western blot analysis was used to examine the expression levels of APP and APP cleavage enzymes, including APP, CTFβ, CTFα, ADAM10, BACE1, and PS1. (B) Levels of APP cleavage fragments, including sAPPα, sAPPβ, C83, and C99 generation, in the brain of three groups were assessed and quantiﬁed by immunoblotting. WT, wild-type mice; Tg: 3×Tg-AD mice; Tg + FLX: ﬂuoxetine treated 3×Tg-AD mice. All data were shown as mean ± SD, n = 12 animals/group. The Promoting Effect of Fluoxetine on BDNF in the Brain of 3×Tg-AD Mice ∗p < 0.05, ∗∗p < 0.01. FIGURE 4 \| Fluoxetine promotes BDNF in the brain of 3×Tg-AD mice. (A) The expression levels of BDNF were examined by western blot analysis. (B) Immunoﬂuorescence of NeuN (red) in the hippocampi of mice from the three groups. Scale bars: 100 µm. WT, wild-type mice; Tg, 3×Tg-AD mice; Tg + FLX, ﬂuoxetine treated 3×Tg-AD mice. Data were shown as mean ± SD, n = 12 animals/group. ∗p < 0.05, ∗∗p < 0.01. FIGURE 4 \| Fluoxetine promotes BDNF in the brain of 3×Tg-AD mice. (A) The expression levels of BDNF were examined by western blot analysis. (B) Immunoﬂuorescence of NeuN (red) in the hippocampi of mice from the three groups. Scale bars: 100 µm. WT, wild-type mice; Tg, 3×Tg-AD mice; Tg + FLX, ﬂuoxetine treated 3×Tg-AD mice. Data were shown as mean ± SD, n = 12 animals/group. ∗p < 0.05, ∗∗p < 0.01. June 2018 \| Volume 10 \| Article 164 Frontiers in Aging Neuroscience \| www.frontiersin.org 7 The Therapeutic Value of Fluoxetine in AD Huang et al. FIGURE 5 \| Fluoxetine treatment inhibited apoptosis in hippocampal primary neurons. (A) The number of TUNEL-positive neurons (green). The number of TUNEL-positive neurons per 200× ﬁeld in each group (n = 6) was averaged. ∗∗p < 0.001 vs. control; Scale bar = 100 µm. (B) The western blot analysis was adopted to determine the Bcl-2, Bcl-xl cleaved-caspase 3, and Bax protein levels. WT, primarily cultured neurons of wild-type mice; Tg, primarily cultured neurons of 3×Tg-AD mice; Tg + FLX, primarily neurons of 3×Tg-AD mice treated with ﬂuoxetine. Data were expressed as fold of control. ∗p < 0.05 compared with control. ∗∗p < 0.01 compared with control. FIGURE 5 \| Fluoxetine treatment inhibited apoptosis in hippocampal primary neurons (A) The number of TUNEL positive neurons (green) The number of FIGURE 5 \| Fluoxetine treatment inhibited apoptosis in hippocampal primary neurons. (A) The number of TUNEL-positive neurons (green). The number of TUNEL-positive neurons per 200× ﬁeld in each group (n = 6) was averaged. ∗∗p < 0.001 vs. control; Scale bar = 100 µm. (B) The western blot analysis was adopted to determine the Bcl-2, Bcl-xl cleaved-caspase 3, and Bax protein levels. WT, primarily cultured neurons of wild-type mice; Tg, primarily cultured neurons of 3×Tg-AD mice; Tg + FLX, primarily neurons of 3×Tg-AD mice treated with ﬂuoxetine. Data were expressed as fold of control. The Promoting Effect of Fluoxetine on BDNF in the Brain of 3×Tg-AD Mice ∗p < 0.05 compared with control. ∗∗p < 0.01 compared with control. activity of PP2A in vivo. It was observed that this ratio was signiﬁcantly higher in 3×Tg AD mice than that in the WT mice (Figure 6A, p < 0.05). This implied that treatment with ﬂuoxetine could reduce the ratio of PP2A pY307/PP2Ac in the 3×Tg AD mice (Figure 6A, p < 0.05). As the activity of GSK3β would require Y216 phosphorylation, the ratio of GSK3β pY216/GSK3β could then imply the activity of GSK3β in vivo. The research provided evidence that ﬂuoxetine could signiﬁcantly decrease the ratio of GSK3β pY216/GSK3β in the 3×Tg AD mice. A signiﬁcant reduction in active β-catenin was observed in the hippocampus of 3×Tg AD mice when compared with the WT mice (Figure 6B, p < 0.01). Treatment with ﬂuoxetine could increase the active β-catenin. The hippocampus of 3×Tg AD mice also demonstrated a signiﬁcant higher level of active β-catenin, compared with that of WT mice (Figure 6B, p < 0.01). Thus, treatment with ﬂuoxetine could decrease the inactive β-catenin. It implied that ﬂuoxetine might be suﬃcient to enhance active β-catenin stabilization. To further conﬁrm the protein expression of active β-catenin in the 3×Tg-AD mice brain, apoptosis (Liu et al., 2005). Thus, to investigate the molecular mechanism of the protective eﬀect of ﬂuoxetine in 3×Tg- AD primary neurons apoptosis, the expression of Bcl-2, Bcl- xl, and Bax was examined. Compared with the WT primary neurons, there could observe signiﬁcant reduction in both Bcl- xl/Bax and Bcl-2/Bax expression ratio in the 3×Tg AD primary neurons. Treatment with ﬂuoxetine signiﬁcantly improved the Bcl-xl/Bax and Bcl-2/Bax expression ratio in the 3×Tg AD primary neurons. The levels of cleaved-caspase 3 were up- regulated in the 3×Tg AD primary neurons compared with the WT primary neurons (Figure 5B, p < 0.01). The results from the 3×Tg AD primary neurons demonstrated that treatment ﬂuoxetine could signiﬁcantly reverse this change (Figure 5B, p < 0.01). Fluoxetine Targeted PP2A to Activate the Wnt/β-Catenin Signaling The activity of PP2A would be inhibited by its phosphorylation at Tyr307 (Y307) residue of PP2A catalytic subunit (PP2Ac). Inversely, the ratio of PP2A-pY307/PP2Ac could reﬂect the June 2018 \| Volume 10 \| Article 164 Frontiers in Aging Neuroscience \| www.frontiersin.org 8 The Therapeutic Value of Fluoxetine in AD Huang et al. FIGURE 6 \| (A) Western blots results that showed the expression levels of GSK3β and PP2A in the brains of mice. (B) Active β-catenin, and inactive β-catenin were studied through Western blot. (C) Immunoﬂuorescence of β-catenin (red) and DAPI (blue) in the hippocampi of mice. WT, wild-type mice; Tg, 3×Tg-AD mice; Tg + FLX, ﬂuoxetine treated 3×Tg-AD mice. (D) Analysis of primarily cultured neurons from mouse hippocampus. WT, primarily cultured neurons of wild-type mice; Tg, primarily cultured neurons of 3×Tg-AD mice; Tg + FLX, primarily neurons of 3×Tg-AD mice treated with ﬂuoxetine. Tg + FLX+LB_100, primarily neurons of 3×Tg-AD mice treated with ﬂuoxetine and LB_100 (an inhibitor of PP2A activity). (E) RT-PCR showed active β-catenin and GSK3β expression in the hippocampi of mice. Data are shown as mean ± SD, n = 12 animals/group. ∗p < 0.05, ∗∗p < 0.01. FIGURE 6 \| (A) Western blots results that showed the expression levels of GSK3β and PP2A in the brains of mice. (B) Active β-catenin, and inactive β-catenin were studied through Western blot. (C) Immunoﬂuorescence of β-catenin (red) and DAPI (blue) in the hippocampi of mice. WT, wild-type mice; Tg, 3×Tg-AD mice; Tg + FLX, ﬂuoxetine treated 3×Tg-AD mice. (D) Analysis of primarily cultured neurons from mouse hippocampus. WT, primarily cultured neurons of wild-type mice; Tg, primarily cultured neurons of 3×Tg-AD mice; Tg + FLX, primarily neurons of 3×Tg-AD mice treated with ﬂuoxetine. Tg + FLX+LB_100, primarily neurons of 3×Tg-AD mice treated with ﬂuoxetine and LB_100 (an inhibitor of PP2A activity). (E) RT-PCR showed active β-catenin and GSK3β expression in the hippocampi of mice. Data are shown as mean ± SD, n = 12 animals/group. ∗p < 0.05, ∗∗p < 0.01. and 3×Tg AD mouse. The cultured neurons from 3×Tg AD mouse were divided into three groups, which are 3×Tg AD, ﬂuoxetine-treated 3×Tg AD, and ﬂuoxetine-treated 3×Tg AD supplemented with LB-100(Tg+Flx+LB-100). LB-100, a speciﬁc inhibitor of PP2A, was used to suppress the activity of PP2A. As shown in Figure 6E, the 3×Tg AD primary neurons showed a signiﬁcantly larger ratio of PP2A pY307/PP2Ac than the WT primary neurons. Frontiers in Aging Neuroscience \| www.frontiersin.org Fluoxetine Protection of Synapses Fluoxetine Protection of Synapses To determine the impact of ﬂuoxetine treatment on synaptic functional protein expression, immunostaining analysis was performed in primary neurons, using synaptic marker PSD95 and MAP2, as shown in Figure 7A. A signiﬁcant reduction in PSD95 was observed in the 3×Tg-AD primary neurons when compared with the WT primary neurons (Figure 7A). After ﬂuoxetine treatment, PSD95 expression was partially recovered in the neuritis of 3×Tg-AD primary neurons. In addition, western blot was used to assay the level of synaptophysin and PSD95. Compared with the culture medium 3×Tg-AD primary neurons, the treatment with ﬂuoxetine could increase the level of both synaptophysin (Figure 7B, p < 0.05) and PSD95 (Figure 7B, p < 0.01). Although ﬂuoxetine is a well-known neuroprotective agent (Qiao et al., 2016), there exists little information about how ﬂuoxetine inﬂuences the Wnt/β-catenin signaling in the hippocampus of AD. Moreover, it remains unconﬁrmed if the activation of the Wnt/β-catenin signaling pathway has any anti-AD eﬀect. Utilizing 3×Tg-AD mice with an early-onset AD-like pathology, the study proved that regular administration of ﬂuoxetine could stem the age-related cognitive impairments, as well as Aβ accumulation (Oddo et al., 2003b). Our research provides supports from multiple aspects that ﬂuoxetine could have beneﬁcial impacts on AD. First, utilizing the URE 7 \| Fluoxetine protection of synapses in 3×Tg-AD neurons. (A) Double-labeling analysis results for neurons from WT mice, 3×Tg-AD, and ﬂuoxetine-treated g-AD mice. Neuronal marker MAP2 (red) with post synaptic density protein 95 (PSD95) (green). Scale bar = 100 µm. (B) The levels of Syna and PSD95 ession were analyzed via Western blot assay. WT, primarily cultured neurons of wild-type mice; Tg, primarily cultured neurons of 3×Tg-AD mice; Tg + FLX, arily neurons of 3×Tg-AD mice treated with ﬂuoxetine. Data were shown as mean ± SD, n = 12 animals/group. ∗p < 0.05, ∗∗p < 0.01. FIGURE 7 \| Fluoxetine protection of synapses in 3×Tg-AD neurons. (A) Double-labeling analysis results for neurons from WT mice, 3×Tg-AD, and ﬂuoxetine-treated 3×Tg-AD mice. Neuronal marker MAP2 (red) with post synaptic density protein 95 (PSD95) (green). Scale bar = 100 µm. (B) The levels of Syna and PSD95 expression were analyzed via Western blot assay. WT, primarily cultured neurons of wild-type mice; Tg, primarily cultured neurons of 3×Tg-AD mice; Tg + FLX, primarily neurons of 3×Tg-AD mice treated with ﬂuoxetine. Data were shown as mean ± SD, n = 12 animals/group. Fluoxetine Targeted PP2A to Activate the Wnt/β-Catenin Signaling Treatment ﬂuoxetine signiﬁcantly decreased the ratio of PP2A pY307/PP2Ac in 3×Tg AD primary neurons, while adding LB-100 extensively eliminated the eﬀect of ﬂuoxetine. The remarkable reduction in active β-catenin between the 3×Tg AD and the WT primary neurons indicated that ﬂuoxetine could raise the active β-catenin. Moreover, the data also demonstrated that adding LB-100 could extensively eliminate this impact from ﬂuoxetine. an immunoﬂuorescence analysis was performed (Figure 6C). Based on the immunoﬂuorescence analysis, the level of active β-catenin in 3×Tg-AD mice was markedly increased in the treatment group (p < 0.01), compared with the 3×Tg-AD mice group (Figure 6C). Quantitative real-time (RT)-PCR analysis also conﬁrmed that β-catenin mRNA expression was increased after ﬂuoxetine treatment (Figure 6D, p < 0.01), while the mRNA levels of GSK3β were reduced (Figure 6D, p < 0.01) after ﬂuoxetine treatment. The results suggested that ﬂuoxetine treatment could eﬃciently activate Wnt/β-catenin signaling through inhibition of GSK3β in the 3×Tg-AD mice brain. To further investigate if ﬂuoxetine aﬀected the Wnt/β-catenin signaling through PP2A activation, we isolated primarily cultured neurons, respectively, from the hippocampi of WT mouse June 2018 \| Volume 10 \| Article 164 Frontiers in Aging Neuroscience \| www.frontiersin.org 9 The Therapeutic Value of Fluoxetine in AD Huang et al. Fluoxetine Protection of Synapses ∗p < 0.05, ∗∗p < 0.01. Frontiers in Aging Neuroscience \| www.frontiersin.org June 2018 \| Volume 10 \| Article 164 10 The Therapeutic Value of Fluoxetine in AD Huang et al. 3×Tg-AD mouse model of AD, the study showed that ﬂuoxetine could impact on brain Aβ levels, cognitive deﬁcits, and amyloid neuropathology. Second, ﬂuoxetine could prevent apoptosis in 3×Tg-AD primary neuronal cell. Third, ﬂuoxetine could increase the expression of BDNF. Fourth, the study also demonstrates that ﬂuoxetine could be able to protect neuron synapses. Moreover, our study proves evidence that ﬂuoxetine would upregulates β-catenin expression and inhibits GSK3β expression in vivo. It suggests that there might be a correlation between neuroprotective eﬀect of ﬂuoxetine and the regulation of the Wnt signaling pathway in the AD brain. of regulating the Aβ level through APP phosphorylation and BACE1 activity. Evidence from various aspects shows that APP could be cleaved within the sequence of the Aβ peptide and generate the sAPPα fragment through the α-secretase pathway (Esch et al., 1990). It would be beneﬁcial for neuronal survival (Mattson et al., 1997; Wallace et al., 1997). However, through the β-secretase pathway, APP would be cleaved form neurotoxic Aβ and play a role in the pathogenesis of AD (Haass et al., 1992). This study focused on examining the role of ﬂuoxetine in the APP process, based on the 3×Tg-AD mouse model of AD. The research found that expression level of APP proteins would be dramatically reduced in the ﬂuoxetine-treated 3×Tg-AD mouse brain. It is widely accepted that Wnt/β-catenin signaling dysfunction plays a critical role in neurodegeneration process in the AD brain (Inestrosa and Toledo, 2008). GSK3β and β-catenin are two key components of the canonical Wnt/β-catenin signaling pathway. Previous research has demonstrated they could be considerably altered in the AD model mice brains (Zhang et al., 1998; Pei et al., 1999). Also, another study shows that activation of Wnt signaling can play a preventive impact on the neurodegeneration induced by Aβ ﬁbrils (De Ferrari et al., 2003). The dysfunction of Wnt/β-catenin signaling induced by Aβ has been detected in AD and proved to be related to the neuron degeneration and synapse impairment. Researchers have detected the dysfunction of Wnt/β-catenin signaling induced by Aβ among AD. And this dysfunction has been proved to be associated with both neuron degeneration and synapse impairment (Thies and Bleiler, 2011; Inestrosa et al., 2012). Fluoxetine Protection of Synapses In this study, we examined the eﬀects of ﬂuoxetine on APP processing in the 3×Tg-AD mouse model of AD. Our data showed that the expression level of APP proteins would be dramatically reduced in the ﬂuoxetine-treated 3×Tg- AD mouse brain. In this study, ﬂuoxetine treatment could signiﬁcantly enhance the expression level of ADAM10, a candidate of α-secretase. This was followed by an increase in the levels of both α-secretase-generated sAPPα and C83 fragments. Activation of the Wnt/β-catenin signaling reduced the transcription of BACE1. In our study, treatment with ﬂuoxetine was shown to activate the Wnt/β-catenin signaling, which in term down regulated the expression of BACE1 in the hippocampus of 3×Tg AD mouse. These processes contributed to the decreased generation of Aβ. The results suggest that the Wnt/β-catenin signaling would play a signiﬁcant part in regulating BACE1 expression, which then make Wnt/β-catenin signaling a core player in the formation of extracellular amyloid plagues. In this study, we showed that, when inactive β-catenin was increased in hippocampus of 3×Tg AD mice, active β-catenin could be reduced signiﬁcantly. And the 3×Tg AD mouse demonstrated a remarkable reverse of these changes after treatment with ﬂuoxetine. Meanwhile, the treatment of ﬂuoxetine inhibited the cell apoptosis in the hippocampus of 3×Tg ADA primary neurons. These ﬁndings suggest that ﬂuoxetine could be capable to activate the Wnt/β-catenin signaling to suppress the pathological hallmarks of AD. Previous study has revealed that mitochondria could play a key role in regulating cell death, especially cell apoptosis (Yu et al., 2015). The dysfunction of mitochondria might become a hallmark of neuronal toxicity in AD (Xi et al., 2012; Zhang et al., 2012, 2016). Several researches also report that Bcl-2 family protein could play a certain role in regulating neuronal apoptotic cell death (Basu et al., 2006; Mancuso et al., 2008; Cartier et al., 2012). One of the key factors for the apoptotic state of cell would be the ratio between pro- apoptotic proteins and anti-apoptotic proteins (Yu et al., 2015). The result of western blot analysis shows that ﬂuoxetine could signiﬁcantly increase both expression ratios (Bcl-2/Bax and Bcl- xl/Bax) and, at the same time, decrease the number of TUNEL- positive cell compared with the culture treated with culture medium only. Thus, it is suggested that ﬂuoxetine could inhibit the apoptosis, which involves the regulation of Bcl-2 family proteins. Frontiers in Aging Neuroscience \| www.frontiersin.org REFERENCES J. (2010). Depression in Alzheimer’s disease. Pathophysiology, diagnosis, and treatment. J. Alzheimers Dis. 21, 1077–1087. doi: 10.3233/JAD- 2010-100153 Haass, C., Schlossmacher, M. G., Hung, A. Y., Vigo-Pelfrey, C., Mellon, A., Ostaszewski, B. L., et al. (1992). Amyloid beta-peptide is produced by cultured cells during normal metabolism. Nature 359, 322–325. doi: 10.1038/359322a0 Mowla, A., Mosavinasab, M., Haghshenas, H., and Borhani Haghighi, A. (2007). Does serotonin augmentation have any eﬀect on cognition and activities of daily living in Alzheimer’s dementia? A double-blind, placebo-controlled clinical trial. J. Clin. Psychopharmacol. 27, 484–487. doi: 10.1097/jcp.0b013e31814 b98c1 Inestrosa, N. C., Montecinos-Oliva, C., and Fuenzalida, M. (2012). Wnt signaling: role in Alzheimer disease and schizophrenia. J. Neuroimmune Pharmacol. 7, 788–807. doi: 10.1007/s11481-012-9417-5 Mueller, S. C., Temple, V., Oh, E., VanRyzin, C., Williams, A., Cornwell, B., et al. (2008). Early androgen exposure modulates spatial cognition in congenital adrenal hyperplasia (CAH). Psychoneuroendocrinology 33, 973–980. doi: 10.1016/j.psyneuen.2008.04.005 Inestrosa, N. C., and Toledo, E. M. (2008). The role of Wnt signaling in neuronal dysfunction in Alzheimer’s disease. Mol. Neurodegener. 3:9. doi: 10.1186/1750- 1326-3-9 Inestrosa, N. C., and Varela-Nallar, L. (2014). Wnt signaling in the nervous system and in Alzheimer’s disease. J. Mol. Cell Biol. 6, 64–74. doi: 10.1093/jmcb/mjt051 Oddo, S., Caccamo, A., Kitazawa, M., Tseng, B. P., and LaFerla, F. M. (2003a). Amyloid deposition precedes tangle formation in a triple transgenic model of Alzheimer’s disease. Neurobiol. Aging 24, 1063–1070. Li, W. L., Cai, H. H., Wang, B., Chen, L., Zhou, Q. G., Luo, C. X., et al. (2009). Chronic ﬂuoxetine treatment improves ischemia-induced spatial cognitive deﬁcits through increasing hippocampal neurogenesis after stroke. J. Neurosci. Res. 87, 112–122. doi: 10.1002/jnr.21829 Oddo, S., Caccamo, A., Shepherd, J. D., Murphy, M. P., Golde, T. E., Kayed, R., et al. (2003b). Triple-transgenic model of Alzheimer’s disease with plaques and tangles: intracellular Abeta and synaptic dysfunction. Neuron 39, 409–421. doi: 10.1016/S0896-6273(03)00434-3 Li, X., Zhu, W., Roh, M. S., Friedman, A. B., Rosborough, K., and Jope, R. S. (2004). In vivo regulation of glycogen synthase kinase-3beta (GSK3beta) by serotonergic activity in mouse brain. Neuropsychopharmacology 29, 1426–1431. doi: 10.1038/sj.npp.1300439 Oulès, B., Del Prete, D., Greco, B., Zhang, X., Lauritzen, I., Sevalle, J., et al. (2012). Ryanodine receptors blockade reduces Amyloid-beta load and memory impairments in Tg2576 mouse model of Alzheimer disease. J. Neurosci. 32, 11820–11834. doi: 10.1523/JNEUROSCI.0875-12.2012 Liu, C. C., Tsai, C. W., Deak, F., Rogers, J., Penuliar, M., Sung, Y. M., et al. (2014). REFERENCES Liu, R., and Wang, J. Z. (2009). Protein phosphatase 2A in Alzheimer’s disease. Pathophysiology 16, 273–277. doi: 10.1016/j.pathophys.2009.02.008 Basu, A., DuBois, G., and Haldar, S. (2006). Posttranslational modiﬁcations of Bcl2 family members–a potential therapeutic target for human malignancy. Front. Biosci. 11, 1508–1521. doi: 10.2741/1900 Liu, Z., Lu, H., Jiang, Z., Pastuszyn, A., and Hu, C. A. (2005). Apolipoprotein l6, a novel proapoptotic Bcl-2 homology 3-only protein, induces mitochondria- mediated apoptosis in cancer cells. Mol. Cancer Res. 3, 21–31. Ma, J., Gao, Y., Jiang, L., Chao, F. L., Huang, W., and Zhou, C. N. (2017). Fluoxetine attenuates the impairment of spatial learning ability and prevents neuron loss in middle-aged APPswe/PSEN1dE9 double transgenic Alzheimer’s disease mice. Oncotarget 8, 27676–27692. doi: 10.18632/oncotarget.15398 Billings, L. M., Oddo, S., Green, K. N., McGaugh, J. L., and LaFerla, F. M. (2005). Intraneuronal Aβ causes the onset of early Alzheimer’s disease-related cognitive deﬁcits in transgenic mice. Neuron 45, 675–688. doi: 10.1016/j.neuron.2005. 01.040 Mancuso, M., Orsucci, D., Siciliano, G., and Murri, L. (2008). Mitochondria, mitochondrial DNA and Alzheimer’s disease. What comes ﬁrst? Curr. Alzheimer Res. 5, 457–468. doi: 10.2174/156720508785908946 Cartier, J., Marivin, A., Berthelet, J., and Dubrez, L. (2012). IAPs: a central element in the NF-κB activating signaling pathway. Med. Sci. 28, 69–75. doi: 10.1051/ medsci/2012281019 Davidson, G., Wu, W., Shen, J. L., Bilic, J., Fenger, U., Stannek, P., et al. (2005). Casein kinase 1 gamma couples Wnt receptor activation to cytoplasmic signal transduction. Nature 438, 867–872. doi: 10.1038/nature04170 Mattson, M. P., Barger, S. W., Furukawa, K., Bruce, A. J., Wyss-Coray, T., Mark, R. J., et al. (1997). Cellular signaling roles of TGF beta, TNF alpha and beta APP in brain injury responses and Alzheimer’s disease. Brain Res. Brain Res. Rev. 23, 47–61. doi: 10.1016/S0165-0173(96)00014-8 De Ferrari, G. V., Chacon, M. A., Barria, M. I., Garrido, J. L., Godoy, J. A., Olivares, G., et al. (2003). Activation of Wnt signaling rescues neurodegeneration and behavioral impairments induced by beta-amyloid ﬁbrils. Mol. Psychiatry 8, 195–208. doi: 10.1038/sj.mp.4001208 Miller-Thomas, M. M., Sipe, A. L., Benzinger, T. L., McConathy, J., Connolly, S., and Schwetye, K. E. (2016). Multimodality review of amyloid-related diseases of the central nervous system. Radiographics 36, 1147–1163. doi: 10.1148/rg. 2016150172 Esch, F. S., Keim, P. S., Beattie, E. C., Blacher, R. W., Culwell, A. R., Oltersdorf, T., et al. (1990). Cleavage of amyloid beta peptide during constitutive processing of its precursor. Science 248, 1122–1124. doi: 10.1126/science.211 1583 Modrego, P. FUNDING This work was supported by the National Natural Science Foundation of China (Grant No. 81503616) and the Project of Administration of Traditional Chinese Medicine of Guangdong Province of China (No.20181064). Fluoxetine Protection of Synapses Furthermore, more exploration was conducted regarding the relation across PP2A activity, Wnt/β-catenin signaling, and AD pathology, via utilizing the primarily cultured neurons from the hippocampus of 3×Tg AD mouse. Based on the results, we found that ﬂuoxetine could signiﬁcantly repair the function of Wnt/β-catenin signaling which had been impaired during the AD progression. LB-100, an inhibitor of PP2A, could extensively eliminated the eﬀect of ﬂuoxetine on β-catenin, which will result in the down-regulation of Wnt/β-catenin signaling and the neuronal apoptosis, Thus, it could be implied that ﬂuoxetine could speciﬁcally activate PP2A to dephosphorylate β-catenin on S33/S37/T41 and GSK3β on Y216 for upregulation of Wnt/β-catenin signaling in 3×Tg AD mice. BDNF is a neurotrophic factor, it is well known that BDNF is involved in the growth of neurites and synaptic plasticity (Sandhya et al., 2013). Fluoxetine might preserve synaptic protein expression, thus improve learning and memory abilities. After ﬂuoxetine treatment, PSD95 and synaptic expression were partially recovered in the 3×Tg-AD neurons. The data above and the in vivo results together strongly implies that ﬂuoxetine Deposition of extracellular amyloid plagues in AD would also be subjected to PP2A regulation. PP2A could be capable June 2018 \| Volume 10 \| Article 164 Frontiers in Aging Neuroscience \| www.frontiersin.org 11 The Therapeutic Value of Fluoxetine in AD Huang et al. could be capable to abolish Aβ generation and preserve synaptic functional proteins. of Alzheimer’s disease through the activation of Wnt/β-catenin signaling. could be capable to abolish Aβ generation and preserve synaptic functional proteins. AUTHOR CONTRIBUTIONS Treatment with ﬂuoxetine would signiﬁcantly enhance the activity of PP2A and repress the pathology of AD signiﬁcantly. After activated by ﬂuoxetine, the PP2A could increase active β-catenin level and inhibit GSK3β activity in the hippocampus of 3×Tg AD mouse, which then could lead to signal the Wnt/β-catenin signaling. The treatment could relieve the APP cleavage and Aβ generation. Taking all these results into consideration, it could be concluded that ﬂuoxetine could activate Wnt/β-catenin signaling via PP2A to repress the cross-talk among Aβ generation and neuronal apoptosis. These results suggest that ﬂuoxetine could have a potential for the therapy Treatment with ﬂuoxetine would signiﬁcantly enhance the activity of PP2A and repress the pathology of AD signiﬁcantly. After activated by ﬂuoxetine, the PP2A could increase active β-catenin level and inhibit GSK3β activity in the hippocampus of 3×Tg AD mouse, which then could lead to signal the Wnt/β-catenin signaling. The treatment could relieve the APP cleavage and Aβ generation. Taking all these results into consideration, it could be concluded that ﬂuoxetine could activate Wnt/β-catenin signaling via PP2A to repress the cross-talk among Aβ generation and neuronal apoptosis. These results suggest that ﬂuoxetine could have a potential for the therapy MH, YL, and HC: conceived, designed, and performed the experiments. MH, YL, BX, CC, and PX: analyzed the data. MH: wrote the paper. MH, YL, and HC: conceived, designed, and performed the experiments. MH, YL, BX, CC, and PX: analyzed the data. MH: wrote the paper. REFERENCES Deﬁciency in LRP6-mediated Wnt signaling contributes to synaptic abnormalities and amyloid pathology in Alzheimer’s disease. Neuron 84, 63–77. doi: 10.1016/j.neuron.2014.08.048 Patapoutian, A., and Reichardt, L. F. (2000). Roles of Wnt proteins in neural development and maintenance. Curr. Opin. Neurobiol 10, 392–399. doi: 10.1016/S0959-4388(00)00100-8 June 2018 \| Volume 10 \| Article 164 Frontiers in Aging Neuroscience \| www.frontiersin.org 12 The Therapeutic Value of Fluoxetine in AD Huang et al. Pei, J. J., Braak, E., Braak, H., Grundke-Iqbal, I., Iqbal, K., Winblad, B., et al. (1999). Distribution of active glycogen synthase kinase 3beta (GSK-3beta) in brains staged for Alzheimer disease neuroﬁbrillary changes. J. Neuropathol. Exp. Neurol. 58, 1010–1019. doi: 10.1097/00005072-199909000-00011 Voronkov, M., Braithwaite, S. P., and Stock, J. B. (2011). Phosphoprotein phosphatase 2A: a novel druggable target for Alzheimer’s disease. Future Med. Chem. 3, 821–833. doi: 10.4155/fmc.11.47 Wallace, W. C., Akar, C. A., and Lyons, W. E. (1997). Amyloid precursor protein potentiates the neurotrophic activity of NGF. Brain Res. Mol. Brain Res. 52, 201–212. doi: 10.1016/S0169-328X(97)00258-1 Pilar-Cuellar, F., Vidal, R., and Pazos, A. (2012). Subchronic treatment with ﬂuoxetine and ketanserin increases hippocampal brain-derived neurotrophic factor, beta-catenin and antidepressant-like eﬀects. Br. J. Pharmacol. 165, 1046–1057. doi: 10.1111/j.1476-5381.2011.01516.x Wang, J., Zhang, Y., Xu, H., Zhu, S., Wang, H., He, J., et al. (2014). Fluoxetine improves behavioral performance by suppressing the production of soluble β-amyloid in APP/PS1 mice. Curr. Alzheimer Res. 11, 672–680. doi: 10.2174/ 1567205011666140812114715 Qiao, J., Wang, J., Wang, H., Zhang, Y., Zhu, S., Adilijiang, A., et al. (2016). Regulation of astrocyte pathology by ﬂuoxetine prevents the deterioration of Alzheimer phenotypes in an APP/PS1 mouse model. GLIA 64, 240–254. doi: 10.1002/glia.22926 Xi, Y. D., Yu, H. L., Ding, J., Ma, W. W., Yuan, L. H., Feng, J. F., et al. (2012). Flavonoids protect cerebrovascular endothelial cells through Nrf2 and PI3Kfrom â-amyloid peptide-induced oxidative damage. Curr. Neurovasc. Res. 9, 32–41. doi: 10.2174/156720212799297092 Rosso, S. B., and Inestrosa, N. C. (2013). WNT signaling in neuronal maturation and synaptogenesis. Front Cell Neurosci. 7:103. doi: 10.3389/fncel.2013.00103 Yu, T. X., Zhang, P., Guan, Y., Wang, M., and Zhen, M. Q. (2015). Protective eﬀects of luteolin against cognitive impairment induced by infusion of Aβ peptide in rats. Int. J.Clin. Exp. Pathol. 8, 6740–6747. Sandhya, V. K., Raju, R., Verma, R., Advani, J., Sharma, R., Radhakrishnan, A., et al. (2013). A network map of BDNF/TRKB and BDNF/p75NTR signaling system. J. Cell Commun. Signal. 7, 301–307. doi: 10.1007/s12079-013-0200-z Scharre, D. REFERENCES W., and Chang, S. I. (2002). Cognitive and behavioral eﬀects of quetiapine in Alzheimer disease patients. Alzheimer Dis. Assoc. Disord. 16, 128–130. doi: 10.1097/00002093-200204000-00011 Zeng, X., Huang, H., Tamai, K., Zhang, X. J., Harada, Y., Yokota, C., et al. (2008). Initiation of Wnt signaling: control of Wnt coreceptor Lrp6 phosphorylation/activation via frizzled, dishevelled and axin functions. Development 135, 367–375. doi: 10.1242/dev.013540 Seeling, J. M., Miller, J. R., Gil, R., Moon, R. T., White, R., and Virshup, D. M. (1999). Regulation of beta-catenin signaling by the B56 subunit of protein phosphatase 2A. Science 283, 2089–2091. doi: 10.1126/science.283.5410. 2089 Zhang, Q., Huang, W. D., Lv, X. Y., and Yang, Y. M. (2012). Puerarin protects diﬀerentiated PC12 cells from H2O2-induced apoptosis through the PI3K/Akt signalling pathway. Cell Biol. Int. 36, 419–426. doi: 10.1042/CBI20100900 Selkoe, D. J. (2004). Cell biology of protein misfolding: the examples of Alzheimer’s and Parkinson’s diseases. Nat. Cell Biol. 6, 1054–1061. doi: 10.1038/ncb1104- 1054 Zhang, X., Liang, D., Lian, X., Jiang, Y., He, H., Liang, W., et al. (2016). Berberine activates Nrf2 nuclear translocation and inhibits apoptosis induced by high glucose in renal tubular epithelial cells through a phosphatidylinositol 3-kinase/Akt-dependent mechanism. Apoptosis 21, 721–736. doi: 10.1007/ s10495-016-1234-5 Sontag, E., Nunbhakdi-Craig, V., Sontag, J. M., Diaz-Arrastia, R., Ogris, E., Dayal, S., et al. (2007). Protein phosphatase 2A methyltransferase links homocysteine metabolism with tau and amyloid precursor protein regulation. J. Neurosci. 27, 2751–2759. doi: 10.1523/JNEUROSCI.3316-06.2007 Zhang, Z., Hartmann, H., Do, V. M., Abramowski, D., Sturchler-Pierrat, C., Staufenbiel, M., et al. (1998). Destabilization of beta-catenin by mutations in presenilin-1 potentiates neuronal apoptosis. Nature 395, 698–702. doi: 10.1038/ 27208 Suh, Y. H., and Checler, F. (2002). Amyloid precursor protein, presenilins, and alphasynuclein: Molecular pathogenesis and pharmacological applications in Alzheimer’s disease. Pharmacol. Rev. 54, 469–525. doi: 10.1124/pr.54.3.469 Zuroﬀ, L., Daley, D., Black, K. L., and Koronyo-Hamaoui, M. (2017). Clearance of cerebral Aβ in Alzheimer’s disease: reassessing the role of microglia and monocytes. Cell Mol. Life. Sci. 74, 2167–2201. doi: 10.1007/s00018-017-2463-7 Sun, D. S., Gao, L. F., Jin, L., Wu, H., Wang, Q., Zhou, Y., et al. (2017). Fluoxetine administration during adolescence attenuates cognitive and synaptic deﬁcits in adult 3 × TgAD mice. Neuropharmacology 126, 200–212. doi: 10.1016/j. neuropharm.2017.08.037 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. Thies, W., and Bleiler, L. (2011). June 2018 \| Volume 10 \| Article 164 Frontiers in Aging Neuroscience \| www.frontiersin.org REFERENCES Alzheimer’s association report 2011 Alzheimer’s disease facts and ﬁgures. Alzheimers Dement. 7, 208–244. doi: 10.1016/j.jalz. 2011.02.004 Copyright © 2018 Huang, Liang, Chen, Xu, Chai and Xing. 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 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. Triaca, V., Sposato, V., Bolasco, G., Ciotti, M. T., Pelicci, P., Bruni, A. C., et al. (2016). NGF controls APP cleavage by downregulating APP phosphorylation at Thr668: relevance for Alzheimer’s disease. Aging Cell 15, 661–672. doi: 10.1111/ acel.12473 Vorhees, C. V., and Williams, M. T. (2006). Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat. Protoc. 1, 848–858. doi: 10.1038/nprot.2006.116 June 2018 \| Volume 10 \| Article 164 Frontiers in Aging Neuroscience \| www.frontiersin.org 13
https://openalex.org/W4308643122	https://bmcurol.biomedcentral.com/counter/pdf/10.1186/s12894-022-01139-9	English	null	Comparison of full-dose gemcitabine/cisplatin, dose-reduced gemcitabine/cisplatin, and gemcitabine/carboplatin in real-world patients with advanced urothelial carcinoma	BMC urology	2,022	cc-by	6,156	Abstract Background: While gemcitabine/cisplatin (GC) is the gold standard regimen for patients with advanced urothelial carcinoma (aUC), either dose-reduced GC or gemcitabine/carboplatin (GCa) is an alternative option for “cisplatin-unfit” patients. However, few studies have compared outcomes with these commonly used regimens in the real-world setting. Methods: We retrospectively reviewed patients with aUC who received full-dose GC, dose-reduced GC, or GCa as first-line salvage chemotherapy at two university hospitals between 2016 and 2020. Progression-free survival, cancer- specific survival, and overall survival, as well as best overall response and adverse event profiles, were compared among these three regimens. Results: Of 105 patients, 41, 27, and 37 patients received full-dose GC, dose-reduced GC, and GCa, respectively. Significant differences were noted in the patients’ baseline age, primary site, and renal function among the three regi- mens. Sixty-nine (65.7%) patients died during a median follow-up period of 14 months. There was no significant differ- ence among the three regimens for all survival outcomes and best overall response. However, the complete response rate of dose-reduced GC (2/27, 7.4%) appeared inferior to that of full-dose GC (9/41, 22.0%) or GCa (6/37, 16.2%). Regarding adverse event profiles, no significant difference was observed among the three regimens, except for signifi- cantly fewer cases with elevated alanine aminotransferase in the GCa group compared with the other groups. Conclusions: This study compared the oncological and toxicological outcomes of full-dose GC, dose-reduced GC, and GCa in real-world patients with aUC. Unlike in the clinical trial setting, there were almost no significant differences among the three regimens. Keywords: Advanced, Carboplatin, Cisplatin, Metastatic, Renal function, Urothelial carcinoma © The Author(s) 2022. 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://creativecom- mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Comparison of full‑dose gemcitabine/ cisplatin, dose‑reduced gemcitabine/cisplatin, and gemcitabine/carboplatin in real‑world patients with advanced urothelial carcinoma Kazuma Sugimoto1, Satoru Taguchi1,2, Kenjiro Kishitani2, Taketo Kawai2,3, Kazuki Masuda1, Yu Nakamura1, Manami Kinjo1, Mitsuhiro Tambo1, Jimpei Miyakawa1,2, Yoshiyuki Akiyama2, Yuta Yamada2, Yusuke Sato2, Daisuke Yamada2, Tohru Nakagawa3, Hiroshi Fukuhara1 and Haruki Kume2 Background For the last three decades, platinum-based chemother- apy has played a key role in the treatment of advanced urothelial carcinoma (aUC). Gemcitabine/cisplatin (GC) is currently the gold standard regimen for patients with aUC who are fit for cisplatin [1, 2], as well as dose-dense 2 Department of Urology, Graduate School of Medicine, The University of Tokyo, 7‑3‑1 Hongo, Bunkyo‑ku, Tokyo 113‑8655, Japan Full list of author information is available at the end of the article Sugimoto et al. BMC Urology (2022) 22:177 https://doi.org/10.1186/s12894-022-01139-9 Sugimoto et al. BMC Urology (2022) 22:177 https://doi.org/10.1186/s12894-022-01139-9 Open Access © The Author(s) 2022. 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://creativecom- mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Sugimoto et al. BMC Urology (2022) 22:177 Page 2 of 9 methotrexate/vinblastine/doxorubicin/cisplatin (ddM- VAC) [3, 4]. However, 30–50% of patients with aUC are ineligible to receive cisplatin because of poor perfor- mance status, impaired renal function (creatinine clear- ance < 60 mL/min), hearing loss, peripheral neuropathy, and heart failure [5, 6]. For such “cisplatin-unfit” patients, gemcitabine/carboplatin (GCa), in which cisplatin in the GC regimen is replaced with another platinum-contain- ing drug, carboplatin, can be an alternative [3, 4]. How- ever, the efficacy of GCa was proven inferior to that of GC in a previous clinical trial [7]. The GC regimen with a reduced dosage of cisplatin (i.e., “dose-reduced GC”) can also be used for patients who are considered ineligible for “full-dose GC” for several reasons (typically, impaired renal function) in the real-world setting [8, 9]. However, few studies have compared the outcomes of these com- monly used regimens (dose-reduced GC and GCa) [9]. Therefore, the present study aimed to investigate the oncological and toxicological outcomes of full-dose GC, dose-reduced GC, and GCa in real-world patients with aUC. (ddM- function, while either dose-reduced GC or GCa was selected for those with poor performance status and/or impaired renal function. The full-dose GC regimen con- sisting of 1000 mg/m2 gemcitabine on days 1, 8, and 15 and 70 mg/m2 cisplatin on day 2 was given every 28 days. Patient characteristicsh The patients’ characteristics at the start of first-line chemotherapy are summarized in Table 1. Of the 105 patients, 41, 27, and 37 patients received full-dose GC, dose-reduced GC, and GCa, respectively. Patients treated with full-dose GC were younger, had less Statistical analysisf Differences in patient characteristics among the three regimens were assessed using Student’s t-test or the χ2 test. Survival curves were generated using the Kaplan– Meier method, and the curves were compared using the log-rank test. A Cox proportional hazards regression model was used for univariate and multivariate analyses of PFS, CSS, and OS. All statistical analyses were per- formed using JMP Pro version 15.0.0 (SAS Institute, Cary, NC, USA), and P < 0.05 indicated a significant difference. Patients and methods Ethical approval and informed consenth Ethical approval and informed consenth The present study was conducted in accordance with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. The study was approved by the Institutional Review Board (IRB) of the Graduate School of Medicine and Faculty of Medicine, The Uni- versity of Tokyo (approval number: 10565) and the IRB of Kyorin University School of Medicine (approval num- ber: 1262). Given the retrospective nature of the study, the requirement for informed consent was waived by the IRB of the Graduate School of Medicine and Faculty of Medicine, The University of Tokyo and the IRB of Kyorin University School of Medicine. Study population We retrospectively reviewed 107 patients with aUC who received full-dose GC, dose-reduced GC, or GCa as first- line salvage chemotherapy at either The University of Tokyo Hospital or Kyorin University Hospital between January 2016 and August 2020. We excluded two patients owing to the lack of appropriate image evaluation after starting chemotherapy, leaving 105 patients for inclusion in the final analysis (The University of Tokyo Hospital, n = 35; Kyorin University Hospital, n = 70). The cisplatin dosage in the dose-reduced GC regimen was determined at the physician’s discretion and ranged from 50 to 80%; however, most (22/27, 81%) patients received the 75% dosage. The GCa regimen consisting of 1000 mg/m2 gemcitabine on days 1 and 8 and area under the curve 5 carboplatin on day 1 was given every 21 days. All patients underwent evaluations every 1–3 months, which comprised routine blood tests and/or computed tomography. The treatment efficacy was assessed in accordance with the Response Evaluation Criteria in Solid Tumours (RECIST) v1.1 [10]. Adverse events were evaluated in accordance with the Common Terminology Criteria for Adverse events (CTCAE) v5.0 [11]. Estimated glomerular filtration rate (eGFR) was calculated using the revised formula for Japanese patients [12]. Patients and methods As oncological endpoints, progression-free survival (PFS), cancer-specific survival (CSS), overall survival (OS), and best overall response according to the RECIST v1.1 criteria [10] were compared among the three regi- mens. As toxicological endpoints, comprehensive adverse event profiles, namely abnormal laboratory data, rash, fatigue, and vomiting, were also assessed in accord- ance with the CTCAE v5.0 criteria [11], as stated above. Follow-up started on the day of initiating first-line chem- otherapy. Follow-up information was obtained as of Sep- tember 2021. Treatment and assessmenth The regimen choice for each patient (full-dose GC, dose- reduced GC, or GCa) was dependent on the attending physician’s decision. Generally, full-dose GC was used for patients with good performance status and normal renal Sugimoto et al. BMC Urology (2022) 22:177 Page 3 of 9 Table 1 Patient characteristics (n = 105) Table 1 Patient characteristics (n = 105) ECOG PS Eastern Cooperative Oncology Group Performance Status, eGFR estimated glomerular filtration rate, GC gemcitabine/cisplatin, GCa gemcitabine/carboplatin, IQR interquartile range Statistically significant, †excluding two patients with missing data, aStudent’s t-test, bχ2 test Parameter Total (n = 105) Full-dose GC (n = 41) Dose-reduced GC (n = 27) GCa (n = 37) P value Age, years, median (IQR) 74 (69–79) 72 (64–77) 76 (69–80) 75 (71–80) 0.0076a* Sex, no. (%) 0.55b Male 80 (76.2) 29 (70.7) 22 (81.5) 29 (78.4) Female 25 (23.8) 12 (29.3) 5 (18.5) 8 (21.6) ECOG PS, no. (%) 0.43b 0 82 (68.6) 31 (75.6) 18 (66.7) 23 (62.2) ≥ 1 33 (31.4) 10 (24.4) 9 (33.3) 14 (37.8) Primary site, no. (%) 0.0040b* Bladder 44 (41.9) 26 (63.4) 10 (37.0) 8 (21.6) Upper urinary tract 46 (43.8) 13 (31.7) 12 (44.4) 21 (56.8) Both 15 (14.3) 2 (4.9) 5 (18.5) 8 (21.6) Serum creatinine, mg/dL, median (IQR)† 1.04 (0.75–1.38) 0.75 (0.67–1.02) 1.11 (0.91–1.39) 1.36 (0.99–1.55) 0.0012a* eGFR, mL/min/1.73 m2, median (IQR)† 49.9 (39.8–67.3) 68.1 (54.8–84.3) 45.2 (39.3–61.2) 39.3 (34.0–48.6) < 0.0001a* Resection of primary site, no. (%) 52 (49.5) 19 (46.3) 10 (37.0) 23 (62.2) 0.12b Prior neoadjuvant/adjuvant chemotherapy, no. (%) 11 (10.5) 4 (9.8) 2 (7.4) 5 (13.5) 0.72b Lymph node metastasis, no. (%) 70 (66.7) 25 (61.0) 23 (85.2) 22 (59.5) 0.060b Visceral metastasis, no. (%) 56 (53.3) 23 (56.1) 12 (44.4) 21 (56.8) 0.56b Lung metastasis, no. (%) 37 (35.2) 18 (43.9) 8 (29.6) 11 (29.7) 0.33b Bone metastasis, no. (%) 14 (13.3) 5 (12.2) 2 (7.41) 7 (18.9) 0.39b Liver metastasis, no. (%) 13 (12.4) 2 (4.9) 3 (11.1) 8 (21.6) 0.79b Follow–up duration, months, median (IQR) 14 (7–24) 14 (5–28.5) 15 (8–24) 11 (6–23) 0.76a Total (n = 105) Full-dose GC (n = 41) Dose-reduced GC (n = 27) GCa (n = 37) P value Statistically significant, †excluding two patients with missing data, aStudent’s t-test, bχ2 test frequent upper urinary tract disease, and better renal function compared with the other groups. Treatment and assessmenth Fifteen of 41 (36.6%) patients in the full-dose GC group had a pri- mary tumor in upper urinary tract or both, whereas 17 of 27 (63.0%) and 29 of 37 (78.4%) patients in the dose-reduced GC and GCa groups did so, respectively (P = 0.0040). Median eGFRs (mL/min/1.73 m2) were 68.1, 45.2, and 39.3, in the full-dose GC, dose-reduced GC, and GCa groups, respectively (P < 0.0001; note: two patients with missing data were excluded in the calcula- tion of eGFR). There was no significant difference in the other parameters, namely Eastern Cooperative Oncol- ogy Group Performance Status (ECOG PS), resec- tion of the primary site, prior neoadjuvant/adjuvant chemotherapy, and metastatic sites, among the three regimens. The median number of chemotherapy cycles was 3 (interquartile range [IQR], 2–4), 3 (IQR, 2–4), and 3 (IQR, 1.5–6.5), with full-dose GC, dose-reduced GC, and GCa, respectively (P = 0.26). During the study period after first-line chemotherapy, 25 (23.8%) patients received pembrolizumab as later-line treatment (full- dose GC, n = 11; dose-reduced GC, n = 9; GCa, n = 5). Statistically significant, †excluding two patients with missing data, aStudent’s t-test, bχ2 test Oncological outcomesh The median follow-up and survival times were 14 (IQR, 7–24) months and 18 (IQR, 8–32) months, respectively. Overall, 91 (86.7%) patients experienced disease pro- gression, 64 (61.0%) died of aUC, and 5 (4.8%) died from other causes (pneumonia, n = 2; congestive heart fail- ure, n = 1; myocarditis, n = 1; and gastric cancer, n = 1). There were no significant differences in PFS, CSS, and OS among the three regimens (Fig. 1). Univariate analyses of PFS associated ECOG PS and resection of the primary site with the outcome, and mul- tivariate analysis identified no resection of the primary site as an independent predictor of shorter PFS (Table 2). Univariate analyses of CSS associated ECOG PS, resec- tion of the primary site, lymph node metastasis, and liver metastasis with the outcome, and multivariate analy- sis revealed ECOG PS ≥ 1, lymph node metastasis, and liver metastasis as independent predictors of shorter CSS (Table 3). Univariate and multivariate analyses of OS identified the same prognosticators as those identified for CSS (Table 4). Figure 2 shows the best overall response results of the three regimens. Overall, 47 (44.8%) patients achieved Sugimoto et al. BMC Urology (2022) 22:177 Page 4 of 9 Fig. 1 Kaplan–Meier curves depicting A PFS, B CSS, and C OS of patients with aUC treated with full-dose GC, dose-reduced GC, or GCa. Abbreviations: aUC Advanced urothelial carcinoma, CSS Cancer-specific survival, GC Gemcitabine/cisplatin, GCa Gemcitabine/carboplatin, OS Overall survival, PFS Progression-free survival Fig. 1 Kaplan–Meier curves depicting A PFS, B CSS, and C OS of patients with aUC treated with full-dose GC, dose-reduced GC, or GCa. Abbreviations: aUC Advanced urothelial carcinoma, CSS Cancer-specific survival, GC Gemcitabine/cisplatin, GCa Gemcitabine/carboplatin, OS Overall survival, PFS Progression-free survival significant differences in the rates of hematological tox- icities (leukopenia, neutropenia, thrombocytopenia, and anemia), most hepatic toxicities (increased bilirubin and aspartate aminotransferase), renal toxicity (increased creatinine), rash, fatigue, and vomiting, among the three regimens. There were significantly fewer cases with increased alanine aminotransferase in the GCa group compared with the other groups (P = 0.025). an objective response (complete response [CR] + par- tial response [PR]). There were no significant differences in the objective response rate, durable response rate (CR + PR + stable disease), or progressive disease rate among the three regimens. Oncological outcomesh Notably, the CR rate of dose- reduced GC (2/27, 7.4%) appeared inferior to that of full- dose GC (9/41, 22.0%) or GCa (6/37, 16.2%), although there was no significant difference (P = 0.28). Discussionh The present study compared the oncological and toxi- cological outcomes of full-dose GC, dose-reduced GC, and GCa in real-world patients with aUC. Unlike in the clinical trial setting, there were almost no significant Figure 3 shows the comprehensive adverse event pro- files among the three regimens, which were evaluated in 97 patients with sufficient data (full-dose GC, n = 41; dose-reduced GC, n = 24; GCa, n = 32). There were no Sugimoto et al. BMC Urology (2022) 22:177 Page 5 of 9 Table 2 Univariate and multivariate Cox proportional hazards regression analyses of PFS CI confidence interval, ECOG PS Eastern Cooperative Oncology Group Performance Status, eGFR estimated glomerular filtration rate, GC gemcitabine/cisplatin, GCa gemcitabine/carboplatin, HR hazard ratio, PFS progression-free survival Statistically significant Parameter Cutoff Univariate Multivariate HR (95% CI) P HR (95% CI) P Age (years) Continuous 1.00 (0.98–1.02) per score 0.98 Sex Male Reference 0.80 Female 0.94 (0.56–1.50) ECOG PS 0 Reference 0.043 Reference 0.13 ≥ 1 1.57 (1.00–2.39) 1.41 (0.90–2.19) eGFR (mL/min/1.73 m2) < 60 Reference 0.15 ≥ 60 1.36 (0.89–2.06) Primary site Bladder Reference 0.52 Upper urinary tract 0.84 (0.54–1.31) Both 1.18 (0.63–2.08) Resection of primary site No Reference 0.018* Reference 0.047* Yes 0.61 (0.41–0.92) 0.66 (0.43–0.99) Prior neoadjuvant/adjuvant chemotherapy No Reference 0.66 Yes 1.15 (0.58–2.08) Lymph node metastasis No Reference 0.31 Yes 1.24 (0.82–1.92) Lung metastasis No Reference 0.68 Yes 1.09 (0.71–1.64) Bone metastasis No Reference 0.48 Yes 0.80 (0.41–1.42) Liver metastasis No Reference 0.22 Yes 1.45 (0.77–2.51) First-line regimen Full-dose GC Reference 0.80 Dose-reduced GC 0.95 (0.56–1.57) GCa 0.85 (0.53–1.36) Table 2 Univariate and multivariate Cox proportional hazards regression analyses of PFS ate Cox proportional hazards regression analyses of PFS CI confidence interval, ECOG PS Eastern Cooperative Oncology Group Performance Status, eGFR estimated glomerular filtration rate, GC gemcitabine/cisplatin, GCa gemcitabine/carboplatin, HR hazard ratio, PFS progression-free survival Statistically significant respectively, while no differences between the regimens were noted for the overall toxicity profiles [7]. Therefore, current guidelines do not recommend the use of GCa for “cisplatin-fit” patients with aUC [3, 4]. In contrast, another phase 2/3 trial comparing GCa and methotrex- ate/carboplatin/vinblastine (M-CAVI) in “cisplatin-unfit” patients (creatinine clearance 30–60 mL/min and/or ECOG PS 2) reported that the best overall response rates were 41.2% for GCa versus 30.3% for M-CAVI (P = 0.08) and that median OS was 9.3 months and 8.1 months for GCa and M-CAVI, respectively (P = 0.64). CI confidence interval, ECOG PS Eastern Cooperative Oncology Group Performance Status, eGFR estimated glomerular filtration rate, GC gemcitabine/cisplatin, GCa gemcitabine/carboplatin, HR hazard ratio, PFS progression-free survival Statistically significant Discussionh However, severe acute toxicity was observed in 9.3% of the patients receiving GCa and 21.2% of those receiving M-CAVI [13, 14]. Therefore, current guidelines recommend the use of GCa for “cisplatin-unfit” patients with aUC [3, 4]. Accordingly, the current European Association of Urol- ogy guideline classifies patients into the following three categories (with recommended first-line regimens): (1) fit for cisplatin (GC or ddMVAC is recommended); differences in all endpoints assessed among the three regimens. Specifically, no significant difference was observed for all survival outcomes (PFS, CSS, and OS) and best overall response among the three regimens. Fur- thermore, the CR rate of dose-reduced GC (2/27, 7.4%) appeared inferior to that of full-dose GC (9/41, 22.0%) or GCa (6/37, 16.2%), albeit without a significant differ- ence (P = 0.28). Similarly, no significant difference was observed for all but one toxicological endpoint among the three regimens, whereas there were significantly fewer cases with elevated alanine aminotransferase in patients undergoing GCa compared with the concentra- tion in those receiving the other regimens (P = 0.025). A randomized phase 2 trial comparing GC and GCa in “cisplatin-fit” patients (creatinine clearance ≥ 60 mL/ min) reported an overall response rate of 49.1% for GC (CR: 14.5%; PR: 34.5%) and 40.0% for GCa (CR: 1.8%; PR: 38.2%). The authors also reported that median OS was 12.8 months and 9.8 months for GC and GCa, Sugimoto et al. Discussionh BMC Urology (2022) 22:177 Page 6 of 9 Table 3 Univariate and multivariate Cox proportional hazards regression analyses of CSS CI confidence interval, CSS cancer-specific survival, ECOG PS Eastern Cooperative Oncology Group Performance Status, eGFR estimated glomerular filtration rate, GC gemcitabine/cisplatin, GCa gemcitabine/carboplatin, HR hazard ratio Statistically significant Parameter Cutoff Univariate Multivariate HR (95% CI) P HR (95% CI) P Age (years) Continuous 1.01 (0.99–1.04) per score 0.31 Sex Male Reference 0.62 Female 0.86 (0.47–1.53) ECOG PS 0 Reference 0.019 Reference 0.013* ≥ 1 1.89 (1.09–3.17) 2.04 (1.14–3.56) eGFR (mL/min/1.73 m2) < 60 Reference 0.57 ≥ 60 1.16 (0.68–1.93) Primary site Bladder Reference Upper urinary tract 1.12 (0.65–1.96) Both 1.70 (0.79–3.42) Resection of primary site No Reference 0.0039* Reference 0.067 Yes 0.48 (0.29–0.79) 0.61 (0.36–1.03) Prior neoadjuvant/adjuvant chemotherapy No Reference 0.47 Yes 1.30 (0.60–2.51) Lymph node metastasis No Reference 0.020* Reference 0.0019* Yes 1.91 (1.13–3.39) 2.51 (1.43–4.58) Lung metastasis No Reference 0.62 Yes 0.88 (0.51–1.46) Bone metastasis No Reference 0.79 Yes 1.10 (0.52–2.07) Liver metastasis No Reference 0.021* Reference 0.0045* Yes 2.16 (1.07–3.99) 2.71 (1.30–5.23) First-line regimens Full-dose GC Reference 0.37 Dose-reduced GC 1.03 (0.52–1.95) GCa 1.45 (0.82–2.56) Table 3 Univariate and multivariate Cox proportional hazards regression analyses of CSS ate Cox proportional hazards regression analyses of CSS CI confidence interval, CSS cancer-specific survival, ECOG PS Eastern Cooperative Oncology Group Performance Status, eGFR estimated glomerular filtration rate, GC gemcitabine/cisplatin, GCa gemcitabine/carboplatin, HR hazard ratio Statistically significant in patients with eGFR < 60 mL/min/1.73 m2 [8]. More recently, Miyake et al. compared first-line full-dose GC, dose-reduced GC, and GCa in terms of the response to subsequent pembrolizumab treatment [9]. The authors reported that the response to pembrolizumab after dose-reduced GC was inferior to that after GCa in cis- platin-unfit patients with aUC and, thus, concluded that dose-reduced GC is not recommended for such patients [9]. (2) unfit for cisplatin but fit for carboplatin (GCa is recommended); and (3) unfit for any platinum-based chemotherapy (immune checkpoint inhibitors, such as pembrolizumab and atezolizumab are considered) [3]. p Our results may be inconsistent with those of the abovementioned clinical trials because the efficacy of GCa was comparable to that of GC, in our study. This dif- ference is probably because of the retrospective design, small sample size, and selection bias. Discussionh BMC Urology (2022) 22:177 Page 7 of 9 Table 4 Univariate and multivariate Cox proportional hazards regression analyses of OS CI confidence interval, ECOG PS Eastern Cooperative Oncology Group Performance Status, eGFR estimated glomerular filtration rate, GC gemcitabine/cisplatin, GCa gemcitabine/carboplatin, HR hazard ratio, OS overall survival Statistically significant Parameter Cutoff Univariate Multivariate HR (95% CI) P HR (95% CI) P Age (years) Continuous 1.02 (0.99–1.04) per score 0.19 Sex Male Reference 0.43 Female 0.78 (0.41–1.39) ECOG PS 0 Reference 0.011* Reference 0.0074* ≥ 1 1.93 (1.15–3.18) 2.09 (1.20–3.57) eGFR (mL/min/1.73 m2) < 60 Reference 0.52 ≥ 60 1.17 (0.71–1.92) Primary site Bladder Reference 0.17 Upper urinary tract 1.06 (0.63–1.82) Both 1.85 (0.91–3.57) Resection of primary site No Reference 0.0043* Reference 0.079 Yes 0.49 (0.30–0.80) 0.64 (0.38–1.05) Prior neoadjuvant/adjuvant chemotherapy No Reference 0.61 Yes 1.20 (0.55–2.31) Lymph node metastasis No Reference 0.038* Reference 0.0032* Yes 1.73 (1.05–2.95) 2.28 (1.34–4.01) Lung metastasis No Reference 0.62 Yes 0.88 (0.53–1.44) Bone metastasis No Reference 0.71 Yes 1.13 (0.56–2.84) Liver metastasis No Reference 0.014* Reference 0.0029* Yes 2.20 (1.12–3.96) 2.72 (1.35–5.09) First-line regimens Full-dose GC Reference 0.73 Dose-reduced GC 1.11 (0.59–2.03) GCa 1.25 (0.72–2.18) Table 4 Univariate and multivariate Cox proportional hazards regression analyses of OS ate Cox proportional hazards regression analyses of OS CI confidence interval, ECOG PS Eastern Cooperative Oncology Group Performance Status, eGFR estimated glomerular filtration rate, GC gemcitabine/cisplatin, GCa gemcitabine/carboplatin, HR hazard ratio, OS overall survival St ti ti ll i ifi t CI confidence interval, ECOG PS Eastern Cooperative Oncology Group Performance Status, eGFR estimated glomerular filtration rate, GC gemcitabine/cisplatin, GCa gemcitabine/carboplatin, HR hazard ratio, OS overall survival Statistically significant CI confidence interval, ECOG PS Eastern Cooperative Oncology Group Performance Status, eGFR estimated glomerular filtration rate, GC gemcitabine/cisplatin, GCa gemcitabine/carboplatin, HR hazard ratio, OS overall survival *Statistically significant assessed (PFS, CSS, and OS) even in the univariate analy- sis (Tables 2, 3, 4). Fig. 2 Best overall response results of the three regimens (full-dose GC, dose-reduced GC, and GCa). Abbreviations: CR complete response, GC gemcitabine/cisplatin, GCa gemcitabine/carboplatin, PD progressive disease, PR partial response, SD stable disease Although not assessed in the present study, gemcit- abine plus split‑dose cisplatin (“GC split”) has been another well-known option for cisplatin-unfit patients with aUC [3, 19–23]. Discussionh Nevertheless, given that GCa might have a higher CR rate than that of dose-reduced GC and that toxicities associated with GCa were similar (or lower) than those associated with dose-reduced GC, GCa might be preferable for “cispl- atin-unfit” patients, rather than dose-reduced GC. The use of GCa could avoid toxicities induced by the long- term use of cisplatin, such as neurotoxicity, ototoxic- ity, and nephrotoxicity [15]. For reference, Ichioka et al. previously reported that the oncological outcomes of dose-reduced GC were inferior to those of full-dose GC Regarding the prognostic factors, poor performance status (ECOG PS ≥ 1) and liver metastasis were shown to be independent predictors of shorter CSS and OS (Tables 3, 4), in this study. These two factors have been reported as critical prognostic factors for patients with aUC [16–18], even in the era of immune checkpoint inhibitors [18]. Our results were similar to findings in the previous literature. In contrast, the type of first-line regimen (full-dose GC, dose-reduced GC, or GCa) was not associated with all of the survival endpoints that we Sugimoto et al. Availability of data and materials B f h l h In real-world patients with aUC, there were almost no significant differences in both oncological and toxico- logical outcomes among patients receiving full-dose GC, dose-reduced GC, and GCa, unlike findings in the clini- cal trial setting. y Because of ethical restrictions, the raw data underlying this study are available from the corresponding author upon reasonable request. Author details 1 Department of Urology, Kyorin University School of Medicine, 6‑20‑2 Shinkawa, Mitaka, Tokyo 181‑8611, Japan. 2 Department of Urology, Gradu- ate School of Medicine, The University of Tokyo, 7‑3‑1 Hongo, Bunkyo‑ku, Competing interests Competing interests The authors declare that they have no competing interests. Acknowledgements We thank Jane Charbonneau, DVM, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript. Consent for publication Not applicable. Consent for publication Not applicable. Ethics approval and consent to participate This study was conducted in accordance with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards, and was approved by the IRB of the Graduate School of Medicine and Faculty of Medicine, The University of Tokyo (Approval No.: 10565) and the IRB of Kyorin University School of Medicine (Approval No.: 1262). Because of the retrospective nature of the study, the requirement for informed consent was waived by the IRB of the Graduate School of Medicine and Faculty of Medicine, The University of Tokyo and the IRB of Kyorin University School of Medicine. Discussionh Several small-scale studies have evaluated “GC split” in UC patients with impaired renal function (creatinine clearance: 40–60 ml/min) using dif- ferent split-dose schedules, all of which reported its feasi- bility and potential efficacy [19–23]. Nevertheless, given the study design of these previous studies (a small phase I/II trial or retrospective study), prospective randomized trials comparing “GC split” with conventional GC are warranted to validate this modified regimen [3]. i The limitations of this study are the retrospective design, small sample size, and selection bias, as stated above. Additionally, owing to the multi-institutional ret- rospective study design, the dose reduction rate of cispl- atin was not uniform. Nevertheless, this study might add Fig. 2 Best overall response results of the three regimens (full-dose GC, dose-reduced GC, and GCa). Abbreviations: CR complete response, GC gemcitabine/cisplatin, GCa gemcitabine/carboplatin, PD progressive disease, PR partial response, SD stable disease Sugimoto et al. BMC Urology (2022) 22:177 Page 8 of 9 Fig. 3 Comprehensive adverse event profiles among the three regimens (full-dose GC, dose-reduced GC, and GCa). The analysis was conducted for 97 patients with sufficient data (GCa, n = 32; full-dose GC, n = 41; dose-reduced GC, n = 24). Abbreviations: ALT alanine aminotransferase, AST aspartate aminotransferase, GC gemcitabine/cisplatin, GCa gemcitabine/carboplatin mong the three regimens (full-dose GC, dose-reduced GC, and GCa). The analysis was conducted full-dose GC, n = 41; dose-reduced GC, n = 24). Abbreviations: ALT alanine aminotransferase, AST platin GCa gemcitabine/carboplatin mong the three regimens (full-dose GC, dose-reduced i Fig. 3 Comprehensive adverse event profiles among the three regimens (full-dose GC, dose-reduced GC, and GCa). The analysis was conducted for 97 patients with sufficient data (GCa, n = 32; full-dose GC, n = 41; dose-reduced GC, n = 24). Abbreviations: ALT alanine aminotransferase, AST aspartate aminotransferase, GC gemcitabine/cisplatin, GCa gemcitabine/carboplatin HK supervised the study, helped draft the manuscript, and were involved in revising the manuscript critically for important intellectual content. All authors read and approved the final manuscript. additional evidence of the real-world outcomes of the commonly used regimens in patients eligible for plati- num-based chemotherapy. Abbreviations UC Ad d aUC: Advanced urothelial carcinoma; CR: Complete response; CSS: Cancer- specific survival; CTCAE: Common Terminology Criteria for Adverse events; ddMVAC: Dose-dense methotrexate/vinblastine/doxorubicin/cisplatin; ECOG PS: Eastern Cooperative Oncology Group Performance Status; eGFR: Estimated glomerular filtration rate; GC: Gemcitabine/cisplatin; GCa: Gemcitabine/ carboplatin; IQR: Interquartile range; IRB: Institutional Review Board; M-CAVI: Methotrexate/carboplatin/vinblastine; OS: Overall survival; PFS: Progression- free survival; PR: Partial response; RECIST: Response evaluation criteria in solid tumours. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 5. Dash A, Galsky MD, Vickers AJ, et al. Impact of renal impairment on eligibility for adjuvant cisplatin-based chemotherapy in patients with urothelial carcinoma of the bladder. Cancer. 2006;107:506–13. 5. Dash A, Galsky MD, Vickers AJ, et al. Impact of renal impairment on eligibility for adjuvant cisplatin-based chemotherapy in patients with urothelial carcinoma of the bladder. Cancer. 2006;107:506–13. 6. Galsky MD, Hahn NM, Rosenberg J, et al. Treatment of patients with metastatic urothelial cancer “unfit” for Cisplatin-based chemotherapy. J Clin Oncol. 2011;29:2432–8. 6. Galsky MD, Hahn NM, Rosenberg J, et al. Treatment of patients with metastatic urothelial cancer “unfit” for Cisplatin-based chemotherapy. J Clin Oncol. 2011;29:2432–8. 7. Dogliotti L, Cartenì G, Siena S, et al. Gemcitabine plus cisplatin versus gemcitabine plus carboplatin as first-line chemotherapy in advanced transitional cell carcinoma of the urothelium: results of a randomized phase 2 trial. Eur Urol. 2007;52:134–41. p 8. Ichioka D, Miyazaki J, Inoue T, et al. Impact of renal function of patients with advanced urothelial cancer on eligibility for first-line chemotherapy and treatment outcomes. Jpn J Clin Oncol. 2015;45:867–73. 8. Ichioka D, Miyazaki J, Inoue T, et al. Impact of renal function of patients with advanced urothelial cancer on eligibility for first-line chemotherapy and treatment outcomes. Jpn J Clin Oncol. 2015;45:867–73. 9. Miyake M, Shimizu T, Nishimura N, et al. Response to pembrolizumab after dose-reduced cisplatin plus gemcitabine chemotherapy is inferior to that after carboplatin plus gemcitabine chemotherapy in cisplatin- unfit patients with advanced urothelial carcinoma. Clin Genitourin Cancer. 2022;20(196):e1-9. 9. Miyake M, Shimizu T, Nishimura N, et al. Response to pembrolizumab after dose-reduced cisplatin plus gemcitabine chemotherapy is inferior to that after carboplatin plus gemcitabine chemotherapy in cisplatin- unfit patients with advanced urothelial carcinoma. Clin Genitourin Cancer. 2022;20(196):e1-9. 10. Schwartz LH, Litière S, de Vries E, et al. RECIST 1.1-Update and clarification: from the RECIST committee. Eur J Cancer. 2016;62:132–7. 11. National Cancer Institute. Common Terminology Criteria for Adverse Events (CTCAE) v5.0. https://ctep.cancer.gov/protocolDevelopment/elect ronic_applications/ctc.htm#ctc_50. Accessed 9 Apr 2022. 12. Matsuo S, Imai E, Horio M, et al. Collaborators developing the Japanese equation for estimated GFR. Revised equations for estimated GFR from serum creatinine in Japan. Am J Kidney Dis. 2009;53:982–92. 13. De Santis M, Bellmunt J, Mead G, et al. References 21. Morales-Barrera R, Bellmunt J, Suárez C, et al. Cisplatin and gemcitabine administered every two weeks in patients with locally advanced or meta- static urothelial carcinoma and impaired renal function. Eur J Cancer. 2012;48:1816–21. 1. von der Maase H, Hansen SW, Roberts JT, et al. Gemcitabine and cisplatin versus methotrexate, vinblastine, doxorubicin, and cisplatin in advanced or metastatic bladder cancer: results of a large, randomized, multina- tional, multicenter, phase III study. J Clin Oncol. 2000;18:3068–77. 1. von der Maase H, Hansen SW, Roberts JT, et al. Gemcitabine and cisplatin versus methotrexate, vinblastine, doxorubicin, and cisplatin in advanced or metastatic bladder cancer: results of a large, randomized, multina- tional, multicenter, phase III study. J Clin Oncol. 2000;18:3068–77. 22. Kim YR, Lee JL, You D, et al. Gemcitabine plus split-dose cisplatin could be a promising alternative to gemcitabine plus carboplatin for cisplatin- unfit patients with advanced urothelial carcinoma. Cancer Chemother Pharmacol. 2015;76:141–53. 2. von der Maase H, Sengelov L, Roberts JT, et al. Long-term survival results of a randomized trial comparing gemcitabine plus cisplatin, with metho- trexate, vinblastine, doxorubicin, plus cisplatin in patients with bladder cancer. J Clin Oncol. 2005;23:4602–8. 2. von der Maase H, Sengelov L, Roberts JT, et al. Long-term survival results of a randomized trial comparing gemcitabine plus cisplatin, with metho- trexate, vinblastine, doxorubicin, plus cisplatin in patients with bladder cancer. J Clin Oncol. 2005;23:4602–8. 23. Izumi K, Iwamoto H, Yaegashi H, et al. Gemcitabine plus cisplatin split versus gemcitabine plus carboplatin for advanced urothelial cancer with cisplatin-unfit renal function. In Vivo. 2019;33:167–72. 23. Izumi K, Iwamoto H, Yaegashi H, et al. Gemcitabine plus cisplatin split versus gemcitabine plus carboplatin for advanced urothelial cancer with cisplatin-unfit renal function. In Vivo. 2019;33:167–72. 3. Cathomas R, Lorch A, Bruins HM, et al. EAU Muscle-invasive, Metastatic Bladder Cancer Guidelines Panel. The 2021 Updated European Associa- tion of Urology Guidelines on Metastatic Urothelial Carcinoma. Eur Urol. 2022;81:95–103. 3. Cathomas R, Lorch A, Bruins HM, et al. EAU Muscle-invasive, Metastatic Bladder Cancer Guidelines Panel. The 2021 Updated European Associa- tion of Urology Guidelines on Metastatic Urothelial Carcinoma. Eur Urol. 2022;81:95–103. 4. Flaig TW, Spiess PE, Agarwal N, et al. Bladder Cancer, Version 3.2020, NCCN Clinical Practice Guidelines in Oncology. J Natl Compr Canc Netw. 2020;18:329–54. 4. Flaig TW, Spiess PE, Agarwal N, et al. Bladder Cancer, Version 3.2020, NCCN Clinical Practice Guidelines in Oncology. J Natl Compr Canc Netw. 2020;18:329–54. Received: 21 June 2022 Accepted: 31 October 2022 20. Hussain SA, Palmer DH, Lloyd B, et al. A study of split-dose cisplatin-based neo-adjuvant chemotherapy in muscle-invasive bladder cancer. Oncol Lett. 2012;3:855–9. Author contributions KS d ST b d KS and ST contributed to the conception and design of the study, analysis and interpretation of the data, and drafted the first manuscript. KK, TK, KM, YN, MK, MT, JM, YA, YY, YS, and DY contributed to acquisition of the data. TN, HF, and Page 9 of 9 Sugimoto et al. BMC Urology (2022) 22:177 Sugimoto et al. BMC Urology (2022) 22:177 Sugimoto et al. BMC Urology (2022) 22:177 Tokyo 113‑8655, Japan. 3 Department of Urology, Teikyo University School of Medicine, 2‑11‑1 Kaga, Itabashi‑ku, Tokyo 173‑8605, Japan. with pembrolizumab: a multicenter retrospective study. Sci Rep. 2021;11:15623. 19. Hussain SA, Stocken DD, Riley P, et al. A phase I/II study of gemcitabine and fractionated cisplatin in an outpatient setting using a 21-day sched- ule in patients with advanced and metastatic bladder cancer. Br J Cancer. 2004;91:844–9. Received: 21 June 2022 Accepted: 31 October 2022 • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: Publisher’s Note Randomized phase II/III trial assess- ing gemcitabine/carboplatin and methotrexate/carboplatin/vinblastine in patients with advanced urothelial cancer “unfit” for cisplatin-based chemotherapy: phase II—results of EORTC study 30986. J Clin Oncol. 2009;27:5634–9. 14. De Santis M, Bellmunt J, Mead G, et al. Randomized phase II/III trial assess- ing gemcitabine/carboplatin and methotrexate/carboplatin/vinblastine in patients with advanced urothelial cancer who are unfit for cisplatin- based chemotherapy: EORTC study 30986. J Clin Oncol. 2012;30:191–9. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: 15. Pabla N, Dong Z. Cisplatin nephrotoxicity: mechanisms and renoprotec- tive strategies. Kidney Int. 2008;73:994–1007. 16. Taguchi S, Nakagawa T, Hattori M, et al. Prognostic factors for metastatic urothelial carcinoma undergoing cisplatin-based salvage chemotherapy. Jpn J Clin Oncol. 2013;43:923–8. 17. Taguchi S, Nakagawa T, Matsumoto A, et al. Pretreatment neutrophil-to- lymphocyte ratio as an independent predictor of survival in patients with metastatic urothelial carcinoma: a multi-institutional study. Int J Urol. 2015;22:638–43. 17. Taguchi S, Nakagawa T, Matsumoto A, et al. Pretreatment neutrophil-to- lymphocyte ratio as an independent predictor of survival in patients with metastatic urothelial carcinoma: a multi-institutional study. Int J Urol. 2015;22:638–43. 18. Taguchi S, Kawai T, Nakagawa T, et al. Prognostic significance of the albumin-to-globulin ratio for advanced urothelial carcinoma treated 18. Taguchi S, Kawai T, Nakagawa T, et al. Prognostic significance of the albumin-to-globulin ratio for advanced urothelial carcinoma treated
https://openalex.org/W3153748153	https://www.e3s-conferences.org/articles/e3sconf/pdf/2021/24/e3sconf_caes2021_01011.pdf	English	null	Gas Steady-state Diffusion in Fractal Porous Media	E3S web of conferences	2,021	cc-by	3,023	1 Introduction media model. Discrete model is more advanced than continuous model. However, there is still a large gap between pore structure of its multi-porous mesosities and that of real porous mesosities (such as coal). In addition, the simulation of gas molecular movement in the hole is a pure mathematical simulation method, which cannot reflect the free movement law of molecules in physical essence. Cao and He proposed a gas diffusion model for fractal porous media based on molecular motion theory [3]. In the field of energy and chemical industry, there are many gas-solid reactions such as coal combustion and limestone desulfurization. Coal and limestone are typical porous media and their pore structure has fractal characteristics[1]. These porous media act as reactants or carriers. The chemical reaction between gas and porous solid is controlled by gas diffusion in the pores. Gas diffusion process in the pore has a great influence on the reaction. Classical gas-solid reaction models such as random hole model and distributed hole model are described by the traditional diffusion law (Fick's diffusion and Knudsen's diffusion). However, it is found through research that the traditional diffusion law is not applicable to gas diffusion in the fractal hole [2]. Therefore, the study of gas diffusion in fractal porous media has important engineering application value and significance. The diffusion model proposed by Cao and He is improved in this paper. First, a random walk method is used to generate a fractal pore model of 400×400×400 grids, and the pore structure is described by porosity, average pore size, and fractal dimension. Then based on the theory of molecular motion of gas, the governing equation of molecular motion is established according to four different situations of pore structure. Through a large number of numerical simulations, it is found that gas diffusion in fractal porous media is strongly affected by the pore structure. Finally, the expression of steady- state diffusion coefficient is obtained. A lot of experiments are needed to obtain the general steady-state diffusion law of fractal porous media. The experiment of gas diffusion in fractal porous media is difficult. The numerical model study has the advantage in this respect. The steady-state diffusion law of gas in fractal porous media can be obtained by a lot of numerical simulation. At present, fractal porous media diffusion models can be divided into continuous model and discrete model. Gas Steady-state Diffusion in Fractal Porous Media Du Zhehua1 1Wuhan Second Ship Design and Research Institute, 430205 Wuhan, China Du Zhehua1 1Wuhan Second Ship Design and Research Institute, 430205 Wuhan, China Abstract. Gas diffusion in fractal pores does not follow the classic Fick’s and Knudsen’s laws, so more research on gas diffusion in fractal porous media is needed. Fractal pore models are generated using the random walk method. The gas diffusion governing equations for the fractal pores are derived from the classic kineti theory of gases. The gas diffusion model is used to study the gas diffusion in fractal porous meida and to determine steady-state diffusion coefficient formulas. The results show that the diffusion coefficient is proportional to the mean proe diameter, porosity, and the exponetial function of the fractal dimension in the Knudsen diffusion regime. The diffusion coefficient is not only related to the three pore parameters but is also related to the molecular mean free path in the configurational diffusion regime. © 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/). * Corresponding author: shunli878@163.com 1 Introduction Continuous model such as dust-gas model and random pore model assume that the porous medium is a continuum with uniform pore structure. However, continuous model cannot reflect the fractal characteristics of pore structure in porous media and is modified with empirical parameters, so continuous model cannot reflect the physical dispersion overpass of real material. With the development of computer, discrete model regards porous media model as composed of discrete grids and adopts Monte-Carlo square method to simulate the movement of gas molecules in the porous https://doi.org/10.1051/e3sconf/202124801011 https://doi.org/10.1051/e3sconf/202124801011 E3S Web of Conferences 248, 01011 (2021) CAES 2021 DP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 es/by/4.0/). 2.1 Fractal hole model The random walk method proposed by Liang et al. [4] is used to generate a porous medium model with fractal characteristics. The seeds (hole grids) are randomly distributed in 400 × 400 × 400 discrete cube grids (solid grids). The fractal pore models with different pore structures can be generated by adjusting the number of seeds and threshold values of random walk. Since pore structure is very complex and has fractal characteristics, it is necessary to comprehensively describe pore connectivity characteristics, pore diameter distribution, E3S Web of Conferences 248, 01011 (2021) CAES 2021 E3S Web of Conferences 248, 01011 (2021) CA S 2021 https://doi.org/10.1051/e3sconf/202124801011 max min 1 1 2 1 ( , , ) ( , , ) (1 ) 6 i h i h i dN i j k N h j k v dt L min 1 min ( , , ) (1 ) 6 i i N i j k v L m 1 max ( , , ) (1 ) 6 ax i i N i j k v L ( , , ) (1 ) (1 )(2 ) 6 N i j k v L (4) pore volume, and pore surface area. Porosity, average pore diameter, and fractal dimension are used to describe the pore structure characteristics, which can more fully describe the above pore characteristics. The porosity ε is the ratio of pore volume to the total volume of particles. The average pore diameter dm is 4 times the ratio of pore volume to pore area. The fractal dimension is defined in the following formula. In the formula, Df is fractal dimension, r is the pore diameter of multi-porous medium, and S(r) is the pore area corresponding to the pore size of porous medium at pore size r. (4) If (i, j, k) is directly connected to the outside large space along the right side of direction i, while the left side is connected to a solid grid, the number of molecules increased in the grid of (i, j, k) in unit time is shown in the following equation. ln( ( ) / ) ln f dS r dr D r (1) (1) 2.2 Governing equation of gas molecule diffusion max min min 1 1 2 2 2 1 ( , , ) ( , , ) (1 ) ( ) 6 i h i i h i h i dN i j k N h j k v dt L max max min 1 2 2 max ( , , ) (1 )( ) 6 i i i i i N i j k v L ( , , ) (1 ) (1 )(2 ) 6 N i j k v L (5) It is assumed that side length of a unit cube grid is L, the number of gas molecules in a certain grid is N0, molecular mean free path is λ, and molecular average velocity is v. According to molecular motion theory, the number of N molecules whose free path is greater than x in N0 molecules can be deduced. (5) 0 x N N e (2) If (i, j, k) is directly connected to the outside large space along the left side of direction i, and the right side is connected to the solid grid, the number of molecules increased in the grid (i, j, k) in unit time is shown in the following equation. (2) Then, the number of molecules that escape from a certain surface of cube grid in unit time without collision between molecules is the following equation. 2.2 Governing equation of gas molecule diffusion 0 (1 ) 6 L N dN v e L (3) max max min 1 1 2 2 2 1 ( , , ) ( , , ) (1 ) ( ) 6 i h i i i h h i dN i j k N h j k v dt L max min min 2 2 1 max ( , , ) (1 )( ) 6 i i i i i N i j k v L ( , , ) (1 ) (1 )(2 ) 6 N i j k v L (6) (3) Based on equation (3), according to the connectivity of hole grid (i, j, k) along the direction i, it can be divided into 4 situations as shown in Fig. 1 (in the figure, white is the hole grid and black is the solid grid). (6) N(imin,j,k) N(imin+1,j,k) N(i-1,j,k) N(i,j,k) N(i+1,j,k) N(imax-1,j,k) N(imax,j,k) (a) Situtation 1 N(imin,j,k) N(imin+1,j,k) N(i-1,j,k) N(i,j,k) N(i+1,j,k) N(imax-1,j,k) N(imax,j,k) (b) Situtation 2 N(imin,j,k) N(imin+1,j,k) N(i-1,j,k) N(i,j,k) N(i+1,j,k) N(imax-1,j,k) N(imax,j,k) (c) Situtation 3 N(imin,j,k) N(imin+1,j,k) N(i-1,j,k) N(i,j,k) N(i+1,j,k) N(imax-1,j,k) N(imax,j,k) (d) Situtation 4 Fig. 1. Four situations for establishing diffusion governing equations in discrete grids If (i, j, k) is connected to the solid grid along the left and right sides of the direction i, the number of molecules increased in the grid of (i, j, k) in unit time is shown in the following equation. If (i, j, k) is connected to the solid grid along the left and right sides of the direction i, the number of molecules increased in the grid of (i, j, k) in unit time is shown in the following equation. 2.2 Governing equation of gas molecule diffusion max min 1 1 2 1 ( , , ) ( , , ) (1 ) 6 i h i h i dN i j k N h j k v dt L max min 2 2( 1) ( , , ) (1 ) (1 ) (1 )(2 ) 6 1 i i N i j k v L max max min max min 1 2 2 3 2 2 3 ( , , ) ( i i h i i i i h i N i j k v ( min,j, ) ( min ,j, ) ( ,j, ) ( ,j, ) ( ,j, ) ( max ,j, ) ( max,j, ) (b) Situtation 2 N(imin,j,k) N(imin+1,j,k) N(i-1,j,k) N(i,j,k) N(i+1,j,k) N(imax-1,j,k) N(imax,j,k) (c) Situtation 3 N(imin,j,k) N(imin+1,j,k) N(i-1,j,k) N(i,j,k) N(i+1,j,k) N(imax-1,j,k) N(imax,j,k) (d) Situtation 4 Fig. 1. Four situations for establishing diffusion governing equations in discrete grids (d) Situtation 4 Fig. 1. Four situations for establishing diffusion governing equations in discrete grids max min 2 2 2 2 ) i i h i h i (7) (7) The above is diffusion governing equation in the i direction. Similarly, the diffusion governing equations in the j direction and k direction can be derived. The governing equation of gas molecular diffusion is established from the basic physical principles according to the specific situation of pore structure, so the diffusion model can reflect the diffusion mechanism. The governing equations of molecular diffusion are established respectively, and the specific process is as follows. If (i, j, k) is directly connected to external large space along direction i, the number of molecules added in the (i, j, k) grid per unit time is as follows. 2 E3S Web of Conferences 248, 01011 (2021) CAES 2021 https://doi.org/10.1051/e3sconf/202124801011 3.2 Influence of pore structure Due to the different diffusion mechanism in different diffusion zones, the influence of pore structure on diffusion coefficient is different, so it is necessary to study the influence of pore structure on diffusion coefficient in sections. In Knudsen diffusion region, diffusion coefficient is proportional to the average aperture. In this paper, when L < 5nm or the ratio of average pore diameter to molecular average free path DM / λ < 0.5, diffusion coefficient in the pore is proportional to the average pore diameter, which can be approximately considered as the diffusion in Knudsen region. In this paper, nitrogen diffusion process of the above 10 groups of porous models at 293K is simulated, and diffusion coefficients of the porous models at L = 1nm - 3nm are calculated. Figure 3 shows the relationship between diffusion coefficient of Knudsen area and the average pore diameter and porosity. The result shows that diffusion coefficient is proportional to the product of the average pore size and porosity. Diffusion coefficient formula of Knudsen region can be obtained by fitting calculated data. Gas diffusion in porous media can be divided into Knudsen diffusion region, main diffusion region and transition region. Gas diffusion of porous media commonly used in engineering mainly occurs in Knudsen diffusion zone and transition zone, so this paper only studies diffusion coefficients of these two zones. Since three parameters are used to comprehensively describe pore structure and its influence on pore diffusion, it is difficult to intuitively study combined effects of three parameters. To solve this problem, the influence of average pore size on diffusion coefficient can be studied under the condition of constant porosity and fractal dimension by increasing grid size. 3.39 2.42 2 f D m v D d e (9) 3.39 2.42 2 f D m v D d e (9) Fig. 3 Influence of average pore size and porosity on diffusion coefficient in Knudsen region 3 Calculation results and discussion In the formula, D is steady-state diffusion coefficient, T is diffusion temperature, and M is molar mass of diffuse gas. In Knudsen zone and transition zone, diffusion coefficient is proportional to average molecular velocity, which is consistent with theoretical analysis and existing experimental results [5]. The diffusion model in this paper is used to simulate gas diffusion in 10 groups of fractal hole models under different diffusion gas and diffusion temperature conditions. The simulated boundary conditions are as follows: one end of gas molar concentration is 41.6 mol/m3, the other end is 20.8 mol/m3. The pore structure parameters of 10-component shaped hole model with unit mesh size L = 1nm are listed in Table 1. Fig. 2 Influence of diffusion gas and temperature on diffusion coefficient (Group 10) Table 1. Pore structure parameters of fractal hole model (L = 1nm) Table 1. Pore structure parameters of fractal hole model (L = 1nm) Serial number ε /͖ dm /nm Df Group 1 31.41 7.96 2.42 Group 2 34.43 8.36 2.01 Group 3 40.09 6.99 2.15 Group 4 46.36 7.07 2.13 Group 5 47.38 7.22 1.99 Group 6 52.57 6.91 1.85 Group 7 57.11 7.05 1.64 Group 8 60.28 6.75 1.71 Group 9 62.04 6.04 1.60 Group 10 68.44 5.85 1.48 Fig. 2 Influence of diffusion gas and temperature on diffusion coefficient (Group 10) 4 Conclusion Gas diffusion coefficient is proportional to the average velocity of gas molecules. In Knudsen diffusion region, the diffusion is obviously affected by pore structure. Under the same fractal dimension, diffusion coefficient is proportional to the product of mean pore size and porosity, but independent of the mean free path of molecule. In transit region, diffusion coefficient is not only related to pore structure parameters, but also related to molecular mean free path. Under different ratio of average pore diameter to average molecular free path, pore structure has different effect on the diffusion rate. Acknowledgments This work was supported by the National Key Research and Development Program漏No.2017YFC0307800漐. 3.1 Influence of diffusion gas and temperature (9) 2 f m D d e (9) The mean free path λ of simulated diffusion molecules is about 90nm. Then for the 10 groups of hole models shown in Table 1, when L=2nm and L=50nm, the diffusion can be considered to be in Knudsen zone and transition zone, respectively. The diffusion of hydrogen, helium, neon, nitrogen, oxygen, and carbon dioxide in L=2nm and L=50nm pore models at 293K and nitrogen at 293K, 343K, 393K, 433K, 493K are calculated. Figure 2 shows the relationship between diffusion coefficient of Group10 hole model at L=2nm and L=50nm, temperature and molar mass of diffusion gas. The result shows that the relationship between diffusion coefficient and molar mass and temperature of diffused gas is as follows. Fig. 3 Influence of average pore size and porosity on diffusion coefficient in Knudsen region / D T M / D T M (8) 3 E3S Web of Conferences 248, 01011 (2021) CAES 2021 https://doi.org/10.1051/e3sconf/202124801011 When 0.5 < dm / λ<10, diffusion is in the transition zone. Similarly, diffusion coefficient formula of transition zone can be calculated by simulating the diffusion of pore model with L=5nm~100nm. Figure 4 shows the relationship between diffusion coefficient of transition zone and the average pore size, which can be approximated by an exponential function. Fig. 4 Influence of average pore size on diffusion coefficient in transition zone Fig. 4 Influence of average pore size on diffusion coefficient in transition zone References 1. A.K. Katz, A.O. Thompson, Physical Review Letters, 54, 3558 (1985) 1. A.K. Katz, A.O. Thompson, Physical Review Letters, 54, 3558 (1985) 2. M. Costa, A. Araujo, Physical Review E, 67, 1296 (2003) 3. L. Cao, R. He, Combust Sci. and Tech., 182, 822 (2010) 4. Z. Liang, R. He, Combust Sci. and Tech., 179, 637 (2007) 5. R. He, X.C. Xu, C.H. Chen, Fuel, 77, 1291 (1998) 5. R. He, X.C. Xu, C.H. Chen, Fuel, 77, 1291 (1998) 4 4
https://openalex.org/W4289816505	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/EA10713097C4DA1801461EE501006660/S1369415422000176a.pdf/div-class-title-sustaining-the-individual-in-the-collective-a-kantian-perspective-for-a-sustainable-world-div.pdf	English	null	Sustaining the Individual in the Collective: A Kantian Perspective for a Sustainable World	Kantian review	2,022	cc-by	9,946	© The Author(s), 2022. Published by Cambridge University Press on behalf of Kantian Review. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (https://creativecommons.org/ licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. Sustaining the Individual in the Collective: A Kantian Perspective for a Sustainable World Zachary Vereb University of Mississippi, University, MS, USA Email: ztvereb@olemiss.edu Zachary Vereb University of Mississippi, University, MS, USA Email: ztvereb@olemiss.edu Kantian Review (2022), 27, 405–420 doi:10.1017/S1369415422000176 Kantian Review (2022), 27, 405–420 doi:10.1017/S1369415422000176 Abstract Individualist normative theories appear inadequate for the complex moral challenges of cli- mate change. In climate ethics, this is especially notable with the relative marginalization of Kant. I argue that Kant’s philosophy, understood through its historical and cosmopolitan dimensions, has untapped potential for the climate crisis. First, I situate Kant in climate ethics and evaluate his marginalization due to perceived individualism, interiority and anthropocen- trism. Then, I explore aspects of Kant’s historical and cosmopolitan writings, which present a global, future-orientated picture of humanity. Ultimately, Kant’s philosophy offers a unique take on the climate deadlock capable of sustaining the individual in the collective. Keywords: Kant; climate change; individualism; history; progress https://doi.org/10.1017/S1369415422000176 Published online by Cambridge University Press 1. Introduction Individualist normative theories appear inadequate for the collective challenges of climate change. The problematization of individualist thinking in climate ethics lit- erature is especially notable with the relative marginalization of Kant’s philosophy.1 Kant’s perceived individualism (along with his non-consequentialism, anthropocen- trism and ‘interiorism’) make him appear to be a bad resource for normative engage- ment with climate change.2 However, this article argues that Kant should not only be part of the philosophical conversation regarding sustainability, but that he has untapped potential for resolution of normative climate deadlocks. I believe that the more usual view of Kant’s philosophy is in fact compatible with several goals in climate ethics. Nonetheless, this more usual approach still falls short with regard to the aforementioned collective dimension. I argue that a consideration of Kant’s historical, religious and cosmopolitan writings provide us with a less individu- alist picture of humanity that requires considering individuals, institutions and nations as part of the ongoing plight of the human species’ progress toward perfection.3 By reconciling the worth of individuals as part of and alongside the transtemporal human species (or regulative ‘macro agent’ as I will call it), this perspective provides an alter- native lens of analysis for climate ethics capable of superseding the usual cost-benefit Zachary Vereb 406 approach there, and the narrow short-termism common to individualist theories. I illustrate this, at the end, with geoengineering as a case example. Just like individual agents, ‘humanity writ-large’ has duties regarding nature and toward itself, and these are relevant for rethinking climate change. A main obstacle blocking entry into climate ethics – namely, Kant’s perceived individualism – is thus side-stepped. At the same time, revealing this alternative pathway is a helpful step toward showing commentators that other emerging literature on Kant’s political, historical and legal philosophy matters. Section 2 begins by situating Kant’s environmental reception, with reflections on why he has been overlooked. Section 3 suggests, in agreement with other commen- tators, that Kant’s philosophy is still compatible with certain environmental aims. Section 4 then explores Kant’s philosophy of history and regulative holism. These help overcome problems noted in section 2 and limitations noted in section 3. Section 5 concludes by addressing objections to this Kantian-inspired reading, with a consideration of global geoengineering. 1. Introduction One liability of geoengineering technologies includes the negligent disregard for the well-being of moral agents who have contrib- uted the least to the problem at hand, such as those in the Global South. 2.1 Early critics of Kant in environmental ethics Holmes Rolston III arguably sets the stage for Kant’s marginalization.4 Long before climate ethics, Rolston argued that environmental ethics requires a collective para- digm shift (1988: 137–59), and Kant’s thought is seen to be constitutive of the old par- adigm. Even ‘greener’ Kantian positions are deemed inadequate for collective responsibility (Rolston 1988: 146). Citing the following passage in Kant’s Anthropology, Rolston rejects his philosophy: The fact that the human being can have the ‘I’ in his representations raises him infinitely above all other living beings on earth. Because of this he is a person, and by virtue of the unity of consciousness through all changes that happen to him, one and the same person – i.e., through rank and dignity an entirely dif- ferent being from things, such as irrational animals, with which one can do as one likes. (Anth, 7: 127) For Rolston, this passage is emblematic of a human-centred tradition with little con- cern for environmental issues. His reaction is unsurprising, given the oft-cited Kantian claim of human superiority (Anth, 7: 127; CPJ, 5: 431–5; CB, 8: 114). There is, of course, the question as to whether this remark generalizes to all of Kant’s phi- losophy, which Rolston assumes.5 Supposing it does, there remains the different ques- tion as to whether an anthropocentric Kantian philosophy inspired by this passage might still not have value for human-related climate concerns. 2. Challenges of climate change Climate ethicists have been tasked to marshall the tools of philosophy to tackle novel challenges. First, climate change is the biggest collective-action problem facing humanity. Individual emission reduction, though admirable, does little on its own. Consequently, many succumb to ‘climate nihilism’ since the problem demands too much of single agents. Second, there are systemic political deadlocks: regulatory cap- ture, or the failure of governance via corporate capturing of politicians and regula- tory agencies, precludes the adoption of sustainable policies and institutions aimed at the public good. Third, climate impacts are indirect and dispersed: from, for example, global warming to drought, and water insecurity to refugee crises and war. This indi- rect and abstract problem short-circuits our usual normative machinery (Garvey 2008; Gardiner 2011). Finally, we require global, collective responses, but are not very good at thinking internationally or long term. Yet humanity finds itself at a fork in the road: In our view, the evidence from tipping points alone suggests that we are in a state of planetary emergency: both the risk and urgency of the situation are acute. We argue that the intervention time left to prevent tipping could already have shrunk towards zero, whereas the reaction time to achieve net zero emissions is 30 years at best. Hence we might already have lost control of whether tip- ping happens. A saving grace is that the rate at which damage accumulates from tipping – and hence the risk posed – could still be under our control to some extent. The stability and resilience of our planet is in peril. International action – not just words – must reflect this. (Lenton et al. 2019) https://doi.org/10.1017/S1369415422000176 Published online by Cambridge University Press 407 Kantian Review Kantian Review Before discussing how Kant might help us, the next section considers why Kant’s his- torical marginalization has seemed to make sense to so many environmental critics. Before discussing how Kant might help us, the next section considers why Kant’s his- torical marginalization has seemed to make sense to so many environmental critics. https://doi.org/10.1017/S1369415422000176 Published online by Cambridge University Press 2.2 Critics of Kant in climate ethics In his book on climate change, Jamieson’s single paragraph on Kant is telling: Kant is dismissed outright, since he is incapable of helping with collective action problems (Jamieson 2014: 173). Little detail is given to defend this claim or how in fact he inter- prets the categorical imperative. Some Kant commentators have begun to take this avenue seriously (Williams 2019, Vereb 2021). Elsewhere, Jamieson highlights what he sees as the two defining features of Kantian ethics: ‘its individualism, and its emphasis on the interior’ (Jamieson 2007: 161). For him, Kantians have little resources to include ‘global environmental change’ as a problem, and do not appreciate ‘that the business of morality is to bring something about’ (Jamieson 2007: 161). He goes on: Some Kantian philosophers have tried to overcome the theory’s individualism but this is difficult since these two features are closely related : : : if our pri- mary concern is how we should act in the face of global environmental change, then we need a theory that is seriously concerned with what people bring about, rather than a theory that is (as we might say) ‘obsessed’ with the purity of the will. (Jamieson 2007: 161) It is curious to note that Jameson seems unaware of (or unwilling to engage) relevant work on Kant’s political philosophy and legal theory (Williams 1983, Ripstein 2009). Several commentators have in fact pursued alternative routes to begin reflecting on climate change (Ataner 2012, Bernstein 2019, Pinheiro Walla 2020). It is also puzzling that Jamieson assumes that emphasis on being motivated by duty precludes actions that could change the world.7 Climate ethicists tend to analyse the climate crisis in two ways: legality and moral- ity. With regard to the former, the crisis is framed in terms of conflicts of interest. With regard to the latter, it is framed in terms of fairness. In either case, classic con- cepts such as liberty and rights settle the foreground, and these concepts are best understood in post-Kantian frameworks. Of course, Kant’s philosophy has been, in part, an inspiration for such approaches. We see this best in the Rawlsian appropria- tion of Kant’s ethical theory, which has in many ways become a paradigm for liberal thought more generally.8 But Kant’s ethical thought is not simply about rights, indi- vidual agents and liberty. It also encompasses positive duties, collective ends, univer- sal limits and constraints on choice (Wood 1999: 316). 2.2 Critics of Kant in climate ethics Even if Kant’s philosophy is problematic for environmental ethics, it still might be useful for climate ethics, for climate ethics often focuses on human concerns and does not typically employ non-anthropocentric arguments. In practice, however, Kant’s early marginalization in environmental ethics left scars.6 The historical and concep- tual connections between environmental and climate ethics, and the relative margin- alization of Kant in the former, prefigure the implicit marginalization of Kant in the latter. In any case, it will not be hard to see why both schools might assume Kant’s philosophy to be a lost cause. p p y Like Rolston, James Garvey is dubious of the value of Kant’s ethics for environmen- tal philosophy, since he is beholden to the anthropocentric canon from Aristotle to Descartes (Garvey 2008: 52). Further, Garvey claims that ‘environmental ethics is largely in the business of expanding our conception of value or at least the number of things that we value’ (p. 51). Kant does not seem to allow for this, given his ratio- centrism, and even non-anthropocentric deontologies are criticized for their inade- quacy for collective problems (pp. 53–5). Garvey does pause on the possibility of conceiving the categorical imperative as a test for sustainability, but then moves quickly on to utilitarianism (pp. 149–50). John Broome assumes utilitarianism as a ‘default’ approach for climate change (Broome 2012: 114). Kantianism is not Zachary Vereb 408 mentioned once in his influential work. If speculation is in order, perhaps the per- ceived individualism makes Kant into a conceptual dead-end here. Henry Shue raises such a complaint: Kant is an apparent obstacle for an intergenerational and global principle of equity (Shue 2010: 104). Kantianism thus seems inadequately limited to present-generation individuals. Stephen Gardiner’s pivotal work includes an extended discussion of utilitarianism as defended by Dale Jamieson (Gardiner 2011: 235–42). Gardiner does not, however, consider Kantianism worthy of serious debate. For he believes the central concerns raised by climate change are collective and intergenerational, not individualist and maxim-centric. Interestingly, Gardiner does suggest, albeit only briefly, that gener- ations could be accounted for by an interconnected conception of humanity. He then specifically references Kant’s kingdom of ends (p. 157), pace Shue. Yet in general, com- mentators briefly and summarily dismiss Kant, or they hardly mention him at all. Jamieson, at the very least, is one exception. https://doi.org/10.1017/S1369415422000176 Published online by Cambridge University Press 3. Value and limitations of Kantianism for climate change: Kant’s problem As noted, it is understandable why environmental critics would reject a framework generally perceived as anthropocentric, individualist and interiorist. Anthropocentrism seems problematic since a large component of the problem involves non-human nature. Individualism seems problematic since climate change must be understood in collective terms, across institutions, nations and generations. Lastly, a focus on maxims and agent-interiority, as Jamieson worries, appears prob- lematic by ignoring climate impacts, such as extreme weather or resource scarcity. y g g p , y Nonetheless, there is a growing literature on a ‘greener’ Kant. Contributions include rehabilitating Kant’s indirect-duty views, where the moral works and third Critique are emphasized. Relatedly, Toby Svoboda defends a Kantian approach to envi- ronmental ethics on the basis of Kant’s account of virtue (Svoboda 2012).9 Martin Schönfeld interprets the categorical imperative naturalistically, as a model for a ‘metaphysics of sustainability’ (Schönfeld 2010). Matthew C. Altman proceeds more pragmatically, arguing that Kant’s practical philosophy – paired with the CPJ’s regu- lative teleology – has strategic value for environmental problems (Altman 2011). Alice Pinheiro Walla, lastly, considers the categorical imperative as a principle for sustain- ability. She concludes that Kant’s legal philosophy is better suited to address climate change (Pinheiro Walla 2020). We have duties to ourselves to avoid wanton destruction of beautiful flora and animal cruelty (MM, 6: 443, Eth-Collins, 27: 459–60, Eth-Vigil, 27: 710), since such actions diminish our moral perfection (MM, 6: 446). Kant also enjoins us to appreciate and love nature as a beautiful and harmonious system (CPJ, 5: 267, 380, Eth-Vigil, 27: 668–9). Climate inaction will involve the moral failing of agents on a mass scale due to the ongoing sixth mass extinction. With widespread biodiversity loss, this failing will also diminish opportunities to appreciate nature’s beauty. And, of course, human destruction, especially of those vulnerable regions of the world, is even more reason to justify action. Thus, even the more usually emphasized aspects of Kantian ethics can help us frame climate change as a moral problem, and in particular make Kant’s anthropocentrism less worrisome. What about his ‘interiority’? 2.2 Critics of Kant in climate ethics Most notably, as we shall see, org/10.1017/S1369415422000176 Published online by Cambridge University Press 409 Kantian Review Kant’s thought also has an evolutionary dimension that is generally underappreci- ated. In particular, it supplements his view of ‘humanity’ in a way crucially relevant to concerns of climate ethics. But before getting to this, I briefly suggest that even the more standardly emphasized aspects of Kant’s ethics can at least justify his inclusion at the climate roundtable. https://doi.org/10.1017/S1369415422000176 Published online by Cambridge University Press 4. A Kantian perspective for a sustainable world I begin by providing a gloss of Kant’s philosophy of history as it is presented in IUH and CB, supported with reference to related remarks in Ped, Anth, MM, CPJ, TPP and R. Then I outline a new perspective on Kant’s account of humanity that can complement emerging approaches. 3. Value and limitations of Kantianism for climate change: Kant’s problem Still apart from what I want most to emphasize in this article, help might be found in Kant’s political phi- losophy, for example, in application of ideas from TPP, the Doctrine of Right, and R (Religion, especially part 3).10 Without entering into global climate agreements and transforming societies into sustainable cooperators, humanity risks civilization collapse, especially for vulnerable nations. Since it is a threat amplifier, climate change breeds war, so we can under- stand climate change to be a hurdle in the way of peace. If we view nations as akin to individual persons (MM, 6: 343; TPP, 8: 344), then their actions would also be subject to sustainability constraints. On the domestic level, failure to reform institutions under- mines the capacity of citizens for long-term independence. Finally, from the Zachary Vereb 410 perspective of nations, disregarding citizens is analogous to persons neglecting health: preservation of health is a duty only indirectly, as a condition for the possi- bility of self-determination or insofar as it assists in resistance to moral temptation (MM, 6: 388). As we have recently seen, citizens cannot act morally if they are inca- pacitated due to heat exhaustion, and nations that deny citizens the proper exercise of external freedom likewise fail their purpose as nations. Health of citizens is a pre- condition for health of nations. Climate change threatens both. Thus arguably, with further development inclusive of his political works, and par- ticularly with respect to perceived challenges from an overly anthropocentric and ‘interiorist’ emphasis in a reading of Kant, Kant might indeed become a significant asset. However, the ‘individualist’ problem for climate change still looms large. And it is in any case unclear whether, even exploiting the resources so far indicated, Kant has anything unique to offer for the questions at hand. I therefore suggest that we pursue a new avenue, exploring a global, future-directed Kant centred on the idea of humanity writ-large – but in particular writ larger than as more traditionally con- ceived, when conceived as a kingdom of individual human ends. https://doi.org/10.1017/S1369415422000176 Published online by Cambridge University Press 4.1 ‘Idea for a Universal History with Cosmopolitan Aim’ Like his pre-critical metaphysical works, Kant’s IUH takes a wider point of view. He reflects on the dynamic development of humanity pressed out across space and time. Kant posits a teleological framework for nature: organic entities in nature are des- tined to develop their natural predispositions (IUH, 8: 18).11 Yet the problem for humanity is that this process cannot be completed with one individual, let alone a generation, as our lives are short and filled with strife (Wood 1999: 211). Thus, we only perfect our full capacities as a species.12 Kant thinks of this as a collective endeavour (8: 19). Human progress requires intergenerational and international struggle, involving large social obstacles like war and political conflict (IUH, 8: 24). Though part of Kant’s claim involves the idea of a cunning mechanism of nature whereby self-interested individuals fortuitously benefit the species, another important aspect of his philoso- phy of history involves the value of reflection for realizing our moral ends. Kant’s teleological framework allows us to see from a broader philosophical perspective in which these two points are connected. As Herman suggests regarding IUH, ‘seeing the social world as tending toward a final end is essential to making it true that it reaches it’ (2009: 164). Louden underscores this in Kant’s philosophical anthropology, remarking that his ‘aim is to offer the species a moral map that they can use to move toward their collective destiny’ (2000: 106). When humanity thinks in the long term through the framing of a regulative teleology, orientating its actions for the sake of perfecting the species, it can begin to actualize its potential. 411 Kantian Review For Kant, historic obstacles assist us in developing our predispositions. Climate change, like war and natural disaster, is an obstacle to progress.13 But at the same time, through conflict and the pull of nature, we come to learn that progress requires the pursuit of enlightened community. Without collaboration, we cannot develop our rational predispositions as a species. Nature can only take us so far, but it does at least take us somewhere. Selfish conquests culminate in war, leading us to see the necessity for peace. Similarly, short-sighted disregard for our finite, collective home (cf. MM, 6: 352) culminates now in anthropogenic climate change, but in turn leads us to see the necessity for international collaboration. 4.2 ‘Conjectural Beginning of Human History’ Like IUH, CB adopts a wide perspective, focusing on humanity rather than individuals. Kant attempts to account for humanity’s origins though a conjectural ‘philosophy of nature’ (CB, 8: 109). He speculates on how it was possible for humanity to begin devel- oping its moral predispositions under the presumption of IUH’s teleological concep- tion of nature. Humanity, he suggests, progresses toward culture, moralization and ultimately political peace. In this human-historical process, humanity can view itself as a transtemporal and transnational collective. Instead of atomistic agents, we are simultaneously, at least in theory, united and future-directed. In the ‘pre-cultural’ stage, humanity amorally obeys the ‘call of nature’ (CB, 8: 111). Once reason begins to stir within it, Kant narrates, humanity starts to make technical and prudential choices (8: 111–12). Humanity then experiences the first glimmers of the power of imagination and self-consciousness. This developmental stage engenders proto-moral figurations of love and beauty, shame and modesty (Sittsamkeit). Curiously, on Kant’s view in CB, feelings of love and beauty with regard to humanity and nature are the earliest signs of moral consciousness (8: 113). Kant will explore this later when he makes connections between morality and love of nature (CPJ, 5: 267, 299, 380). The next stage in humanity’s moral development is the stage of culture. With cul- ture and education (cf. Ped, 9: 443–4), humanity becomes aware of itself as a temporal, directional being. Moral consciousness arouses death-anxiety and a concern for pos- terity; the communal sense of care for humanity’s future is, Kant reflects, important for awareness of our moral vocation (CB, 8: 113; Ped, 9: 449). Finally, humanity becomes cognizant of itself qua reason, deserving of dignity and respect as end-in- itself (CB, 8: 114). Kant very stoically asserts that we have not fully achieved this stage. That is, we have not realized culture in its enlightened, moralized form (8: 121; cf. Wood 1999: 298). As a collective-cultural obstacle, perhaps climate change is the impe- tus – the epoch-changing fork in the road – on that pathway. 4.1 ‘Idea for a Universal History with Cosmopolitan Aim’ If Kant’s historical framing narrative is adopted, the aspiration to a peaceful and sustainable world can be seen as not only possible, but required to realize our predispositions. https://doi.org/10.1017/S1369415422000176 Published online by Cambridge University Press 4.3 Climate change as a collective problem In these works, Kant views humanity unlike the way a typical ethical individualist would. Instead, he frames it in a broader collective context. Since the ideas central to these works are only regulative, this collectivism should of course be understood as org/10.1017/S1369415422000176 Published online by Cambridge University Press Zachary Vereb 412 a useful heuristic, namely, for realizing our moral duty to perfect humanity in har- mony with nature (IUH, 8: 21; CB, 8: 123).14 Now as moral agents, individual humans have duties of self-perfection. However, because perfection is obviously unattainable in a single lifetime, Kant’s perfectibility injunction is pressed for the human species as a whole (IUH, 8: 18–19). From Kant’s historical perspective, humanity is thus framed as a collectively united subject, and through this lens present generations ought to care for posterity (8: 27–8). In short, humanity can be seen as having one single, over- arching telos that transcends individuals (cf. Ameriks 2009: 46), unites all humankind and enjoins our species to cultivate its dispositions toward goodness (8: 115, 120). Humanity must direct itself toward a future of cosmopolitan solidarity (IUH, 8: 26), strive for global peace (TPP, 8: 362) and end institutions of domination (Herman 2009: 157). We must, moreover, work together to realize this task with long-term vision. IUH, suggests Ameriks, ‘implies an ideal end that is pre-given for all of us, one that, in several senses, we “must” all work to bring about’ (2009: 49). Every human has a duty to contribute to the progress of the species from worse to better: ‘and each of us, for his part, is called upon by nature itself to contribute as much as lies in his power to this progress’ (CB, 8: 123). Our historical responsibility is thus to contribute toward the developing good of the species and for the betterment of the world, which Kant affirms in the Pedagogy. We must ‘try to bring posterity further than [we ourselves] have gone’ and take an interest in what is ‘best for the world even if it is not to the advantage of [our] father- land or to [our] own gain’ (Ped, 9: 449, 499). States, we can infer, have similar respon- sibilities and can address institutional obstacles where individuals cannot (cf. Pinheiro Walla 2020). For, as quasi-moral agents, they have duties to their subjects and to the species. 4.3 Climate change as a collective problem This injunction includes consideration to future generations, since each generational instantiation of humanity relates to the collective telos, and each has an obligation to contribute to its perfection. Such a task commands our respect and fills us with hope for future generations, despite the difficult reality of the situa- tion (CB, 8: 113; IUH, 8: 28). Viewed through Kant’s historical philosophy, humanity is framed as a transtem- poral, transnational agent: a ‘macro end-in-itself’ struggling with obstacles impeding its perfection. This macro agent, mediated through the present generation, must act for posterity as a necessary precondition for its ‘health’ as a continually developing entity; and this macro agent must not shirk its duties with regard to non-rational nature, relevant to its self-perfection. Both require reforming unsustainable institu- tions. In short, by adopting Kant’s view in these texts, we can think of humanity as a whole, as both historic agent and patient. Kant’s regulative lens, with its correspond- ing heuristic ‘moral map’, has value for rethinking climate change’s collective prob- lems, such as the institutional inertia discussed in section 2. I expand on this below. https://doi.org/10.1017/S1369415422000176 Published online by Cambridge University Press 4.4 A new Kantian perspective on sustainability As repeatedly emphasized in this article, climate change is a collective problem, requiring international and intergenerational effort. For this reason, it often sparks a sense of overwhelming hopelessness. Fortunately, one strength of Kant’s teleologi- cal perspective is its emphasis on rational hope (Cureton 2018). Kant claims that, politically, we have ‘a very rough project’ ahead (IUH, 8: 28). Indeed, climate change, org/10.1017/S1369415422000176 Published online by Cambridge University Press 413 Kantian Review like war, is a unique challenge and opportunity for testing humanity’s mettle. War exaggerates humanity’s unsociable tendencies but creates conditions for peace through its revelations. Conflicts often arise because we put our ‘narrow and short-term interests before the long-term interests of mankind in general’ (Williams 1983: 135). But realization of the unsustainability of these destructive ten- dencies by private citizens challenges humanity to become innovative and coopera- tive, and presses us to seek a rightful global condition. Similarities between war and climate in this regard give us hope that humanity may come to related technical and moral solutions.15 Climate ethicist Stephen Gardiner’s hopeful call for a ‘global con- stitutional convention’ sounds remarkedly Kantian in this respect. Kant’s views on hope and political cooperation underscore an often- underappreciated long-term view of humanity. His views on progress, likewise, help us rethink his perceived individualism.16 Kant warns that our intergenerational proj- ect will be arduous, yet we must remain courageous in the face of our responsibilities to improve culture and perfect the species (CB, 8: 115–17, 121). Furthermore, our moral vocation can only be realized if we strive together (Anth, 7: 323–5, Ped, 9: 445). Humanity’s vocation includes not only self- and other-regarding duties, but pur- suit of shared, collective ends (R, 6: 100–1; see also Wood 1999: 316). Thus, Kant’s dis- cussion of hope and moral progress naturally leads to considerations of humanity’s overarching moral vocation, which, in light of climate change, can be directed toward ecological stewardship. Critical stewardship enjoins us to rethink our relationship to nature, not as dom- inators but as stewards or members embedded in it.17 Perhaps surprisingly, Kant rethinks humanity along these lines in several of his teleological works (CPJ, 5: 380, UNH, 1: 353–4). Stewardship does not concern mere reflection, however, but also actions and attitudes. For Kant, this involves duties of humaneness and love regarding non-rational nature (MM, 6: 443; Eth-Vigil, 27: 668–9). https://doi.org/10.1017/S1369415422000176 Published online by Cambridge University Press 4.4 A new Kantian perspective on sustainability Without a stewardly shift – from both private citizens and institutions via more sustainable development – humanity will likely exacerbate climate impacts affecting others across the globe. Even worse, those contributing least to the acceleration of climate change will have their auton- omy stifled most. This is because many regions subject to the worst impacts – e.g. droughts in Africa or sea-level rise in the Carteret Islands – have less responsibility in the way of historic and annual climate emissions. These impacts will at best con- strain the external freedom of their citizens, and will at worst involve large-scale death and dependence. By contrast, geographically lucky wealthy nations, ones that have contributed more total and per capita emissions such as the US and the UK, will have an easier chance to adapt to maintain similar levels of autonomy. Failures of stewardship and sustainable development fail our collective duty as articulated in Kant’s historical and religious texts. Such failures also flout individual duties to agents whose autonomy will be blunted from climate impacts. Progress for Kant is two-fold: greater respect for agents and broader views of humanity as a spe- cies, including the need for moral, cultural and institutional maturation. Kant’s unique vision – by refocusing humanity’s vocation in collective terms and enjoining beneficent actions to promote progress – can thus be of use to facilitate the needed sustainable shift. On Allen Wood’s view, the kingdom of ends formulation demands that we unite our ends to attain a ‘harmonious, organic system’ (1999: 185). The end of one agent is tied Zachary Vereb 414 up with the aim of the species. Furthermore, perfecting the species necessarily con- cerns consideration of future generations, since they too are part of that living sys- tem. Though this aim is an ideal, approximating it remains unlikely with business-as- usual. Failure to address climate change precludes the moral harmonization of ends on a finite earth, and threatens a global state of nature. One way to avoid this is to think of humanity as like a single moral agent and patient, having duties toward itself as a whole. Since present generations are instantiations of this whole, they must uphold their duties not only toward themselves and others, but toward humanity as a whole. By ignoring the crisis, present generations thwart their macro self- regarding duties of perfection. 4.4 A new Kantian perspective on sustainability Additionally, since duties of humanity as a whole toward itself are self-regarding duties for perfection, we can appreciate these as wide duties. So when there is a conflict between perfect duties to individuals and duties to humanity as a whole (e.g. regarding future generations), while the former have a claim to primacy, the latter should not be overlooked. A virtue of this perspective is that it evades classic entanglements in ethical or metaphysical holism. For example, critics of environmental and Hegelian holism warn of reductionism. These may entail, worst of all, eco- and political fascist tendencies. Holists, by valuing natural or political systems over individual agents, could justify reducing those individuals to mere means. For individuals are like the organism’s expendable cells. The regulative status of Kant’s moral map helps evade this concern, as agents are not reduced, theoretically, to the cells of humanity writ-large. Rather, the Kantian picture sustains the practical worth of individuals, and appreciates them as embedded in historically evolving institutions and nations.18 To summarize, it is possible to read Kant’s philosophy as enjoining collective duties toward the human species writ-large. This reading, first, helps us to reconcile the value of individuals framed in a collective context: we see ourselves as relational con- stituents of a species attempting to actualize its predispositions, mediated through cultures, institutions and nations. Second, this perspective can be mobilized as a heu- ristic for justifying institutional and lifestyle reform, and so can add to the new Kantian literature on climate change. Pinheiro Walla 2020) discusses the Kantian need to create sustainable institutions, Williams (2019) develops a new Kantian perspective on collective harms, and Bernstein (2019) discusses the role of Kantian hope and cos- mopolitanism for climate change; all these accounts converge with my own in showing that it is fruitful to move beyond the Groundwork and Critique of Practical Reason, to (as already argued by others) his political, legal19 and (as emphasized here) historical phi- losophy. Doing so is an important step for showing sceptics that Kant is an asset. https://doi.org/10.1017/S1369415422000176 Published online by Cambridge University Press 5. Geoengineering a better world? Objections and concluding remarks In the Anthropocene, humanity now understands itself as a pivotal global force. Kant’s humanistic anthropocentrism – a bane to critics of old – is a boon in this unprecedented age. On the political side, debate centres on humanity, with non- anthropocentric accounts dismissed. An approach that takes seriously the power and promise of humanity, such as Kant’s, is therefore relevant. It is nonetheless rea- sonable to raise concerns. First, Kant never talks about climate change or sustainability. Does not this inter- pretation bend Kant in exegetically inappropriate ways? But recall that philosophical 415 Kantian Review interpretation need not be confined to strict exegesis. There remains an alternative purpose for interpretation: the mobilization of philosophical ideas for their heuristic assistance with present practical problems. Both approaches are indeed connected, and the applied approach is consistent with the exegetical one insofar as the philo- sophical ideas must be there, present in Kant’s works. As long as they are, the inter- preter is free to engage them. This is no problem, because this interpretation has different aims than exegesis. Just as commentators employ Kant’s philosophy to address issues he did not discuss, like healthcare and refugees (Altman 2011, 2017), it is also legitimate to pursue Kantian-inspired philosophical application for sustainability. Interestingly, Kant does discuss climate in his early natural philosophy works. There, he is sensitive to climatological dynamics, humanity’s dependency on them and our capacity for environmental modification.20 Taking Kant’s philosophy further by relating its practical dimensions to our predicament is not as problematic as might appear. g Despite this, one may seriously worry about drawing from these works given implications that follow from Kant’s view of progress.21 One concern relevant to envi- ronmental philosophy and climate justice has to do with the implications of Kant’s historical philosophy for geoengineering. This is prima facie worrisome for a variety of reasons, including the promotion of human ‘mastery values’ and an instrumentalist attitude toward nature, along with, as I emphasize below, neglect of those in the Global South.22 It is true that Kant sees human progress in terms of incremental, qual- itative developments in science, art and culture, or civilization.23 Yet as a consequence, the more we progress culturally, the more we are obliged to modify nature for human ends. For Kant, progress does not consist in simplifying our lives, as Rousseau or Thoreau would urge. https://doi.org/10.1017/S1369415422000176 Published online by Cambridge University Press 5. Geoengineering a better world? Objections and concluding remarks Rather, progress entails becoming more skilful at modifying nature.24 It is an easy step and a sensible worry: if the Kantian view encourages the modifi- cation of nature, it might be used to justify problematic geoengineering solutions. Most broadly, geoengineering is ‘the intentional manipulation of the environment on a global scale’ (Gardiner 2011: 340). However, as discussed in policy debates, geo- engineering means extreme global intervention to preserve or prolong business-as- usual practices. These include technologies to absorb emissions (e.g. sea iron fertili- zation); to block solar radiation (e.g. space mirrors or atmospheric sulphate injec- tions); and to remove carbon (atmospheric scrubbing machines). As global technological ‘experiments’, these will have many unforeseeable side-effects, since the planet is an interconnected system only partially understood; manipulating one part affects the earth-system in complicated ways, unlike our usual familiarity with technical manipulation. Understood in the extreme sense outlined above, the worry is partly justified. Much like Marx after him, Kant thinks modifying nature is invaluable for humanity. To be sure, it does not follow that such modifications – to create a ‘habitat for human- ity’ as Herman puts it (2009) – mean we are justified in treating nature poorly, blind to probable deleterious side-effects. For our obligations remain regarding non-rational nature, to not treat animals cruelly and beautiful flora instrumentally. However, although many geoengineering solutions are in principle compatible with Kant’s view, on closer examination it becomes obvious that geoengineering tunnel-vision Zachary Vereb 416 conceals deeper maladaptive relationships with our human habitat and could itself create further catastrophes. It seems reasonable that Kant, arch-nemesis of fanaticism, would never uncriti- cally endorse such magical (or at least wishful) thinking. Instead, he would critically tend to its roots, framing possible solutions alongside their limitations in a wider con- text. Indeed, the Kantian way to address problems – a recurring lesson taught in the critical theoretical and practical works – is to reflect on the sources and preconditions of the issue being analysed. Such reflection may reveal that a contradiction is only apparent, or that a framework is hopelessly one-sided. For example, under the Humean perspective many problems with metaphysics make sense, while rationalistic perspectives are enticing for other reasons, despite their dogmatism. But as Kant shows, these are not the only plausible frameworks. https://doi.org/10.1017/S1369415422000176 Published online by Cambridge University Press Notes 1 Kant has been historically marginalized in animal and environmental ethics (Singer 1990, Regan 2004, Hoff 1983, Rolston 1988). Defences of Kant include Altman 2011, Svoboda 2012, and Korsgaard 2018. For works on Kant and climate, see Schönfeld 2010, Rentmeester 2010, Frierson 2014, Bernstein 2019, Williams 2019, Pinheiro Walla 2020. 2 Climate ethicists often favour consequentialist analyses (e.g. Broome 2012). Treatments of Kantianism are typically dismissive (Jamieson 2007, Garvey 2008, Shue 2010, Gardiner 2011). 3 References to Kant’s writings refer to volume and page numbers of the Academy edition, with trans- lations from the Cambridge Edition of the Works of Immanuel Kant (Kant 1996, 2000, 2007). Abbreviations are the following: CB = ‘Conjectural Beginning of Human History’, IUH = ‘Idea for a Universal History with a Cosmopolitan Aim’, MM = Metaphysics of Morals, Anth = Anthropology from a Pragmatic Point of View, Ped = Lectures on Pedagogy, CPJ = Critique of the Power of Judgement, TPP = Toward Perpetual Peace, R = Religion within the Boundaries of Mere Reason, Eth-Collins = Collins Lectures on Ethics, Eth-Vigil = Vigilantius Lectures on Ethics, UNH = Universal Natural History and Theory of the Heavens. 4 Hoff (1983) presents a similar critique of Kant’s anthropocentrism but has been less influential. h ll f d d b ll d ll ( ) f h 4 Hoff (1983) presents a similar critique of Kant’s anthropocentrism but has been less influential. 5 See the recent collection of essays, edited by Callanan and Allais (2020), on Kant’s views of non-human 4 Hoff (1983) presents a similar critique of Kant’s anthropocentrism but has been less influential. 5 See the recent collection of essays, edited by Callanan and Allais (2020), on Kant’s views of non-human animals for ways to problematize this view. 5 See the recent collection of essays, edited by Callanan and Allais (2020), on Kant’s views of non-human animals for ways to problematize this view. 6 Consequently, those sympathetic to non-human concerns in environmental philosophy will find it easier to reject Kant without considering his value for present problems. See Vereb (2021) on critics of Kant in environmental philosophy, and an attempted rehabilitation of the pre-critical metaphysics. 7 Wood highlights Kant’s ‘consequentialism’ regarding public communication (Wood 1999: 306). Cummiskey (1996) challenges Kant’s ‘nonconsequentialism’, and Garrath Williams (2019) discusses col- lective harms associated with climate change. Notes The last is similar in spirit to my reading in that it focuses on Kantian non-‘individualism’, though I highlight the historical and teleological works, especially Kant’s idea of ‘humanity’, rather than institutions and moral complicity. 8 Though Rawls draws from Kant’s thought, he rarely mentions Kant’s political philosophy. This narrow view of Kant furthered by Rawls and others continues to be challenged (Habermas 1996, Waldron 1996, Ellis 2008, Ripstein 2009). Since Gardiner argues that Rawlsian individualism supports present institu- tions that engendered the crisis in the first place (2011: 230), it is reasonable to assume that commen- tators critical of Kant might see his thought in a related way. 9 Svoboda’s indirect-duty account does not engage climate change, but this is understandable since most early objections originate in environmental, not climate ethics. 10 For good entry-points to Kant’s legal and political philosophy, see Williams 1983, Ellis 2008, Ripstein 2009, Byrd and Hruschka 2010, Wood 2017. Bernstein 2019 also takes what she calls a ‘broadly Kantian approach’ in considering, on her view, the ‘heuristic’ value of Kant’s historical and political philosophy for the crisis (2019: 98, 88). Unlike Bernstein, however, my approach focuses on humanity. 11 Human natural predispositions (Anlagen), fleshed out later in the Religion, include ‘animality’, ‘human- ity’ and ‘personality’ (R, 6: 26). Animality as an existential predisposition concerns survival and welfare; humanity relates to the cultivation of culture and sociality; lastly, personality relates to autonomy and morality. These predispositions are to be perfected by the establishment of stable societies, scientific and artistic progress, and the moralization of citizens. As we will see, climate change, as an existential threat to human welfare, relates to animality; as a social threat, via civilization collapse and refugee crises, relates to humanity; and as a moral threat, tempting us to neglect our duties to others and humanity as a whole, relates to personality. See Wood on the ‘technical’ and ‘pragmatic’ predispositions in Anth, 7: 321–9 and their relation to the collective destiny of humanity (1999: 118–19, 210–12). y y 12 Individuals have duties of perfection that include cultivating physical, intellectual and moral faculties (MM, 6: 445–7). However, as Kant hints in MM and asserts in IUH, this obligation to perfection can only be completed with the species. 5. Geoengineering a better world? Objections and concluding remarks Similarly, a technocratic mindset tends toward geoengineering by reinforcing the assumption of nature’s instrumen- tality, but such a mindset conceals other equally plausible perspectives (for example, viewing nature less mechanistically and more organically). One-sided frameworks, even critical ones, readily succumb to the dangers of dogmatism. The wishful thinking of extreme geoengineering exemplifies this. Of course, Kant would still aim to understand the increasing tendencies toward geoengineering in the context of his philosophy of history. Just as militaristic think- ing functions to facilitate a learning process vis-à-vis war and peace, so also does tech- nological thinking teach when appreciated in the right light. Humans are resourceful, especially in challenging environments, but there is only one planet; geoengineering solutions alone are unlikely, at least at present, to solve the crisis. They will, in the coming decades, be an inevitable factor in climate emission mitigation.25 However, undue reliance on geoengineering betrays a cognitive blind spot, and this failed per- spective has normative implications. These include epistemic obfuscation of the roots of climate change and, as it were, kicking the can down the road. The latter would exacerbate harms to those in the Global South and future generations. The window is closing, yet means for international collaboration are still available. Pursuing these may promote progress on a higher qualitative plane than intergenerationally and geographically short-sighted solutions. g g y g Since the Kantian view encourages benevolent modification of nature, incorporat- ing the conception of humanity writ-large can in any case add to current discussions of geoengineering in climate ethics. For on this position, it is only possible to justify geoengineering solutions if they account for the moral status of future humanity and those in the Global South. Many extreme geoengineering solutions threaten to gam- ble away the well-being of portions of humanity, and so at the very least require addi- tional careful consideration. Such solutions are sometimes accepted as necessary on consequentialist modes of analysis. The Kantian approach could also justify geoengin- eering, but only insofar as it were sufficiently rational and mindful: rational, in not being ignorant of the relevant political, historical and institutional contexts, and mindful, in being considerate of the collective plight of humanity worldwide. Drawing from the long-range perspectives in Kant’s historical philosophy takes the seeming liability of Kantian individualist ‘mastery values’ and transforms it into an asset. 417 Kantian Review Kantian Review https://doi.org/10.1017/S1369415422000176 Published online by Cambridge University Press Notes With regard to application of concepts like ‘perfection’ and ‘progress’ to the crisis, it is perhaps best to leave their definitions open to interpret in context-specific ways. What it means to perfect humanity looks different from sub-Saharan Africa than in the UK, given differential resource constraints. This article underscores the spirit of Kant’s philosophy, and not the exact letter, so I use the terms ‘perfection’ and ‘progress’ loosely. 13 The concept of ‘climate change’ viewed from a Kantian perspective is curious. Talk about climate change qua nature (from the teleological view of history) bears on the production of the unity of Zachary Vereb 418 humanity through international collaboration. On the other hand, climate change qua anthropogenesis is part of humanity (we created it, and hence have a responsibility to undo it, as those in conflict ought to pursue peace). p p 14 The harmony of nature Kant has in mind is likely not the view that environmentalists envisage, it i l h i t t it l d lt l d I dd thi i ti 5 14 The harmony of nature Kant has in mind is likely not the view that environmentalists envisage, since it involves shaping nature to suit our moral and cultural needs. I address this in section 5. The harmony of nature Kant has in mind is likely not the view that environmentalists envisage, sinc nvolves shaping nature to suit our moral and cultural needs. I address this in section 5. The analogy only goes so far, however, since climate change presses us further: whereas Kant sees i 15 The analogy only goes so far, however, since climate change presses us further: whereas Kant sees in war an active force of nature driving the species and our predispositions for animality and humanity, climate change differs since it requires not only the development and cultivation of civilization (which ‘artificer nature’ can do alone, TPP, 8: 361), but also moralization; technical solutions produced from unsociable competition alone will not cut it. p 16 Kant’s conception of progress emphasizes the value of challenges. Section 5 addresses the concern that the crisis could lead to extreme geoengineering rather than ecological stewardship. 17 By ‘stewardship’ I mean an attitude encouraging cultivation and growth, rather than mastery and subordination. It is possible for humanity to steward while still seeing itself as morally distinct. Notes Still, I am optimistic that the moralization and aesthetic development of humanity will tend toward non- human protectionism. Recall, additionally, Kant’s disinterested or non-prudential valuing of beautiful flora, or of the gentleness and love demanded with regard to non-human animals and natural objects (Vereb 2019, Eth-Vigil, 27: 668–9, CPJ, 5: 267). It is true that Kant talks of mastery in the Doctrine of Right as the basis for property relations. It would be interesting to see whether Kant’s legal and juridical phi- losophy is compatible with stewardship, but I cannot pursue that here. p y p p p 18 See Wood on related non-individualist aspects of Kant (1999: 204, 281, 289, 316). 19 See Ataner’s impressive MA thesis (2012) for a thorough exploration of Kant’s theory of property vis- à-vis environmental protectionism. Ataner focuses on MM and Kant’s ‘juridical postulate of practical rea- son’, whereas I focus on the historical works and Kant’s view of humanity. It would be interesting to contrast these works, though I cannot do this here. g 20 For a good place to start, see Kant’s 1755 essay on the earthquake in Lisbon (1: 456) as well as the Physical Geography (9: 298). 21 I bracket questions considering the extent to which Kant’s system supports racism. ( 22 It has been well-documented that those in the Global South (including countries such Nigeria, Haiti, India and Thailand) are in a uniquely bad situation vis-à-vis climate. From the normative perspective, not only have they emitted less, historically speaking, but they are – geographically and infrastructurally speaking – in tough spots with regard to human vulnerability. Further, their voices are often not afforded equal consideration to those in the Global North. Klein (2014: 256–61) recounts a harrowing instance of this at a 2011 geoengineering meeting convened by the UK Royal Society. There, an African delegate voiced concerns of droughts likely to follow from the disruption of monsoon seasons in Africa and Asia as a result of a solar geoengineering proposal. 23 See Ped, 9: 449–50 and 475 for sustained discussions of ‘culture’ relevant to the historical works. f h h l d d h l f ( h ) 23 See Ped, 9: 449–50 and 475 for sustained discussions of ‘culture’ relevant to the historical works. 24 Cf. humanity’s ‘technical’ predisposition regarding the manipulation of nature (Anth, 7: 322–3). Notes 25 Not all geoengineering is as experimental as ocean fertilization or stratospheric injections. Humanity has always engaged in natural modifications, as Kant suggests in CB. ‘Benign’ geoengineering strategies are certainly compatible with the Kantian injunction to treat nature humanely. Examples include ‘green’ burials, reforestation and ‘green’ roof cultivation. These strategies encourage reconsideration of our rela- tionship to nature, promise new opportunities for aesthetic reflection and buy us time to come to inter- national policy agreements. References Altman, Matthew C. (2011) Kant and Applied Ethics: The Uses and Limits of Kant’s Practical Philosophy. Malden, MA: Wiley-Blackwell. —— (2017) ‘The Limits of Kant’s Cosmopolitanism: Theory, Practice, and the Crisis in Syria’. Kantian Review, 22(2), 179–204. Ameriks, Karl (2009) ‘The Purposive Development of Human Capacities’. In Amélie Oksenberg Rorty and James Schmidt (eds), Kant’s Idea for a Universal History with a Cosmopolitan Aim: A Critical Guide (Cambridge: Cambridge University Press), 46–67. Ameriks, Karl (2009) ‘The Purposive Development of Human Capacities’. In Amélie Oksenberg Rorty and James Schmidt (eds), Kant’s Idea for a Universal History with a Cosmopolitan Aim: A Critical Guide (Cambridge: Cambridge University Press), 46–67. https://doi.org/10.1017/S1369415422000176 Published online by Cambridge University Press https://doi.org/10.1017/S1369415422000176 Published online by Cambridge University Press 419 Kantian Review Ataner, Atilla (2012) ‘Kant on Freedom, Property Rights, and Environmental Protection’. Master’s thesis, McMaster University Retrieved from http://hdl handle net/11375/12678 taner, Atilla (2012) ‘Kant on Freedom, Property Rights, and Environmental Protection’. Master’s the McMaster University. Retrieved from http://hdl.handle.net/11375/12678. McMaster University. Retrieved from http://hdl.handle.net/11375/12678. Bernstein, Alyssa R. (2019) ‘Cosmopolitanism and the Climate Crisis’. Con-Textos Kantianos, 10, 84–101. McMaster University. Retrieved from http://hdl.handle.net/11375/12678. Bernstein, Alyssa R. (2019) ‘Cosmopolitanism and the Climate Crisis’. Con-Textos Kantia Bernstein, Alyssa R. (2019) ‘Cosmopolitanism and the Climate Crisis’. Con-Textos Kantianos, 10, Broome, John (2012) Climate Matters: Ethics in a Warming World. New York: W. W. Norton. Byrd, B. Sharon, and Joachim Hruschka (2010) Kant’s Doctrine of Right: A Commentary. Cam Byrd, B. Sharon, and Joachim Hruschka (2010) Kant’s Doctrine of Right: A Commentary. Cambridge: Cambridge University Press. Cambridge University Press. Callanan, John J., and Lucy Allais (eds) (2020) Kant and Animals. Oxford: Oxford University Press. Cummiskey, David (1996) Kantian Consequentialism. New York: Oxford University Press. Cureton, Adam (2018) ‘Reasonable Hope in Kant’s Ethics’. Kantian Review, 23(2), 181–203. g y allanan, John J., and Lucy Allais (eds) (2020) Kant and Animals. Oxford: Oxford University Press. Callanan, John J., and Lucy Allais (eds) (2020) Kant and Animals. Oxford: Oxford University Press. Cummiskey David (1996) Kantian Consequentialism New York: Oxford University Press Cummiskey, David (1996) Kantian Consequentialism. New York: Oxford University Press. Cureton, Adam (2018) ‘Reasonable Hope in Kant’s Ethics’. Kantian Review, 23(2), 181–203. Ellis, Elisabeth (2008) Provisional Politics: Kantian Arguments in Policy Context. London: Yale University Press. Frierson, Patrick (2014) ‘Kant, Individual Responsibility, and Climate Change’. Ethics, Policy and Ellis, Elisabeth (2008) Provisional Politics: Kantian Arguments in Policy Context. London: Yale University Press. Frierson, Patrick (2014) ‘Kant, Individual Responsibility, and Climate Change’. Ethics, Policy and Environment, 17(1), 35–8. Environment, 17(1), 35–8. Gardiner, Stephen Mark (2011) A Perfect Moral Storm: The Ethical Tragedy of Climate Change. New York: O f d U i it P Gardiner, Stephen Mark (2011) A Perfect Moral Storm: The Ethical Tragedy of Climate Change. New York: Oxford University Press. Garvey, James (2008) The Ethics of Climate Change: Right and Wrong in a Warming World. London and New York: Continuum. Habermas, Jürgen (1996) Between Facts and Norms. Cambridge, MA: MIT Press. Herman, Barbara (2009) ‘A Habitat for Humanity’. https://doi.org/10.1017/S1369415422000176 Published online by Cambridge University Press In Amélie Oksenberg Rorty and James Schmidt (eds), Kant’s Idea for a Universal History with a Cosmopolitan Aim: A Critical Guide (Cambridge: Cambridge University Press), 150–70. y Hoff, Christina (1983) ‘Kant’s Invidious Humanism’. Environmental Ethics, 5(1), 63–70. Jamieson, Dale (2007) ‘When Utilitarians should be Virtue Theorists’. Utilitas, 19(2): 160–83. —— (2014) Reason in a Dark Time: Why the Struggle Against Climate Change Failed – and What it Means for our Future. Oxford and New York: Oxford University Press. Kant, Immanuel (1996) Practical Philosophy. Trans. and ed. Mary J. Gregor. Cambridge: Cambridge University Press. Kant, Immanuel (1996) Practical Philosophy. Trans. and ed. Mary J. Gregor. Cambridge: Cambridge University Press. —— (2000) Critique of the Power of Judgment. Trans. and ed. Paul Guyer and Eric Matthews. Cambridge and New York: Cambridge University Press. —— (2007) Anthropology, History, and Education. Ed. Günter Zöller and Robert B. Louden. Cambridge: Cambridge University Press. Klein, Naomi (2014) This Changes Everything: Capitalism vs the Climate. New York: Simon & Schuster. University Press. —— (2000) Critique of the Power of Judgment. Trans. and ed. Paul Guyer and Eric Matthews. Cambridge and New York: Cambridge University Press. —— (2007) Anthropology, History, and Education. Ed. Günter Zöller and Robert B. Louden. Cambridge: C b id i i P —— (2000) Critique of the Power of Judgment. Trans. and ed. Paul Guyer and Eric Matthews. Cambridge and New York: Cambridge University Press. —— (2007) Anthropology, History, and Education. Ed. Günter Zöller and Robert B. Louden. Camb Cambridge University Press. Klein Naomi (2014) This Changes Everything: Capitalism vs the Climate New York: Simon & Schuste (2007) Anthropology, History, and Education. Ed. Günter Zöller and Robert B. Louden. Cambridge: mbridge University Press. (2007) Anthropology, History, and Education. Ed. Günter Zöller and Robert B. Louden. Cambridge: Cambridge University Press. Klein, Naomi (2014) This Changes Everything: Capitalism vs the Climate. New York: Simon & Schuster. Cambridge University Press. lein, Naomi (2014) This Changes Everything: Capitalism vs the Climate. New York: Simon & Schuster. ( ) Korsgaard, Christine M. (2018) Fellow Creatures: Our Obligations to Other Animals. Oxford: Oxford University Press. Lenton, Timothy M., Johan Rockström, Owen Gaffney, Stefan Rahmstorf, Katherine Richardson, Will Steffen and Hans Joachim Schnellnhuber (2019) ‘Climate Tipping Points: Too Risky to Bet Against’. Nature, 575, 592–5. Louden, Robert B. (2000) Kant’s Impure Ethics: From Rational Beings to Human Beings. New York: Oxford University Press. University Press. https://doi.org/10.1017/S1369415422000176 Published online by Cambridge University Press Pinheiro Walla, Alice (2020) ‘Kant and Climate Change’. In Ben Eggleston and Dale E. Miller (eds), Moral y Pinheiro Walla, Alice (2020) ‘Kant and Climate Change’. In Ben Eggleston and Dale E. Miller (eds), Moral Theory and Climate Change: Ethical Perspectives on a Warming Planet (New York: Routledge), 99–115. Regan, Tom (2004) The Case for Animal Rights. Berkeley, CA: University of California Press. Rentmeester Casey (2010) ‘A Kantian Look at Climate Change’ Essays in Philosophy 11(1) 76 86 Theory and Climate Change: Ethical Perspectives on a Warming Planet (New York: Routledge), Regan, Tom (2004) The Case for Animal Rights. Berkeley, CA: University of California Press. ( ) ( ) y g p g ( g ) Regan, Tom (2004) The Case for Animal Rights. Berkeley, CA: University of California Press. Rentmeester, Casey (2010) ‘A Kantian Look at Climate Change’. Essays in Philosophy, 11(1), 76–86 Ripstein, Arthur (2009) Force and Freedom. Cambridge, MA: Harvard University Press. l l ( ) i l hi i d l i h l ld Ripstein, Arthur (2009) Force and Freedom. Cambridge, MA: Harvard University Press. Rolston, Holmes III (1988) Environmental Ethics: Duties to and Values in the Natural World. Philad p g y olston, Holmes III (1988) Environmental Ethics: Duties to and Values in the Natural World. Philadelphia, Holmes III (1988) Environmental Ethics: Duties to and Values in the Natural World. Philadelphia, PA: le University Press Holmes III (1988) Environmental Ethics: Duties to and Values in the Natural World. Philadelphia, PA: le University Press. mple University Press. f ld M ti (2010) ‘Th M t h i f S t i bilit K t’ C t i l I ti ’ I J k L Temple University Press. chönfeld Martin (2010) ‘The Metaphysics of Sustainability: Kant’s Categorical Imperative’ In Jack L p y Schönfeld, Martin (2010) ‘The Metaphysics of Sustainability: Kant’s Categorical Imperative’. In Jack Lee (ed ) Sustainability and Health (Stanford CA: Ria University Press) 1–18 p y nfeld, Martin (2010) ‘The Metaphysics of Sustainability: Kant’s Categorical Imperative’. In Jack Lee (ed.), Sustainability and Health (Stanford, CA: Ria University Press), 1–18. Shue, Henry (2010) ‘Global Environment and International Inequality’. In Stephen M. Gardiner, Simon Caney, Dale Jamieson and Henry Shue (eds), Climate Ethics: Essential Readings (New York: Oxford University Press) 101–11 University Press), 101–11. Singer, Peter (1990) Animal Liberation. New York: New York Review of Books/Random House. Cite this article: Vereb, Z. (2022). Sustaining the Individual in the Collective: A Kantian Perspective for a Sustainable World. Kantian Review 27, 405–420. https://doi.org/10.1017/S1369415422000176 —— (2021) ‘Kant’s Pre-Critical Ontology and Environmental Philosophy’, Environmental Philosophy, 18(1), 81–102. Waldron, Jeremy (1996) ‘Kant’s Legal Positivism’. Harvard Law Review, 109(7), 1535–66. Williams, Garrath (2019) ‘The Social Creation of Morality and Complicity in Collective Harms: A Kantian Account’. Journal of Applied Philosophy 36(3), 457–70. Williams, Howard (1983) Kant’s Political Philosophy. New York: St Martin’s Press. Wood, Allen (1999) Kant’s Ethical Thought. Cambridge and New York: Cambridge University Press. —— (2017) ‘Marx and Kant on Capitalist Exploitation’. Kantian Review, 22(4), 641–59. Waldron, Jeremy (1996) ‘Kant’s Legal Positivism’. Harvard Law Review, 109(7), 1535–66. Williams, Garrath (2019) ‘The Social Creation of Morality and Complicity in Collective Har —— (2021) ‘Kant’s Pre-Critical Ontology and Environmental Philosophy’, Environmental Philosophy, 18(1), 81–102. Waldron, Jeremy (1996) ‘Kant’s Legal Positivism’. Harvard Law Review, 109(7), 1535–66. , ( ) p y Wood, Allen (1999) Kant’s Ethical Thought. Cambridge and New York: Cambridge University Press. —— (2017) ‘Marx and Kant on Capitalist Exploitation’. Kantian Review, 22(4), 641–59. —— (2021) ‘Kant’s Pre-Critical Ontology and Environmental Philosophy’, Environmental Philosophy, 18(1), 81–102. https://doi.org/10.1017/S1369415422000176 Published online by Cambridge University Press Account’. Journal of Applied Philosophy 36(3), 457–70. Williams, Howard (1983) Kant’s Political Philosophy. New York: St Martin’s Press. https://doi.org/10.1017/S1369415422000176 Published online by Cambridge University Press https://doi.org/10.1017/S1369415422000176 Published online by Cambridge University Press Svoboda, Toby (2012) ‘Duties Regarding Nature: A Kantian Approach to Environmental Ethics’. Kant University Press), 101 11. Singer, Peter (1990) Animal Liberation. New York: New York Review of Books/Random House. Svoboda, Toby (2012) ‘Duties Regarding Nature: A Kantian Approach to Environmental Ethics’. Kant g ( ) / Svoboda, Toby (2012) ‘Duties Regarding Nature: A Kantian Approach to Environmental Ethics’. Kant Y b k 4(1) 143 63 Svoboda, Toby (2012) ‘Duties Regarding Nature: A Kantian Approach to Environmental Ethics’. Kant Yearbook, 4(1), 143–63. Svoboda, Toby (2012) ‘Duties Regarding Nature: A Kantian Approach to Environmental Ethics’. Kant Yearbook, 4(1), 143–63. Yearbook, 4(1), 143–63. Vereb, Zachary (2019) ‘Moral Views of Nature: Normative Implications of Kant’s Critique of Judgment’. Public Reason, 10(2)/11(1), 127–42. Vereb, Zachary (2019) ‘Moral Views of Nature: Normative Implications of Kant’s Critique of Judgment’. Public Reason, 10(2)/11(1), 127–42. Vereb, Zachary (2019) ‘Moral Views of Nature: Normative Implications of Kant’s Critique of Judgment’. Public Reason, 10(2)/11(1), 127–42. https://doi.org/10.1017/S1369415422000176 Published online by Cambridge University Press Zachary Vereb 420 Cite this article: Vereb, Z. (2022). Sustaining the Individual in the Collective: A Kantian Perspective for a Sustainable World. Kantian Review 27, 405–420. https://doi.org/10.1017/S1369415422000176
https://openalex.org/W2283728539	https://orca.cardiff.ac.uk/id/eprint/91885/1/Zacharopoulos_et_al-2016.pdf	English	null	The genetics of neuroticism and human values	Genes, brain and behavior	2,016	cc-by	6,316	George Zacharopoulos†,∗, Thomas M. Lancaster†,‡,§, Gregory R. Maio† and David E. J. Linden†,‡,§ contrasts motives to follow the status quo (conservation) against motives to pursue personal intellectual and emo- tional interests in uncertain directions (openness). One impor- tant characteristic of this circumplex model is that it makes speciﬁc predictions about sinusoidal associations between social values and external variables. As shown in Fig. 1b, this sinusoidal waveform becomes evident if the values are ordered according to their positions along the value circle: an external variable that is most positively related to a par- ticular value should manifest less positive and progressively more negative correlations until reaching the opposing value type. This prediction has received support in many studies ﬁnding that values at opposite ends of the circular model exhibit opposing relations to other judgements and behaviour (see Schwartz 1996) and in one study observing a sinusoidal pattern in relations between values and personality traits (Parks-Leduc et al. 2015). This sinusoidal waveform supports the model’s assumptions about latent motivational conﬂicts between values. †School of Psychology, ‡Neuroscience and Mental Health Research Institute, and §MRC Centre for Neuropsychiatric Genetics and Genomics, Cardiff University, Cardiff, UK *Corresponding author: G. Zacharopoulos, School of Psychology, Cardiff University, Tower Building 70 Park Place, Cardiff CF10 3AT, UK. E-mail: zacharopoulosg@cardiff.ac.uk Human values and personality have been shown to share genetic variance in twin studies. However, there is a lack of evidence about the genetic components of this association. This study examined the interplay between genes, values and personality in the case of neuroti- cism, because polygenic scores were available for this personality trait. First, we replicated prior evidence of a positive association between the polygenic neuroticism score (PNS) and neuroticism. Second, we found that the PNS was signiﬁcantly associated with the whole human value space in a sinusoidal waveform that was consistent with Schwartz’s circular model of human values. These results suggest that it is useful to consider human val- ues in the analyses of genetic contributions to personal- ity traits. They also pave the way for an investigation of the biological mechanisms contributing to human value orientations. However, the exact genetic loci driving this association between values from Schwartz’s model and personality have remained obscure. This association can be investigated by utilizing a growing body of knowledge on personality genet- ics. As complex psychological dispositions, human values and personality traits are both likely to be affected by numerous genes simultaneously (in addition to strong environmental inﬂuences). George Zacharopoulos†,∗, Thomas M. Lancaster†,‡,§, Gregory R. Maio† and David E. J. Linden†,‡,§ To capture the genetic inﬂuence of complex traits and values, it is therefore useful to focus on genetic indices that reﬂect the contribution of a great number of single nucleotide polymorphisms (SNPs), such as polygenic scores derived from Genome-Wide Association Studies (GWAS). Keywords: Genetics, human values, neuroticism, personality, polygenic score Thus far, a polygenic score has been identiﬁed only for one trait: neuroticism. A polygenic neuroticism score (PNS) is available through a recent meta-analysis of GWAS of personality traits (N = 63 661) (Genetics of Personality Consortium et al. 2015). Neuroticism is a personality factor ranging from emotional stability to high nervousness, ten- sion and moodiness. In the meta-analysis, a neuroticism score (NS) was derived from a number of measures includ- ing the NEO Personality Inventory, the Eysenck Personality Questionnaire, the International Personality Item Pool inven- tory, harm avoidance scores in Cloninger’s Tridimensional Personality Questionnaire and negative emotionality scores in the Multidimensional Personality Questionnaire. The meta-analysis showed that 0.6% of the variance in this NS was explained by the PNS. Although this low percentage suggests only a small genetic component, it was reliable and potentially important, making it a relevant candidate for studying genetic contributions to neuroticism and other individual differences related to neuroticism. Received 28 November 2015, revised 27 January 2016, 19 February 2016, accepted for publication 21 February 2016 Received 28 November 2015, revised 27 January 2016, 19 February 2016, accepted for publication 21 February 2016 The beliefs people have about ideals that are important in life, their ‘values’, are reliably associated with certain personality traits (Parks-Leduc et al. 2015; Rim 1984). Extending this con- nection, studies of twins have found that the shared variance between human values and personality has a signiﬁcant her- itable component (Schermer et al. 2008, 2011). Schermer et al.’s (2008, 2011) analyses of shared genetic variance between traits and values utilized Schwartz’s (1992) circular model of values. This model is supported by data from over 70 nations with a range of cross-sectional, lon- gitudinal and experimental methods (Maio 2010; Schwartz et al. 2012). The model posits the existence of 10 types of social values (Fig. 1a), with each expressing speciﬁc motives. These motives are organized along two dimen- sions. © 2016 The Authors. Genes, Brain and Behavior published by International Behavioural and Neural Genetics Society and John Wiley & Sons Ltd. 361 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Genes, Brain and Behavior (2016) 15: 361–366 doi: 10.1111/gbb.12286 Genes, Brain and Behavior (2016) 15: 361–366 doi: 10.1111/gbb.12286 Genes, Brain and Behavior (2016) 15: 361–366 doi: 10.1111/gbb.12286 The genetics of neuroticism and human values George Zacharopoulos†,∗, Thomas M. Lancaster†,‡,§, Gregory R. Maio† and David E. J. Linden†,‡,§ Subjects A l f j A total of 81 right-handed Caucasian university students aged between 19 and 42 (50 females; mean ± SD age = 23.85 ± 3.71) par- ticipated in the study, which was approved by the Ethics Committee in the School of Psychology, Cardiff University. Participants were informed that the study examined the connection between value–morality judgements and biological indices. They took part individually in the laboratory, wherein they completed the measures of human values and personality, provided a saliva sample, and were then debriefed. The sample used consisted of an existing sample collected for behavioural analysis. In this study we included all the participants from the existing sample for which the human value score, personality score and the genetic score were available. However, a different possibility emerges if we consider relevant research examining links between neuroticism and relevant affective states and attitudes. Neuroticism is asso- ciated with a higher likelihood of anxiety and depression, which are two hallmarks of emotional instability that lead people to withdraw from the world around them (Angst et al. 2003; Thompson et al. 2011). This pattern suggests that emotional instability may cause people to be less open to new experiences, ideas and feelings, because of the poten- tial threats to their fragile emotional state. Convergent with these observations, lower levels of neuroticism are associ- ated with more liberal, curious and open-minded attitudes (Carney et al. 2008; Van Hiel & Mervielde 2004). Strong links between such attitudes and Schwartz’s openness value type (Ashton et al. 2005) suggest that an inverse relation between openness values (see Fig. 1a) and neuroticism is viable. George Zacharopoulos†,∗, Thomas M. Lancaster†,‡,§, Gregory R. Maio† and David E. J. Linden†,‡,§ One dimension contrasts motives to promote the self (self-enhancement) against motives that transcend personal interests (self-transcendence), whereas the other dimension The shared genetic associations between personality traits and human values provide a foundation for expecting that the polygenic association with neuroticism may also relate to 361 Zacharopoulos et al. Figure 1: (a) The circumplex structure of personal values. (b) Plot of hypothesized relationships between three external variables (A, B and C) and the 10 values (SD, self-direction; ST, stimulation; HE, hedonism; AC, achievement; PO, power; SE, security; CO, conformity; TR, tradition; BE, benevolence; UN, universalism). Each dot point could represent a correlation coefﬁcient (modiﬁed from Schwartz 1992). Figure 1: (a) The circumplex structure of personal values. (b) Plot of hypothesized relationships between three external variables (A, B and C) and the 10 values (SD, self-direction; ST, stimulation; HE, hedonism; AC, achievement; PO, power; SE, security; CO, conformity; TR, tradition; BE, benevolence; UN, universalism). Each dot point could represent a correlation coefﬁcient (modiﬁed from Schwartz 1992). Figure 1: (a) The circumplex structure of personal values. (b) Plot of hypothesized relationships between three external variables (A, B and C) and the 10 values (SD, self-direction; ST, stimulation; HE, hedonism; AC, achievement; PO, power; SE, security; CO, conformity; TR, tradition; BE, benevolence; UN, universalism). Each dot point could represent a correlation coefﬁcient (modiﬁed from Schwartz 1992). in a sinusoidal pattern congruent with Schwartz’s circumplex model of values. value orientations. Human values are particularly interesting in connection to neuroticism. A recent meta-analysis of the relations between human values and the big ﬁve traits found reliable trait–value associations, except when looking at neu- roticism (Parks-Leduc et al. 2015). The authors explained this non-association using Cloninger’s (1994) proposition that neu- roticism is more appropriately described as a temperament (i.e. an automatic associative response to emotional stimuli) than as a character trait (i.e. a self-aware volitional concept related to behavioural intentions). This indicates a stronger biological component to neuroticism than to other traits, which, like human values, may be amenable to higher levels of cognitive processing and control. Thus, from this perspec- tive, neuroticism may manifest a genetic component, but lit- tle association with human values. in a sinusoidal pattern congruent with Schwartz’s circumplex model of values. Genes, Brain and Behavior (2016) 15: 361–366 Human values P i i l Participants completed the 56-item Schwartz Value Survey (Schwartz 1992). Participants rated the importance of each of the 56 values as a guiding principle in their lives, using a quasi-bipolar 9-point scale rang- ing from −1 (opposed to my values), 0 (not important), 4 (important) to 7 (of supreme importance). Examples of Schwartz Value Survey items are as follows: ‘equality: equal opportunity for all’ (universal- ism); ‘pleasure: gratiﬁcation of desires’ (hedonism); ‘obedient: dutiful meeting obligations’ (conformity). The average score across the 56 items was then calculated and subtracted from each of the 56 initial raw scores. Schwartz recommends this procedure to help control for superﬂuous individual variations in rating styles (Schwartz 1992). The individual centred item scores were then averaged to form scores for each type of value examined in Schwartz’s model (see Fig. 1a). The internal consistency of these indices was moderate to good (see Table 1). The present research was therefore motivated by the shared genetic variance between human values and person- ality, the existence of a polygenic score for neuroticism, and the ambiguity about neuroticism value relations. We sought to test whether the potential genetic contribution to neu- roticism has similar patterns of the association with human values and the trait on a phenotypic level. To be clear, we were not predicting that values mediate the link between genes and traits or that traits mediate the link between genes and values. In theory, values and traits should recip- rocally inﬂuence each other, as stable individual differences over time, leading to an association that is bidirectional. Our principal aim was to test whether associations with genes emerged for both the trait and values. Moreover, we wished to detect whether any observed associations arose DNA extraction and genotyping Genomic DNA was obtained from saliva using Oragene OG-500 saliva kits. Genotyping was performed using custom genotyping arrays (Illumina HumanCoreExome-24 BeadChip), which contain 570 038 genetic variants (Illumina, Inc., San Diego, CA, USA). Quality con- trol was implemented in PLINK (Purcell et al. 2007) to ensure that the genotypes did not display ambiguous sex, cryptic relatedness (up to third-degree relatives by the identity of descent), genotyping com- pleteness <97% and non-European ethnicity admixture (detected as outliers in iterative EIGENSTRAT analyses of an LD-pruned data set) (Price et al. 2006). The SNPs were excluded where the minor allele frequency was <1%, if the call rate <98% or if the 𝜒2-test for Hardy–Weinberg Equilibrium had a P-value <1 e-04. Individuals’ genotypes were imputed using the pre-phasing/imputation step- wise approach implemented in IMPUTE2/SHAPEIT (Delaneau et al. 2012; Howie et al. 2009) and 1000Genomes (December 2013, release 1000 Genome haplotypes Phase I integrated variant set) as the reference data set. (2) In this eqn (2), K represents the number of correlation coefﬁcients, yk represents the correlation coefﬁcients, yk represents the esti- mated correlation coefﬁcient through the optimization function and yk represents the mean of the correlation coefﬁcients. The denominator is the formula for the variance. Hanel et al. (2016) tested the number of false-positive results for the SFI, using three simulations of m = 100 000 samples each in R. To simulate a random pattern of correlation coefﬁcients, they relied upon two assumptions about the distribution of the correlation coef- ﬁcients. First, they sampled 10 numbers (i.e. number of human val- ues) between −0.5 and 0.5, with k being the number of correlation coefﬁcients (k = 10), assuming a uniform distribution. The numbers −0.5 to 0.5 represent the interval in which most correlation coef- ﬁcients of values with external variables usually fall. Second, they sampled 10 numbers from a normal distribution with ∼N(0, 0.1) and ∼N(0, 0.3). Numbers >\|1\| were restricted to −1 or 1, respectively. For the obtained values of SFI <0.20, the percentages of false positives were below 1% for all the three simulations of 100 000 samples. The percentage of false positives for an SFI <0.20 was 0.49% (i.e. less than ﬁve false-positive results per 1000 comparisons) assuming a nor- mal distribution and 0.76% assuming a uniform distribution. Similarly, assuming normal and uniform distributions, respectively, the false positives were 0.20% and 0.30% for SFI <0.15. y = f (x) = a + b × sin (c × x + d) , (1) Generation of risk proﬁle scores p The PNS was calculated using the method described by the International Schizophrenia Consortium (International Schizophrenia Consortium et al. 2009). The PNS was estimated using publicly available data from the international GWAS (Genetics of Personality Consortium et al. 2015). The SNPs were subsequently pruned for linkage disequilibrium (r2 < 0.2). This method ensured that all SNPs included in the PNS model were fairly independent. The PNSs were calculated using the ‘score’ command in PLINK, which averages the number of risk alleles for each index SNP, weighted by the natural logarithm of the SNP’s odds ratio extracted from the GWAS results (Genetics of Personality Consortium et al. 2015). From the 6 949 612 SNPs, a total of 206 516 quasi-independent SNPs were considered in the PNS (PT < 0.5). We calculated PNS at the liberal P-threshold (PT < 0.5), because it best predicted NS in the GWAS reference data (Genetics of Personality Consortium et al. 2015). There were no outliers in the PNSs, and the scores were normally distributed (Shapiro–Wilk: P > 0.3). The genetics of neuroticism and human values Table 1: Cronbach’s 𝛼for each of the 10 values Table 1: Cronbach’s 𝛼for each of the 10 values Value Number of items Cronbach’s 𝛼 Universalism 7 0.76 Benevolence 9 0.76 Tradition 6 0.63 Conformity 4 0.63 Security 6 0.68 Power 5 0.79 Achievement 6 0.67 Hedonism 2 0.74 Stimulation 3 0.79 Self-direction 6 0.65 Firstly, all four of the parameters (a, b, c and d) of the sinusoidal function were optimized with the R command optim. The parameter a, the y-offset, which moves the function up and down along the ordinate, was restricted to between −1 and 1, as were the correlation coefﬁcients. The same restrictions were applied to parameter b, which determines the differences between the turning points of the sinusoidal function (amplitude). The parameter c, the period of the sine wave, was allowed to range from 85% to 95% of a full sine wave. This restriction was based on the circular model’s assumption that ‘the distances between the values around the circle may not be equal’ (Schwartz et al. 2012). Given that the ﬁrst value type was plotted at x = 1, the parameter d (x-offset), which moves the sinusoidal function along the abscissa, was set to the interval [1 + k/2, 1 −k/2]. Therefore, parameter d was unrestricted because there was no hypothesis regarding the exact starting point of the sine wave for each of the two measures, PNS and NS. To deﬁne a lower and upper bound given these constraints, a method developed by Byrd et al. (1995) was used. We calculated the sum of the squared residuals divided by the variance to estimate the model ﬁt indices for the sinusoidal function. This ﬁt is called the Sinusoidal Fit Index (SFI) (Hanel et al. 2016) and is presented below (eqn 2). et al. 2003; Thompson et al. 2011), and HEXACO’s emotionality dimension is well suited to detecting the links with values (Pozzebon & Ashton 2009). SFI = 1 K −1 K ∑ k=1 (yk −̂yk )2 1 K −1 K ∑ k=1 (yk −yk )2 (2) SFI = 1 K −1 K ∑ k=1 (yk −̂yk )2 1 K −1 K ∑ k=1 (yk −yk )2 (2) Replicating the link between PNS and NS Replicating the link between PNS and NS Our ﬁrst aim was to provide further evidence on the associa- tion between emotionality (NS from HEXACO-PI-R) and PNS. As expected, we obtained a positive association between these variables, r79 = 0.22, P = 0.048 (Fig. 2), replicating the ﬁndings of the personality GWAS (Genetics of Personality Consortium et al. 2015). Personality measure W iﬁd NS i h y We quantiﬁed NS using the 100-item self-reported version of the HEXACO Personality Inventory-Revised (HEXACO-PI-R) (Lee & Ashton 2004). In the HEXACO-PI-R, NS is termed emotionality, and it features subscales for fearfulness, anxiety, dependence and sentimentality. These subscales are combined together as the total emotionality score (𝛼= 0.64). Furthermore, many inﬂuential research programmes have interpreted and labelled neuroticism from the big ﬁve as emotional stability (De Raad et al. 2010; Goldberg 1990; Saucier 1994). It was previously shown that the HEXACO emotional- ity represents an alternative rotation of big ﬁve neuroticism (Ashton et al. 2014) and that they are similar constructs (Ashton et al. 2014; Romero et al. 2015). Furthermore, the emotionality score provides a particularly interesting and important rendition of neuroticism in this context because of its relative emphasis on emotional instability, which leads people to withdraw from the world around them (Angst 362 Genes, Brain and Behavior (2016) 15: 361–366 y = f (x) = a + b × sin (c × x + d) , (1) y = f (x) = a + b × sin (c × x + d) , DNA extraction and genotyping For SFI <0.10, the false positives were 0.05% and 0.08%, and for SFI <0.05, the false positives were 0.005% and 0.007%. Genes, Brain and Behavior (2016) 15: 361–366 Sinusoidal relationship analysis p To test for a sinusoidal pattern of association between values, NS and PNS, we calculated the correlation coefﬁcients of the 10 value types with NS and PNS. The ﬁt of the sinusoidal function presented below (eqn 1) was calculated using the programme R. Structure of values 3b) but the ﬁt to the Figure 3: Correlation coefﬁcients between the 10 value types (x-axis, conformity, tradition, benevolence, universalism, self-direction, stimulation, hedonism, achievement, power and security) and PNS (a) and NS (b). human value benevolence deviated from the sine wave; run- ning the sinusoidal test while excluding benevolence yields a signiﬁcant SFI = 0.08 (Fig. S1). Overall, NS and PNS map onto the human value space in similar, sinusoidal waveforms. Furthermore, in addition to testing the patterns of correlations using the SFI method, we replicated the PNS and NS ﬁnd- ings using two previously established methods, with even more robust results (Boer & Fischer 2013; Roccas et al. 2002) (Appendix S1). Discussion Figure 2: Scatter-plot depicting the positive association between NS and PNS. Both NS (derived from the HEXACO-PI-R, see Material and methods) and PNS were standardized with a z-score transformation. Each dot represents a participant. Figure 2: Scatter-plot depicting the positive association between NS and PNS. Both NS (derived from the HEXACO-PI-R, see Material and methods) and PNS were standardized with a z-score transformation. Each dot represents a participant. Schwartz’s hypothesized circular structure in our sample. This test used two multi-dimensional scaling (MDS) as recommended by Schwartz (Bilsky et al. 2011). The ﬁrst analysis plotted the 56-value items, and the second anal- ysis plotted the 10 higher order values. Both the analyses use the respective correlation matrix to plot the values in a two-dimensional space. The ﬁrst analysis yielded S-Stress = 0.167 and Stress I = 0.274, whereas the second analysis yielded S-Stress = 0.032 and a Stress-I = 0.115. The stress value is an index of how well the data ﬁt the hypothesized conﬁguration; higher stress values signify a poorer conﬁguration. The stress values and the patterns in the MDS (see Table 1) were consistent with the structure hypothesized by Schwartz (1992). In addition, the openness values, self-direction and stimulation, were signiﬁcantly negatively related to NS, but these associations did not reach signiﬁcance when related to the PNS. Figure 3: Correlation coefﬁcients between the 10 value types (x-axis, conformity, tradition, benevolence, universalism, self-direction, stimulation, hedonism, achievement, power and security) and PNS (a) and NS (b). human value benevolence deviated from the sine wave; run- ning the sinusoidal test while excluding benevolence yields a signiﬁcant SFI = 0.08 (Fig. S1). Overall, NS and PNS map onto the human value space in similar, sinusoidal waveforms. Fitting the sinusoidal model to the NS and PNS Fitting the sinusoidal model to the NS and PNS Given our replication of Schwartz’s circular structure in the MDS analyses, we turned to testing whether there are sinu- soidal patterns of association between values and NS and PNS. To address this question, we plotted the correlation coefﬁcients between NS and PNS on the y-axis and each of the 10 lower order values on the x-axis in an order that follows their circular structure. The patterns are shown in Fig. 3. A pattern of sinusoidal association was found between human values and PNS, particularly near the inﬂec- tion points (Fig. 3a), which was signiﬁcant, SFI = 0.19; false positives = 0.6%. Similarly, our analysis of NS show a sinu- soidal association of a similar form (Fig. 3b) but the ﬁt to the sine wave was not reliable, SFI = 29; P > 0.05. Visual inspec- tion of Fig. 3a shows that the correlation between NS and the Structure of values Before testing for a sinusoidal waveform in the pattern of associations between values and NS, and PNS, we validated where x is a vector containing the correlation coefﬁcients of the 10 values with either PNS or NS. 363 Zacharopoulos et al. Figure 3: Correlation coefﬁcients between the 10 value types (x-axis, conformity, tradition, benevolence, universalism, self-direction, stimulation, hedonism, achievement, power and security) and PNS (a) and NS (b). Zacharopoulos et al. Figure 2: Scatter-plot depicting the positive association between NS and PNS. Both NS (derived from the HEXACO-PI-R, see Material and methods) and PNS were standardized with a z-score transformation. Each dot represents a participant. Schwartz’s hypothesized circular structure in our sample. This test used two multi-dimensional scaling (MDS) as recommended by Schwartz (Bilsky et al. 2011). The ﬁrst analysis plotted the 56-value items, and the second anal- ysis plotted the 10 higher order values. Both the analyses use the respective correlation matrix to plot the values in a two-dimensional space. The ﬁrst analysis yielded S-Stress = 0.167 and Stress I = 0.274, whereas the second analysis yielded S-Stress = 0.032 and a Stress-I = 0.115. The stress value is an index of how well the data ﬁt the hypothesized conﬁguration; higher stress values signify a poorer conﬁguration. The stress values and the patterns in the MDS (see Table 1) were consistent with the structure hypothesized by Schwartz (1992). In addition, the openness values, self-direction and stimulation, were signiﬁcantly negatively related to NS, but these associations did not reach signiﬁcance when related to the PNS. Fitting the sinusoidal model to the NS and PNS Given our replication of Schwartz’s circular structure in the MDS analyses, we turned to testing whether there are sinu- soidal patterns of association between values and NS and PNS. To address this question, we plotted the correlation coefﬁcients between NS and PNS on the y-axis and each of the 10 lower order values on the x-axis in an order that follows their circular structure. The patterns are shown in Fig. 3. A pattern of sinusoidal association was found between human values and PNS, particularly near the inﬂec- tion points (Fig. 3a), which was signiﬁcant, SFI = 0.19; false positives = 0.6%. Similarly, our analysis of NS show a sinu- soidal association of a similar form (Fig. Genes, Brain and Behavior (2016) 15: 361–366 Structure of values Furthermore, in addition to testing the patterns of correlations using the SFI method, we replicated the PNS and NS ﬁnd- ings using two previously established methods, with even more robust results (Boer & Fischer 2013; Roccas et al. 2002) (Appendix S1). The genetics of neuroticism and human values Second, the NS variance explained from the PNS was much higher in this study (4%) than in the initial discovery sample (0.6%). A number of factors may account for the larger rela- tion in our study. First, this study measured neuroticism using a single scale in a single homogeneous cohort, whereas the meta-analytic study assessed neuroticism from multiple instruments (even in the same cohort). Second, this study used a single measure of neuroticism with subscales (fear- fulness, anxiety, dependence and sentimentality) that are different and more emotional in focus than in the replica- tion cohort in the meta-analytic study (NEO-FFI’s neuroticism: anxiety, hostility, depression, self-consciousness, impulsive- ness, vulnerability to stress and Amsterdam Biographical Questionnaire). Third, the power of this study merely allows the detection of a moderate effect, and future replication studies may yield a smaller effect; therefore, future research should interpret the current effect size with caution. Despite these possibilities, the current replication of the NS–PNS relation is promising for future research attempting to learn more about this relation and its implications. important dimension of personality, neuroticism. We used empirically robust measures of human values, neuroticism and genetic neuroticism. The results replicated the associ- ation between NS and PNS despite using a different mea- sure of neuroticism than in prior research (i.e. emotionality from HEXACO-PI-R). This result adds to the evidence that the PNS derived by GWAS helps to explain individual varia- tion in neuroticism (Genetics of Personality Consortium et al. 2015). Moreover, it laid the foundation for testing whether human values are linked to both NS and PNS. Results indi- cated that human values were indeed associated with NS and PNS, following the sinusoidal pattern predicted by Schwartz et al. (2012) cross-cultural model. important dimension of personality, neuroticism. We used empirically robust measures of human values, neuroticism and genetic neuroticism. The results replicated the associ- ation between NS and PNS despite using a different mea- sure of neuroticism than in prior research (i.e. emotionality from HEXACO-PI-R). This result adds to the evidence that the PNS derived by GWAS helps to explain individual varia- tion in neuroticism (Genetics of Personality Consortium et al. 2015). Moreover, it laid the foundation for testing whether human values are linked to both NS and PNS. Results indi- cated that human values were indeed associated with NS and PNS, following the sinusoidal pattern predicted by Schwartz et al. (2012) cross-cultural model. References Angst, J., Gamma, A. & Endrass, J. (2003) Risk factors for the bipolar and depression spectra. Acta Psychiatr Scand Suppl, 418, 15–19. Ashton, M.C., Danso, H.A., Maio, G.R., Esses, V.M., Bond, M.H. & Keung, D.K.Y. (2005) Two dimensions of political attitudes and their individual difference correlates: a cross-cultural perspective. Ont Symp P 10, 1–29. Ashton, M.C., Lee, K. & de Vries, R.E. (2014) The HEXACO honesty-humility, agreeableness, and emotionality factors: a review of research and theory. Pers Soc Psychol Rev 18, 139–152. Two other aspects of our results merit further discussion. First, it is informative to contrast the sinusoidal pattern, which is a test of association across all values, with the strength of the correlations with speciﬁc values. This is interesting in part because most of the correlations between speciﬁc val- ues and PNS or NS were weak and unreliable, aside from the signiﬁcant theoretically congruent correlations discussed above (see Table S1). Nonetheless, the sinusoidal ﬁt shows a crucial pattern that is missing from univariate tests that focus on one value at a time. It is possible for individual relations to be weak at the same time as their combined pattern is meaningful and reliable. In the analyses of values, this differ- ence between individual correlations and the net pattern is crucial, because the relative roles of different values are psy- chologically more important and meaningful than the roles of any single value type in isolation, because of the competing implications between values (Rokeach 1973; Schwartz 1992). Bilsky, W., Janik, M. & Schwartz, S.H. (2011) The structural organiza- tion of human values-evidence from three rounds of the European social survey (ESS). J Cross-Cult Psychol 42, 759–776. Boer, D. & Fischer, R. (2013) How and when do personal values guide our attitudes and sociality? explaining cross-cultural variability in attitude-value linkages. Psychol Bull 139, 1113–1147. Byrd, R.H., Lu, P.H., Nocedal, J. & Zhu, C.Y. (1995) A limited memory algorithm for bound constrained optimization. Siam J Sci Comput 16, 1190–1208. Carney, D.R., Jost, J.T., Gosling, S.D. & Potter, J. (2008) The secret lives of liberals and conservatives: personality proﬁles, interaction styles, and the things they leave behind. Polit Psychol 29, 807–840. Cloninger, C.R. (1994) Temperament and personality. Curr Opin Neu- robiol 4, 266–273. De Raad, B., Barelds, D.P., Levert, E., Ostendorf, F., Mlacic, B., Di Blas, L., Hrebickova, M., Szirmak, Z., Szarota, P., Perugini, M., Church, A.T. & Katigbak, M.S. The genetics of neuroticism and human values These ﬁndings fundamentally extend the understanding of human values. Previous twin studies (Keller et al. 1992; Schermer et al. 2008, 2011; Waller et al. 1990) have docu- mented that human values may have a genetic component, but this has occurred without simultaneously pinpointing rel- evant patterns of genes, the pattern of associations with the values and the nature of the common association to the behavioural phenotype for personality. Here, we document a novel sinusoidal relationship between human values and a speciﬁc genetic marker, the PNS – a relationship that was very similar to that found between NS and values. In summary, the present research (1) replicated the prior evidence of a polygenic contribution to neuroticism using a novel measure of the trait, (2) showed an association between speciﬁc genetic components and human values for the ﬁrst time and (3) found a pattern of associations with values that is congruent with Schwartz’s (1992) and Schwartz et al.’s (2012) circular model of values. Together, these results show that it is useful to include value orien- tations as relevant individual differences in polygenic contri- butions to neuroticism-related traits, suggesting that future research should consider values in investigations of polygenic contributions to other traits. Furthermore, as expected, Fig. 3 shows that the sinusoidal waveforms were anchored at one end by negative relations between values promoting stimulation or self-direction on one hand and NS or PNS on the other hand. This pattern ﬁts links between neuroticism and anxiety and depres- sion. As noted earlier, anxiety and depression lead people to withdraw from the world around them (Angst et al. 2003; Thompson et al. 2011). In addition, higher levels of neuroticism are associated with less liberal, curious and open-minded attitudes (Carney et al. 2008; Van Hiel & Mervielde 2004). Neuroticism may contribute to lower open- ness to new experiences, ideas and feelings because of the threats posed by novelty. At the same time, the pattern of withdrawal elicited by lower stimulation and self-direction values may contribute to emotional instability by increasing rumination, perseveration in an isolated environment and self-absorption. Further evidence is needed to explore these possibilities. Discussion The present research investigated the genetic components connected to the relations between human values and an 364 Genes, Brain and Behavior (2016) 15: 361–366 Genes, Brain and Behavior (2016) 15: 361–366 References (2010) Only three factors of personality description are fully replicable across languages: a comparison of 14 trait taxonomies. J Pers Soc Psychol 98, 160–173. 365 Zacharopoulos et al. Delaneau, O., Marchini, J. & Zagury, J.F. (2012) A linear complex- ity phasing method for thousands of genomes. Nat Methods 9, 179–181. Schermer, J.A., Vernon, P.A., Maio, G.R. & Jang, K.L. (2011) A behavior genetic study of the connection between social values and person- ality. Twin Res Hum Genet 14, 233–239. Genetics of Personality Consortium, de Moor, M.H., van den Berg, S.M. et al. (2015) Meta-analysis of genome-wide association stud- ies for neuroticism, and the polygenic association with major depressive disorder. JAMA Psychiatry 72, 642–650. y Schwartz, S.H. (1992) Universals in the content and structure of values - theoretical advances and empirical tests in 20 countries. Adv Exp Soc Psychol 25, 1–65. Schwartz, S. (1996) Value priorities and behavior: applying a theory of integrated value systems. In Seligman C., Olson J. M., & Zanna M. P. (eds), Psychology of Values: The Ontario Symposium, Lawrence Erlbaum Assoc., Mahwah, NJ. Vol. 8, pp. 1–24. Goldberg, L.R. (1990) An alternative "description of personality": the big-ﬁve factor structure. J Pers Soc Psychol 59, 1216–1229. Goldberg, L.R. (1990) An alternative "description of personality": the big-ﬁve factor structure. J Pers Soc Psychol 59, 1216–1229. Hanel, P.H.P., Zacharopoulos, G., Mégardon, G., & Maio, G.R. (2016) Detecting sinusoidal patterns of prediction from circumplex models of psychological constructs. g y , Hanel, P.H.P., Zacharopoulos, G., Mégardon, G., & Maio, G.R. (2016) Detecting sinusoidal patterns of prediction from circumplex models of psychological constructs. Erlbaum Assoc., Mahwah, NJ. Vol. 8, pp. 1–24 Schwartz, S.H., Cieciuch, J., Vecchione, M., Davidov, E., Fischer, R., Beierlein, C., Ramos, A., Verkasalo, M., Lonnqvist, J.E., Demirutku, K., Dirilen-Gumus, O. & Konty, M. (2012) Reﬁning the theory of basic individual values J Pers Soc Psychol 103 Schwartz, S.H., Cieciuch, J., Vecchione, M., Davidov, E., Fischer, R., Beierlein, C., Ramos, A., Verkasalo, M., Lonnqvist, J.E., Demirutku, K., Dirilen-Gumus, O. & Konty, M. (2012) Reﬁning the theory of basic individual values. J Pers Soc Psychol 103, 663–688. Howie, B.N., Donnelly, P. & Marchini, J. (2009) A ﬂexible and accu- rate genotype imputation method for the next generation of genome-wide association studies. PLoS Genet 5, e1000529. the theory of basic individual values. J Pers Soc Psychol 103, 663–688. Acknowledgments Pozzebon, J.A. & Ashton, M.C. (2009) Personality and values as predictors of self- and peer-reported behavior. J Individ Differ 30, 122–129. This study was supported by the National Centre for Men- tal Health (NCMH) at Cardiff University, with funds from the National Institute for Social Care and Health Research (NISCHR), Welsh Government, Wales (grant number BR09) and by grant MR/K004360/1 from the Medical Research Council (MRC) and by the MRC Centre for Neuropsychiatric Genetics and Genomics (G0800509). We are grateful to all professionals, patients and volunteers involved with the National Centre for Mental Health (NCMH). Price, A.L., Patterson, N.J., Plenge, R.M., Weinblatt, M.E., Shadick, N.A. & Reich, D. (2006) Principal components analysis corrects for stratiﬁcation in genome-wide association studies. Nat Genet 38, 904–909. Purcell, S., Neale, B., Todd-Brown, K., Thomas, L., Ferreira, M.A., Bender, D., Maller, J., Sklar, P., de Bakker, P.I., Daly, M.J. & Sham, P.C. (2007) PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet 81, 559–575. Rim, Y. (1984) Importance of values according to personality, intelli- gence and sex. Pers Individ Differ 5, 245–246. References International Schizophrenia Consortium, Purcell, S.M., Wray, N.R., Stone, J.L., Visscher, P.M., O’Donovan, M.C., Sullivan, P.F. & Sklar, P. (2009) Common polygenic variation contributes to risk of schizophrenia and bipolar disorder. Nature 460, 748–752. Thompson, R.J., Berenbaum, H. & Bredemeier, K. (2011) Cross-sectional and longitudinal relations between affective instability and depression. J Affect Disord 130, 53–59. Van Hiel, A. & Mervielde, I. (2004) Openness to experience and boundaries in the mind: relationships with cultural and economic conservative beliefs. J Pers 72, 659–686. Keller, L.M., Arvey, R.D., Dawis, R.V., Bouchard, T.J. & Segal, N.L. (1992) Work values - genetic and environmental-inﬂuences. J Appl Psychol 77, 79–88. Waller, N.G., Kojetin, B.A., Bouchard, T.J., Lykken, D.T. & Tellegen, A. (1990) Genetic and environmental-inﬂuences on religious interests, attitudes, and values - a study of twins reared apart and together. Psychol Sci 1, 138–142. Lee, K. & Ashton, M.C. (2004) Psychometric properties of the HEX- ACO Personality Inventory. Multivar Behav Res 39, 329–358. Maio, G.R. (2010) Mental representations of social values. Adv Exp Soc Psychol 42, 1–43. Parks-Leduc, L., Feldman, G. & Bardi, A. (2015) Personality traits and personal values: a meta-analysis. Pers Soc Psychol Rev 19, 3–29. Genes, Brain and Behavior (2016) 15: 361–366 Supporting Information Additional supporting information may be found in the online version of this article at the publisher’s web-site: Roccas, S., Sagiv, L., Schwartz, S.H. & Knafo, A. (2002) The big ﬁve personality factors and personal values. Pers Soc Psychol B 28, 789–801. Table S1: Pearson correlation coefﬁcients between PNS and NS with the 10 human values (conformity, benevolence, tradition, universalism, self-direction, stimulation, hedonism, achievement, power and security). Rokeach, M. (1973) The Nature of Human Values. Free Press Collier-Macmillan, New York and London. Romero, E., Villar, P. & Lopez-Romero, L. (2015) Assessing six factors in Spain: validation of the HEXACO-100 in relation to the ﬁve factor model and other conceptually relevant criteria. Pers Individ Differ 76, 75–81. Figure S1: Correlation coefﬁcients between the nine value types (x-axis, conformity, tradition, universalism, self-direction, stimulation, hedonism, achievement, power and security) and NS. Saucier, G. (1994) Mini-markers: a brief version of Goldberg’s unipolar big-ﬁve markers. J Pers Assess 63, 506–516. Schermer, J.A., Feather, N.T., Zhu, G. & Martin, N.G. (2008) Pheno- typic, genetic, and environmental properties of the portrait values questionnaire. Twin Res Hum Genet 11, 531–537. Appendix S1: Replication of the sinusoidal ﬁndings of PNS and NS using two previously established methods. Appendix S1: Replication of the sinusoidal ﬁndings of PNS and NS using two previously established methods. 366 Genes, Brain and Behavior (2016) 15: 361–366
https://openalex.org/W4391784614	https://www.frontiersin.org/journals/psychiatry/articles/10.3389/fpsyt.2024.1352641/pdf	English	null	Exploring global and local processes underlying alterations in resting-state functional connectivity and dynamics in schizophrenia	Frontiers in psychiatry	2,024	cc-by	11,349	TYPE Original Research PUBLISHED 13 February 2024 DOI 10.3389/fpsyt.2024.1352641 TYPE Original Research PUBLISHED 13 February 2024 DOI 10.3389/fpsyt.2024.1352641 TYPE Original Research PUBLISHED 13 February 2024 DOI 10.3389/fpsyt.2024.1352641 KEYWORDS schizophrenia, resting-state fMRI, computational model, large-scale networks, functional connectivity, temporal dynamics OPEN ACCESS OPEN ACCESS EDITED BY Massoud Stephane, Oregon Health and Science University, United States REVIEWED BY Lin Liu, Peking University, China Pierluigi Selvaggi, University of Bari Aldo Moro, Italy CORRESPONDENCE Christoph Metzner cmetzner@ni.tu-berlin.de RECEIVED 08 December 2023 ACCEPTED 19 January 2024 PUBLISHED 13 February 2024 CITATION Metzner C, Dimulescu C, Kamp F, Fromm S, Uhlhaas PJ and Obermayer K (2024) Exploring global and local processes underlying alterations in resting-state functional connectivity and dynamics in schizophrenia. Front. Psychiatry 15:1352641. doi: 10.3389/fpsyt.2024.1352641 Christoph Metzner 1,2,3, Cristiana Dimulescu 1,4, Fabian Kamp 1,5,6, Sophie Fromm 1,7, Peter J. Uhlhaas 2,8 and Klaus Obermayer 1,4 Christoph Metzner 1,2,3, Cristiana Dimulescu 1,4, Fabian Kamp 1,5,6, Sophie Fromm 1,7, Peter J. Uhlhaas 2,8 and Klaus Obermayer 1,4 1Neural Information Processing Group, Institute of Software Engineering and Theoretical Computer Science, Technische Universität Berlin, Berlin, Germany, 2Department of Child and Adolescent Psychiatry, Charite´ – Universitätsmedizin Berlin, Berlin, Germany, 3School of Physics, Engineering and Computer Science, University of Hertfordshire, Hatﬁeld, United Kingdom, 4Bernstein Center for Computational Neuroscience Berlin, Berlin, Germany, 5Max Planck School of Cognition, Max Planck Institute for Human Cognitive and Brain Science, Leipzig, Germany, 6Center for Lifespan Psychology, Max Planck Institute for Human Development, Berlin, Germany, 7Department of Psychiatry and Psychotherapy, Charite´ – Universitätsmedizin Berlin, Berlin, Germany, 8Institute of Neuroscience and Psychology, University of Glasgow, Glasgow, United Kingdom Introduction: We examined changes in large-scale functional connectivity and temporal dynamics and their underlying mechanisms in schizophrenia (ScZ) through measurements of resting-state functional magnetic resonance imaging (rs-fMRI) data and computational modelling. schizophrenia, resting-state fMRI, computational model, large-scale networks, functional connectivity, temporal dynamics Exploring global and local processes underlying alterations in resting-state functional connectivity and dynamics in schizophrenia OPEN ACCESS EDITED BY Massoud Stephane, Oregon Health and Science University, United States REVIEWED BY Lin Liu, Peking University, China Pierluigi Selvaggi, University of Bari Aldo Moro, Italy CORRESPONDENCE Christoph Metzner cmetzner@ni.tu-berlin.de RECEIVED 08 December 2023 ACCEPTED 19 January 2024 PUBLISHED 13 February 2024 CITATION Metzner C, Dimulescu C, Kamp F, Fromm S, Uhlhaas PJ and Obermayer K (2024) Exploring global and local processes underlying alterations in resting-state functional connectivity and dynamics in schizophrenia. Front. Psychiatry 15:1352641. doi: 10.3389/fpsyt.2024.1352641 2.1 Patient Sample The study sample was collected through the Center for Biomedical Research Excellence (COBRE) led by Dr. Vince Calhoun (more information here: http://fcon1000.projects.nitrc.org/ indi/retro/cobre.html) and obtained from the SchizConnect database (http://schizconnect.org). This sample has previously been used by our group to explore structural deﬁcits in patients with ScZ [Dimulescu et al. (25)]. From the sample of 43 patients and 43 healthy control participants, we excluded 5 patients due to missing resting-state functional MRI (rs-fMRI) data or artefacts/excessive motion identiﬁed during the pre-processing. We thus analyzed a ﬁnal sample of 43 healthy control subjects and 38 patients with schizophrenia, which we will refer to as the COBRE sample. All patients were receiving antipsychotic medication (see Table 1). Symptom severity in patients was assessed using the Positive and Negative Syndrome Scale (PANSS) [Kay et al. (26)]. Written informed consent was obtained from all participants, and the study was reviewed and approved by the Institutional Review Board of the University of New Mexico. Yet, the origin of functional dysconnectivity patterns in ScZ is still unclear. One hypothesis is that cellular and synaptic changes associated with ScZ disrupt local processing and thus impact on large-scale connectivity. Indeed changes at the microcircuit level have been identiﬁed in ScZ. Excitatory and inhibitory neurotransmission is disturbed, for example a reduced excitatory drive onto GABAergic inhibitory neurons [Chung et al. (15, 16) and a decreased inhibitory output (Hashimoto et al. (17); Morris et al. (18); Moyer et al. (19)]. Changes to the glutamatergic system, such as increased recurrent excitation, have been suggested to lead to deﬁcits in large-scale connectivity with a gradient along the cortical hierarchy [Yang et al. (20)]. TABLE 1 Demographics and clinical characteristics. HC ScZ Statistics, p value Group size 43 38 – Age (y) 36.70 (11.04) 38.97(13.67) t=0.82, p=0.41 Gender 11F/32M 10F/28M c2 = 0.02, p=0.88 PANSS positive – 14.92(5.04) – PANSS negative – 14.81(5.31) – PANSS general – 29.49(8.37) – PANSS total – 59.22(78) – CPZ- equivalent dosage – 396.26 (330.91) – Illness duration (y) – 17.19(12.61) – Data are shown as mean(standard deviation). Age differences between groups were compared using an independent samples t-test and differences in gender distribution using a chi-square test. Antipsychotic medication is reported as chlorpromazine (CPZ)-equivalent dosage. Computational models of large-scale brain circuits can be used to investigate dynamical circuit mechanisms linking local ScZ- associated alterations to global changes in the functional organisation of the brain. COPYRIGHT © 2024 Metzner, Dimulescu, Kamp, Fromm, Uhlhaas and Obermayer. 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. Methods: The rs-fMRI measurements from patients with chronic ScZ (n=38) and matched healthy controls (n=43), were obtained through the public schizConnect repository. Computational models were constructed based on diffusion-weighted MRI scans and ﬁt to the experimental rs-fMRI data. Results: We found decreased large-scale functional connectivity across sensory and association areas and for all functional subnetworks for the ScZ group. Additionally global synchrony was reduced in patients while metastability was unaltered. Perturbations of the computational model revealed that decreased global coupling and increased background noise levels both explained the experimentally found deﬁcits better than local changes to the GABAergic or glutamatergic system. Discussion: The current study suggests that large-scale alterations in ScZ are more likely the result of global rather than local network changes. schizophrenia, resting-state fMRI, computational model, large-scale networks, functional connectivity, temporal dynamics 01 01 Frontiers in Psychiatry frontiersin.org 10.3389/fpsyt.2024.1352641 Metzner et al. 1 Introduction patients. We then implemented local microcircuit and global network parameter changes in a computational model of large- scale cortical dynamics and compare the resulting connectivity changes to the experimental data. Furthermore, we also explored the temporal dynamics of the resting-state brain and characterised potential deﬁcits in large-scale synchrony and metastability in ScZ patients and compared them to the different computational models, thus identifying mechanistic links underlying these changes. ScZ is a severe mental disorder with a high burden of disease [Lopez and Murray (1); Charlson et al. (2)]. However, the underlying mechanisms remain elusive. While no single brain area accounting for the heterogeneous symptom proﬁles has been identiﬁed, the notion that ScZ can be understood in terms of a general dysconnectivity has emerged [Friston et al. (3); Friston (4), Bullmore et al. (5); Pettersson-Yeo et al. (6)]. Experimental evidence for the dysconnection hypothesis comes from neuroimaging studies. Analyses of resting-state fMRI connectivity have shown widespread changes of functional connectivity. However, there is still a debate whether correlations of neural activity between regions are decreased [Liang et al. (7); Bluhm et al. (8)] or increased in ScZ [Zhou et al. (9)]. There is also growing evidence for possible longitudinal changes of functional connectivity over the course of the disorder. Anticevic et al. (10) demonstrated that prefrontal cortical connectivity is increased in early-course ScZ while the opposite pattern was observed in chronic ScZ patients. Going beyond pairwise correlations between brain regions, graph theoretic measurements have identiﬁed reductions in integration, hierarchy, clustering, efﬁciency and small-worldness [Bassett et al. (11); Liu et al. (12); Bullmore and Sporns (13); Lynall et al. (14)]. Frontiers in Psychiatry Data are shown as mean(standard deviation). Age differences between groups were compared using an independent samples t-test and differences in gender distribution using a chi-square test. Antipsychotic medication is reported as chlorpromazine (CPZ)-equivalent dosage. 2.3 Resting-state functional MRI data GBC(i) = 1 n (o j FC(i, j)), COBRE data was acquired using single-shot full k-space echo- planar imaging (EPI) with ramp sampling correction using the intercomissural line (AC-PC) as a reference (TR: 2 s, TE: 29 ms, matrix size: 64x64, 32 slices, voxel size: 3x3x4 mm3). The resting- state scans were acquired in the axial plane with with an ascending slice order (multi slice method; interleaved). For more information see Aine et al. (28). For the COBRE data set, we preprocessed the rsfMRI data using the FSL FEAT toolbox [Woolrich et al. (29)]. For each data set, we discarded the ﬁrst ﬁve volumes. We analyzed the relative mean framewise displacement as the root mean square (RMS) of the translation parameters. We found an average RMS of 0.15(± 0.09) for the control group and an RMS of 0.20 (± 0.10) for the patient group (t=2.0884, p=0.04). These results are in line with previous studies indicating that ScZ patients have higher framewise motion displacement than healthy controls [Guo et al. (30)]. We thus corrected head motion using the FSL McFLIRT algorithm and subsequently high-pass ﬁltered the data with a ﬁlter cutoff of 100 s. We linearly registered each functional image to the corresponding anatomical image of that subject using FLIRT. We then used the mean volume of the data to create a brain mask using BET. Using the ICA FIX FSL toolbox [Griffanti et al. (31); Salimi-Khorshidi et al. (32)], we conducted MELODIC ICA and removed artefactual components (motion, non-neuronal physiological artefacts, scanner artefacts, and other nuisance sources). Finally, we transformed the where n is the number of regions. The average global GBC can then be deﬁned as the average GBC over all cortical regions i. To calculate the average GBC for a functional subnetwork or generally a set of regions, one simply averages over the regional GBC values for the respective regions. To assess the temporal dynamics of the functional networks, we used the Kuramoto order parameter as a measure of synchrony and its standard deviation as a measure of metastability, i.e. the variability of the states of phase conﬁgurations over time [see for example Deco et al. (36)]. Here the Kuramoto order parameter R(t) is deﬁned as: R(t) = 1 n jo n k=1 eifk(t)j, where again n is the number of regions and fk(t) is the instantaneous phase of the BOLD signal in region k. 2.2 Anatomical data high-resolution mask volumes from MNI to individual subject functional space and extracted the average BOLD time courses for each cortical region in the AAL2 parcellation scheme using the fslmeants command from Fslutils. Data collection for the COBRE sample was performed using a Siemens Magnetom Trio 3T MR scanner. Structural images (high resolution T1-weighted) were acquired using a ﬁve-echo MPRAGE sequence with the following parameters: repetition time (TR) = 2530ms; echo time (TE) = 1.64, 3.5, 5.36, 7.22, 9.08ms; inversion time (TI) = 1200ms; ﬂip angle (FA) = 7°; ﬁeld of view (FOV) = 256mm × 256mm; matrix = 256 × 256; slice thickness = 1mm; 192 sagittal slices. Diffusion tensor imaging (DTI) data were acquired using a single-shot EPI sequence with TR/TE = 9000/84ms; FA = 90°; FOV =256mm × 256mm; matrix = 128 × 128; slice thickness = 2mm without gap; 72 axial slices; 30 non-collinear diffusion gradients (b = 800s/mm2) and 5 non-diffusion-weighted images (b = 0s/mm2) equally interspersed between the 30 gradient directions. For more information see also Cetin et al. (27). Acquisition details for the functional MRI data from the HCP S1200 release can be found here: https://www.humanconnectome.org/ study/hcp-young-adult/document/1200-subjects-data-release. For the HCP data set, we used the data preprocessed according to Glasser et al. (33) and extracted the average BOLD time courses for each cortical region as described above. 2.1 Patient Sample Leveraging such computational models, studies have shown that decreases in global inter-regional connectivity strengths can lead to wide-spread functional disruptions [Cabral et al. (21)], increased global signal variance [Yang et al. (22)] and altered topological characteristics of functional brain networks (Cabral et al. (23, 24) resembling ScZ. However, except for Yang et al. (22), these studies only investigated a global scaling of the inter-regional connectivity. Yang et al. (22) manipulated local and global neuronal coupling and demonstrated that both could increase signal variance as seen in ScZ but did not explore their potentially differential effects on large-scale functional connectivity. Thus, so far the effect of ScZ-associated local changes to glutamatergic and GABAergic neurotransmission and the effect of increased background noise on large-scale functional connectivity has not been explored. To address this question, we quantiﬁed functional connectivity differences in a data set of healthy controls and chronic ScZ 02 frontiersin.org Metzner et al. 10.3389/fpsyt.2024.1352641 2.4 Measures of connectivity and temporal dynamics We used the average global brain connectivity (GBC) measure (Cole et al. (34, 35) to assess the changes in connectivity strength. To assess alterations in temporal dynamics we used synchrony and metastability [Deco et al. (36)]. Because of the computational model being restricted to cortical areas, we also restricted our connectivity analysis to cortical areas. However, including subcortical regions did not substantially change the ﬁndings (see Supplementary Material). For model validation we additionally used a subset of 156 healthy participants from the human connectome project (HCP), which we will refer to as the HCP sample. The diffusion-weighted data were collected with multiband diffusion sequence (HCP version available at http://www.cmrr.umn.edu/multiband). Three different gradient tables are used, each with 90 diffusion weighting directions and six b = 0 acquisitions. More information can be found at https://www.humanconnectome.org/study/hcp-young- adult/document/1200-subjects-data-release. Speciﬁcally, we deﬁne the functional connectivity matrix (FC) as the matrix of Pearson correlations of the BOLD signal between two brain areas over the whole time range of acquisition. From the FC matrices we calculate the global brain connectivity (GBC) of a single brain region i as follows (see also Cole et al. (34, 35): 2.5.1 Single-Node model Furthermore, Kgl scales the global coupling in the network, and Cij and Dij deﬁne the connection strengths and the connection delays between regions, estimated from the ﬁbre count and ﬁbre length matrices, respectively. Finally, dabE = 1 for a = b = E and 0 otherwise restricting coupling between regions to be exclusively from excitatory to excitatory populations. given a certain delay for the spike transmission dab. Here cab represent the amplitude of the post-synaptic current resulting from one individual spike (for sab = 0). Furthermore, Kgl scales the global coupling in the network, and Cij and Dij deﬁne the connection strengths and the connection delays between regions, estimated from the ﬁbre count and ﬁbre length matrices, respectively. Finally, dabE = 1 for a = b = E and 0 otherwise restricting coupling between regions to be exclusively from excitatory to excitatory populations. The adaptive exponential integrate-and-ﬁre model explicitly accounts for the evolution of a slow adaptation currents that represents both subthreshold and spike-triggered adaptation currents. The subthreshold adaptation current is described by the adaptation conductance a and the spike-triggered adaptation current is denoted by b. In the limit of inﬁnite population sizes, an adiabatic approximation can be employed to describe the mean adaptation current in terms of the mean population ﬁring rate. The mean adaptation current IA can be understood as an inhibitory membrane current whose dynamics are governed by: A single network node in the whole-brain model is represented by the population activity of two interconnected neural populations, an excitatory population E and an inhibitory population I. The dynamics of the membrane currents of a population a ∈{E, I}, are governed by the following equations: ta dma dt = msyn a (t) + mext a (t) + mou a (t) −ma(t) msyn a = JaEsaE(t) + JaIsaI(t) dIA dt = t−1 A (a(VE(t) −EA) −IA) −brE(t) : s 2 a (t) = o b∈E,I f g 2J2 abs 2 s,ab(t)ts,btm (1 + rab(t))tm + ts,b + s 2 ext,a The individual populations a of a single region of the whole- brain network receive an external input current with a given mean mext a and a standard deviation s ext a (t). This background input current can be thought to represent baseline input from extracortical areas in the brain. 2.5 Computational network model time constant. Furthermore, s 2 a is the variance of the membrane currents, and Jab represent the maximum synaptic current when all synapses from population b to population a are active. The dynamics of the synapses are described by: We use a whole-brain network model, where the connectivity, connection strength and delay between network nodes (i.e. brain regions) is derived from brain imaging data (Figure 2). As a model of single-node activity dynamics we employ a mean-ﬁeld description of a spiking neural network of an excitatory and an inhibitory neural population, where the individual neurons are described by the adaptive exponential integrate-and-ﬁre model [AdEx model; Brette and Gerstner (37)], developed in our group [Augustin et al. (38); Cakan and Obermayer (39)]. The following section describes the model in detail. dsab dt = t−1 s,b ((1 −sab(t))rab(t) −sab(t)) ds 2 sab dt = t−2 s,b (1 −sab(t))2rab(t)) + (rab(t) + 2ts,b(rab(t) + 1)s2 sab(t)) where sab represents the mean of the fraction of all active synapses, which lies in the range [0,1], with the extreme cases being no active synapses and no inactive synapses, respectively. Furthermore, s 2 sab is the variance of sab. Frontiers in Psychiatry 2.3 Resting-state functional MRI data It measures the global level of synchronization of the BOLD signals from all regions, where a low level close to 0 reﬂects an almost uniform distribution of the signal phases, and a high value close to 1 reﬂects near equality of the signal phases. To calculate R, we band-pass ﬁltered the signal in the narrowband 0.04-0.07Hz [see Deco et al. (36)] and then extracted the instantaneous phases of the signals at every time step using the Hilbert transform. Frontiers in Psychiatry 03 frontiersin.org Metzner et al. 10.3389/fpsyt.2024.1352641 2.5.1 Single-Node model The timescale ta = FT(µa, sa) of the input-dependent adaptation, the average membrane potential VE = FE(µE, sE), and the instantaneous population spike rate ra = Fr(µa, sa) are computed every time step by means of precomputed transfer functions. The mean rab and the variance rab of the effective input rate from population b to population a can be described by: A mean-ﬁeld neural mass model based on a spiking network of coupled excitatory and inhibitory populations, the so-called ALN model [Augustin et al. (38)], was implemented. The mean-ﬁeld description offers a drastic speed-up of simulations on the order of about 4 orders of magnitude compared to the spiking model while still retaining its dynamical states and its biophysical parameters. The model has been extensively validated against simulations with the detailed spiking network and overall shows very good agreement [Cakan and Obermayer (39)]. rab = cab Jab ts,b Kb · rb(t −dab) + dabE · Kglo N j=0 Cij(rb −Dij) ! and rab = c2 ab J2 ab t2 s,b Kb · rb(t −dab) + dabE · Kglo N j=0 C2 ij(rb −Dij) ! rab = cab Jab ts,b Kb · rb(t −dab) + dabE · Kglo N j=0 Cij(rb −Dij) ! and The mean-ﬁeld reduction of the spiking neural network utilises the Fokker-Planck approach, i.e. the fact that in the limit of an inﬁnite network size and under the assumption of a sparse, random connectivity, the distribution p(V) of the membrane potentials and the mean ﬁring rate ra of a population a, can be described by a Fokker-Planck equation [Brunel (40)]. However, to calculate the potential distribution a partial differential equation has to be solved, which is computationally costly. Therefore, the dynamics of a population is captured by a low-dimensional linear-nonlinear cascade model, and can be described by a set of ordinary differential equations [Fourcaud-Trocmé et al. (41); Ostojic and Brunel (42)]. The mathematical derivation and the underlying assumptions have been detailed in [Augustin et al. (38)], and we will only provide the ﬁnal set of model equations in this manuscript. rab = c2 ab J2 ab t2 s,b Kb · rb(t −dab) + dabE · Kglo N j=0 C2 ij(rb −Dij) ! given a certain delay for the spike transmission dab. Here cab represent the amplitude of the post-synaptic current resulting from one individual spike (for sab = 0). 2.5.1 Single-Node model Additionally, the regions also receive a noise input current mou a (t) modelled as an Ornstein- Uhlenbeck process with a mean of 0 described by In the above equations µa describes the total mean membrane currents, msyn a the currents from synaptic activity, mext a the currents from any sources of external input, mou a the external noise input, tmthe membrane time constant (calculated from the membrane capacitance C and the leak conductance gL), and ts,b the synaptic 04 frontiersin.org frontiersin.org 10.3389/fpsyt.2024.1352641 Metzner et al. Metzner et al. Metzner et al. dmou a dt = −mou a tou + soux(t) : TABLE 2 Network parameters. Parameter Value Description µext E µext I 1.63 mV/ms 0.05 mV/ms Mean external input to E Mean external input to I sou 0.19 Noise strength tou 5.0 ms Noise time constant Ke 800 Number of excitatory inputs per neuron Ki 200 Number of inhibitory inputs per neuron CEE,CIE 0.3 mV/ms Maximum AMPA PSC amplitude CEI,CII 0.5 mV/ms Maximum GABA PSC amplitude JEE 2.4 mV/ms Maximum synaptic current from E to E JIE 2.6 mV/ms Maximum synaptic current from E to I JEI -3.3 mV/ms Maximum synaptic current from I to E JII -1.6 mV/ms Maximum synaptic current from I to I ts,E 2 ms Excitatory synaptic time constant ts,I 5 ms Inhibitory synaptic time constant dE 4 ms Synaptic delay to excitatory neurons dI 2 ms Synaptic delay to inhibitory neurons C 200 pF Membrane capacitance gL 10 nS Leak conductance tm C/gL Membrane time constant EL -65 mV Leak reversal potential of the AdEx model DT 1.5 mV Threshold slope factor of the AdEx model VT -50 mV Threshold voltage of the AdEx model Vs -40 mV Spike voltage threshold of the AdEx model Tnet 1.5 ms Refractory time of the AdEx model sext 1:5 mV= ﬃﬃﬃﬃﬃﬃ ms p Standard deviation of external input EA -80 mV Adaptation reversal potential a 28.26 nS Subthreshold adaptation conductance b 24.04 pA Spike-triggered adaptation increment tA 200 ms Adaptation time constant Kgl 250.0 Global coupling strength vgl 20.0 m/s Global signal speed Overview of the different parameter values for the whole-brain model employed here. Here x(t) is a white noise process drawn from a normal distribution with a mean of 0 and a variance of 1. sou determines the ﬂuctuation amplitude of the noise around its mean. 2.5.3 Network connectivity Structural images were preprocessed employing a semi- automatic pipeline implemented in the FSL toolbox (www.fmrib.ox.ac.uk/fsl, FMRIB, Oxford). For the anatomical T1- weighted images we used the brain extraction toolbox (BET) in FSL to remove non-brain tissue and to generate the brain masks. After manual quality checks, 80 cortical regions were deﬁned according to the automatic anatomical labelling (AAL2) atlas [Rolls et al. (47)]. For the diffusion-weighted images, we performed a brain extraction as well and corrected the images for head motion and eddy current distortions afterwards. Probabilistic ﬁbre tracking, using the Bayesian Estimation of Diffusion Parameters Obtained using Sampling Techniques (BEDPOSTX) and the PROBTRACKX algorithms implemented in FSL [Behrens et al. (48)], was then used with 5,000 random seeds per voxel to extract individual connectomes. Since the tractography does not yield directionality information and the connectome matrices are non-symmetric, we explicitly enforced symmetry by averaging the entries from region i to region j and from region j to region i for all pairs of regions. Furthermore, we normalised each connectome by dividing each matrix entry by the maximum matrix entry, thus ensuring compatibility between participants. The resulting connectome then determines the relative coupling strength between regions in the above described computational whole-brain model. The ﬁbre Overview of the different parameter values for the whole-brain model employed here. tracking also yielded matrix ﬁbre lengths for each participant, which, when multiplied with the signal speed, determines the delay of signal propagation between any two regions in the model. 2.5.1 Single-Node model To determine the mean external input to the E (µEext) and I (µIext) populations, the noise strength sou, the subthreshold adaptation conductance a and spike-triggered adaptation increment b parameters for the model in the control condition, we used an evolutionary optimization procedure as described in Cakan et al. (43). We compared the simulated BOLD FC to the empirical rsfMRI data. We initialized the algorithm with a random population of Ninit = 160 individuals and repeated the evolutionary block with Npop = 80 individuals for 100 generations. Initial parameter values were selected from a uniform distribution across the following intervals for the model parameters: µEext ∈[0.0,4.0] mV/ms, µIext ∈[0.0,4.0] mV/ms, sou ∈[0.0,0.3], a ∈[0.0,40.0] nS, and b ∈[0.0,40.0] pA. The global coupling strength was set as in Figure 2 of Cakan et al. (43). All other model parameters were set as given in Table 1 in Cakan et al. (43) and they are summarised in Table 2. 2.5.2 BOLD model In order to compare the model output, i.e. the neural activity of the regions, to the BOLD signal of the rs-fMRI data, the ﬁring rates of the excitatory population of each region had to be converted into model BOLD signal timecourses. Here, we used the well-established Balloon-Windkessel model [Friston et al. (44); Deco et al. (45)], for speciﬁc parameters see Friston et al. (46). Frontiers in Psychiatry 2.5.4 Modelling ScZ-associated changes We implemented four different sets of parameter changes that are thought to represent the following four ScZ-associated alterations: 1) local GABAergic inhibition, 2) local glutamatergic Frontiers in Psychiatry 05 frontiersin.org Metzner et al. 10.3389/fpsyt.2024.1352641 excitation of inhibitory cells, 3) global interregional coupling, and 4) global noise levels. change in symptomatology or type/dose of antipsychotic medication during the three months before the assessment [for more details see Aine et al. (28)]. First, we systematically reduced GABAergic inhibition in the model. Postmortem gene expression studies have robustly demonstrated reduced levels of parvalbumine (PV) and somatostatin [SST) expression in PV (Hashimoto et al. (17)] and SST [Morris et al. (18)] interneurons together with a reduction of GAD65 and GAD67 [Hashimoto et al. (17)], in cortical regions in ScZ. We implemented these changes as a reduction of the inhibitory weights JEI and JII in the ALN model of the regional dynamics. We varied the strength of the inhibition onto the excitatory population JEI and onto the inhibitory population JII simultaneously in the range from 100% to 60% in steps of 5%. 2.5.5 Simulation details Simulations were implemented using the neurolib Python framework [Cakan et al. (52)]. The differential equations of the model were solved numerically using an Euler forward scheme with a time step of 0.1ms. For all described simulations the duration was 70s and we discarded the transient response in the ﬁrst 5 s before calculating any of the above described measures. To assess the robustness of our results, we created 40 virtual subjects by changing the seed for the random number generator underlying the Ornstein- Uhlenbeck noise process. These 40 virtual subjects were then kept ﬁxed for all implemented changes allowing for a direct comparison to the default, ‘healthy’ condition. 3.3.1 Control model 3.1 Demographic and clinical characteristics 3.2 Global differences in connectivity strength and temporal dynamics between ScZ patients and healthy controls Global dysconnectivity might also be explained by a simple reduction of the global connectivity strength. Therefore, to test whether the differences we found experimentally could alternatively be explained by an overall network decoupling, we reduced the global coupling strength Kgl in the range from 100% to 60% in steps of 5%. We further tested whether the GBC differences we found were speciﬁc to association areas as indicated by a previous study [Yang et al. (20)]. We grouped the default mode subnetwork, the control subnetwork and the ventral attention subnetwork together as the association areas and the somatomotor subnetwork, the visual subnetwork and the dorsal attention subnetwork as the sensory areas. We found reduced GBC for ScZ patients in both groupings, with the sensory areas showing an even stronger effect than the association areas (effect sizes g = −0.78 for sensory areas versus g = −0.61 for association areas, see Figure 1E and Table 3). Finally, the global alterations of functional connectivity might also be the result of an increase in background noise disrupting functional connectivity in the network [Winterer et al. (49); Winterer and Weinberger (50); Winterer et al. (51)]. Consequently, we increased the global background noise sou in a range from 100% to 140% in steps to 5%, to test whether a global increase in noise level can account for the connectivity differences found in the experimental data. Since our sample showed a signiﬁcant difference in head motion between controls and patients, we investigated whether the changes in connectivity and dynamics were still present when applying a very strict threshold for head motion. Speciﬁcally, we had 4 control participants and 5 patients with a framewise displacement > 0.3. We repeated the analysis of FC and temporal dynamics after removing these subjects. Details of this analysis can be found in the Supplementary Table S3. Removal of the participants did not alter the results signiﬁcantly with one exception. For the synchrony measure the mean difference and effect size both slightly decreased and did not reach statistical signiﬁcance anymore. 3.2 Global differences in connectivity strength and temporal dynamics between ScZ patients and healthy controls Global GBC was signiﬁcantly reduced in patients with ScZ (effect size g = −0.65; see Figure 1A and Table 3). Comparing both groups a substantial shift from high GBC towards medium to low GBC values occurs in ScZ patients (Figure 1B and Table 3). Synchrony, as measured by the Kuramoto order parameter was lower in the patient group (effect size g = −0.44; see Figure 1C and Table 3). However, variability in synchrony, measured by metastability, did not signiﬁcantly differ between groups (Figure 1D and Table 3). Next, we systematically reduced the glutamatergic, excitatory drive onto inhibitory neurons in our model of regional activity. These changes reﬂected the reduced and more varied colocalization of glutamatergic pre- and postsynaptic markers on PV interneurons in dorsolateral prefrontal cortex (DLPFC) (Chung et al. (15, 16). Speciﬁcally, we reduced the excitatory weight onto inhibitory neurons JIE in the ALN model in a range from 100% to 60% in steps of 5%. Next, we systematically reduced the glutamatergic, excitatory drive onto inhibitory neurons in our model of regional activity. These changes reﬂected the reduced and more varied colocalization of glutamatergic pre- and postsynaptic markers on PV interneurons in dorsolateral prefrontal cortex (DLPFC) (Chung et al. (15, 16). Speciﬁcally, we reduced the excitatory weight onto inhibitory neurons JIE in the ALN model in a range from 100% to 60% in steps of 5%. Global dysconnectivity might also be explained by a simple reduction of the global connectivity strength. Therefore, to test whether the differences we found experimentally could alternatively be explained by an overall network decoupling, we reduced the global coupling strength Kgl in the range from 100% to 60% in steps of 5%. Finally, the global alterations of functional connectivity might also be the result of an increase in background noise disrupting functional connectivity in the network [Winterer et al. (49); Winterer and Weinberger (50); Winterer et al. (51)]. Consequently, we increased the global background noise sou in a range from 100% to 140% in steps to 5%, to test whether a global increase in noise level can account for the connectivity differences found in the experimental data. Reductions of functional connectivity strengths affected all seven subnetworks (effect sizes ranging from g = −0.57 to g = −0.83; see Table 3), with the dorsal-attention, the somato-motor and the visual subnetworks showing the strongest effects (Figure 1F). Frontiers in Psychiatry 3.1 Demographic and clinical characteristics We derived a model of healthy large-scale cortical activity that matched the behaviour of the control group data from the COBRE study well in terms of functional connectivity (Figures 2B, D). The correlation between simulated FC (simFC) and empirical FC (empFC) (r = 0.33 ± 0.09; Figure 2E) was higher than the The control and the patient group did not differ signiﬁcantly in terms of age and gender (see Table 1). Patients also did not show a 06 frontiersin.org 10.3389/fpsyt.2024.1352641 Metzner et al. A B D E F C FIGURE 1 Global differences in functional connectivity and temporal dynamics between healthy controls and ScZ patients. (A) Comparison of average GBC per participant for the two groups. Individual dots represent average GBC for one participant. The difference plot on the right shows the difference between the groups in terms of effect size. (B) Histogram of region-wise GBC values for the two groups. The histogram displays the region-wise GBC data pooled for all participants in each group. (C) Synchrony comparison between the two groups. Each dot represents the mean Kuramoto order parameter (a measure of synchrony) for one participant. The difference plot on the right shows the group difference in terms of effect size. (D) Metastability comparison between the two groups. Each dot represents the metastability of one participant. The difference plot on the right shows the group difference in terms of effect size. (E) Comparison of global brain connectivity for association areas (Asso. comprising: DMN, Cont, Sal/ VAttn) and sensory areas (Sen. comprising: Sommot, Vis, DAttn). (F) Comparison of global brain connectivity for the seven functional networks from Yeo et al. (53): SomMot, Somato-motor subnetwork; Cont, Control subnetwork; Def, Default mode subnetwork; Sal/VAttn, Salience/Ventral attention subnetwork; DAttn, Dorsal attention subnetwork; Lim, Limbic subnetwork; Vis, Visual subnetwork. A A B C D D E E F FIGURE 1 Global differences in functional connectivity and temporal dynamics between healthy controls and ScZ patients. (A) Comparison of average GBC per participant for the two groups. Individual dots represent average GBC for one participant. The difference plot on the right shows the difference between the groups in terms of effect size. (B) Histogram of region-wise GBC values for the two groups. The histogram displays the region-wise GBC data pooled for all participants in each group. (C) Synchrony comparison between the two groups. 3.1 Demographic and clinical characteristics Each dot represents the mean Kuramoto order parameter (a measure of synchrony) for one participant. The difference plot on the right shows the group difference in terms of effect size. (D) Metastability comparison between the two groups. Each dot represents the metastability of one participant. The difference plot on the right shows the group difference in terms of effect size. (E) Comparison of global brain connectivity for association areas (Asso. comprising: DMN, Cont, Sal/ VAttn) and sensory areas (Sen. comprising: Sommot, Vis, DAttn). (F) Comparison of global brain connectivity for the seven functional networks from Yeo et al. (53): SomMot, Somato-motor subnetwork; Cont, Control subnetwork; Def, Default mode subnetwork; Sal/VAttn, Salience/Ventral attention subnetwork; DAttn, Dorsal attention subnetwork; Lim, Limbic subnetwork; Vis, Visual subnetwork. Frontiers in Psychiatry 3.3.2 Modelling ScZ-associated alterations correlation between empirical structural (empSC) and empFC (r = 0.19 ± 0.07; Figure 2E). We systematically performed perturbations to four key aspects of the model that have been associated with schizophrenia: 1) local GABAergic inhibition, 2) local glutamatergic excitation of inhibitory cells, 3) global interregional coupling, and 4) global noise levels. To further assert that the default model captures the resting- state functional connectivity of healthy subjects well, we also validated the model behaviour against a set of 156 subjects from the HCP S1200 release. Here, we also found a good ﬁt for functional connectivity (Figures 2C, D). We found that changing the inhibitory weights (model perturbation 1) did not alter the global GBC and the GBCs for sensory and association areas signiﬁcantly. Furthermore, the changes did not alter the synchrony and the metastability (see Supplementary Table S3). As for the local changes to the inhibitory system, changes to the glutamatergic excitatory drive to the inhibitory population (model perturbation 2) did not result in signiﬁcant changes in GBC on all levels, synchrony and metastability (see Supplementary Table S4). Overall, the model functional connectivity correlated well with the empirical functional connectivity of individual HCP subjects (r = 0.43 ± 0.08; see Figure 2E). This correlation was again substantially higher than the correlation of structural connectivity and empirical functional connectivity (r = 0.20 ± 0.08; see Figure 2E). 07 frontiersin.org 10.3389/fpsyt.2024.1352641 Metzner et al. TABLE 3 Local and global group differences. Mean difference Hedges’ 95% CI p value g Global cortical GBC -0.11 -0.65 [-1.11 -0.18] p=0.0056 Global cortical synchrony -0.12 -0.44 [-0.89 0.04] p=0.0488 Global cortical metastability -0.005 -0.39 [-0.81 0.07] p=0.0850 GBC Sensory areas -0.12 -0.78 [-1.26 -0.29] p=0.0004 GBC Association areas -0.09 -0.61 [-1.07 -0.14] p=0.0076 GBC Somato-motor (SomMot) -0.12 -0.74 [-1.20 -0.26] p=0.0008 GBC Control (Cont) -0.09 -0.57 [-1.03 -0.10] p=0.0110 GBC Default mode (Def) -0.09 -0.57 [-1.03 -0.11] p=0.0110 GBC Salience/Ventral attention (Sal/VAttn) -0.11 -0.69 [-1.14 -0.21] p=0.0024 GBC Dorsal attention (DAttn) -0.12 -0.83 [-1.31 -0.33] p=0.0001 GBC Limbic (Lim) -0.08 -0.59 [-1.02 -0.13] p=0.0102 GBC Visual (Vis) -0.11 -0.77 [-1.26 -0.29] p=0.0010 Overview of the global and local differences in functional connectivity and temporal dynamics between the healthy control and the ScZ patient group. 3.3.2 Modelling ScZ-associated alterations An increase in noise levels (model perturbation 4) yielded a strong decrease in global brain connectivity as well as connectivity within the sensory and association systems, even stronger than for the global coupling changes (Table 5). Additionally, synchrony decreased strongly and metastability increased for larger noise strengths (Table 5). A reduction of global coupling (model perturbation 3) resulted in a strong decrease in global brain connectivity as well as connectivity within the sensory and association systems (Table 4). Additionally, synchrony decreased strongly and metastability increased for larger reductions (Table 4). A B D E C FIGURE 2 Computational model (A) Modelling approach combining a model for the regional dynamics with anatomical input that deﬁnes the structural network. (B) Average FC matrix for the COBRE sample (C) Average FC matrix for the HCP sample (D) Model FC matrix (E) comparison of the correlation of empSC to empFC (blue) and the correlation of simFC and empFC (yellow) for the COBRE (left) and the for the HCP (right) data sets. A A A B D D D C B B C B E E FIGURE 2 Computational model (A) Modelling approach combining a model for the regional dynamics with anatomical input that deﬁnes the structural network. (B) Average FC matrix for the COBRE sample (C) Average FC matrix for the HCP sample (D) Model FC matrix (E) comparison of the correlation of empSC to empFC (blue) and the correlation of simFC and empFC (yellow) for the COBRE (left) and the for the HCP (right) data sets 08 Frontiers in Psychiatry frontiersin.org 10.3389/fpsyt.2024.1352641 Metzner et al. TABLE 4 ScZ-associated changes of global coupling. 95% 90% 85% 80% Avg. global GBC -0.035 [-0.45] -0.089 [-1.12] -0.153 [-1.88] -0.205 [-2.49] Avg. GBC sen. -0.039 [-0.47] -0.097 [-1.16] -0.169 [-1.99] -0.231 [-2.68] Avg. GBC ass. -0.032 [-0.40] -0.0.089 [-1.10] -0.159 [-1.91] -0.215 [-2.49] Synchrony -0.008 [-0.10] -0.040 [-0.53] -0.093 [-1.29] -0.155 [-2.20] Metastability -0.001 [-0.05] 0.001 [0.03] 0.001 [0.04] 0.007 [0.26] 75% 70% 65% 60% Avg. global GBC -0.238 [-3.16] -0.258 [-3.63] -0.265 [-3.89] -0.264 [-4.02] Avg. GBC sen. -0.275 [-3.45] -0.302 [-3.98] -0.311 [-4.32] -0.311 [-4.45] Avg. GBC ass. 4 Discussion equally and that was present for all functional subnetworks together with a moderate decrease of temporal synchrony. Using a biophysical network model, we found that a decrease in global coupling or an increase in global noise levels could explain the connectivity reduction and the increase in synchrony best, whereas local changes to the glutamatergic or GABAergic system did not produce changes matching our experimental ﬁndings. However, both changes also yielded an increase in metastability in our model, which we did not ﬁnd in the experimental data. Comparison of average global GBC, average GBC in sensory areas, average GBC in association areas, average synchrony, and average metastability with increased noise (from 105% to 140% in steps of 5%). Shown are the mean differences, i.e. the mean of the default condition minus the respective increased noise condition and in brackets the effect size (Hedge’s g). The mean in each condition is calculated over the 40 virtual subjects. Signiﬁcant differences, i.e. a permutation p value of< 0.001, are highlighted in bold. Permutation tests were performed using 5,000 permutations of labels. 3.3.2 Modelling ScZ-associated alterations -0.250 [-3.15] -0.273 [-3.66] -0.282 [-3.94] -0.282 [-4.13] Synchrony -0.215 [-3.01] -0.266 [-4.00] -0.298 [-4.76] -0.313 [-5.23] Metastability 0.018 [0.70] 0.026 [1.22] 0.032 [1.32] 0.035 [1.47] Comparison of average global GBC, average GBC in sensory areas, average GBC in association areas, average synchrony and average metastability for reduced global coupling (from 95% to 60% in steps of 5%). Shown are the mean differences, i.e. the mean of the default condition minus the respective reduced global coupling condition and in brackets the effect size (Hedge’s g). The mean in each condition is calculated over the 40 virtual subjects. Signiﬁcant differences, i.e. a permutation p value of< 0.001, are highlighted in bold. Permutation tests were performed using 5,000 permutations of labels. al GBC, average GBC in sensory areas, average GBC in association areas, average synchrony and average metastability for reduced global coupling (from 95% to 60% he mean differences, i.e. the mean of the default condition minus the respective reduced global coupling condition and in brackets the effect size (Hedge’s g). The mean ed over the 40 virtual subjects. Signiﬁcant differences, i.e. a permutation p value of< 0.001, are highlighted in bold. Permutation tests were performed using 5,000 Comparison of average global GBC, average GBC in sensory areas, average GBC in association areas, average synchrony and average metastability for reduced global coupling (from 95% to 60% in steps of 5%). Shown are the mean differences, i.e. the mean of the default condition minus the respective reduced global coupling condition and in brackets the effect size (Hedge’s g). The mean in each condition is calculated over the 40 virtual subjects. Signiﬁcant differences, i.e. a permutation p value of< 0.001, are highlighted in bold. Permutation tests were performed using 5,000 permutations of labels. Frontiers in Psychiatry 4.2 Mechanistic explanations of global changes in ScZ Reduced global coupling and increased global noise levels are in line with earlier modelling studies. For example, several studies, using both simple phase oscillator models and dynamic mean-ﬁeld models, have shown that a decrease of global coupling compared to the best model ﬁt to human resting-state data led to a decrease in connectivity and a more random, less integrated graph structure (21, 23, 24). Similar to the model presented here, the operating point is chosen close to a bifurcation point from a silent down state to a limit-cycle which produces oscillating activity. In this regime, both functional connectivity and temporal dynamics best match empirical data. Therefore, the reduced coupling or the increased global noise disturbs this speciﬁc state and thus reduces global connectivity, synchrony and more complex network properties. Another limitation of the participant sample analysed here is its moderate size. For the group comparisons of GBC a post-hoc analysis of achieved power [performed with GPower 3.1 Faul et al. (70, 71)] resulted in sufﬁcient achieved power, however, group comparisons of temporal dynamics suffered from lower power (see Supplementary Material for more details). Therefore, replication of our ﬁndings in larger independent samples is warranted. The computational model that we have employed in this study, while generally showing a very good ﬁt to the experimental data, is not fully biophysically realistic. Moreover, the model used an average connectome and was not able to provide subject-speciﬁc, individual results for each participant. Furthermore, the anatomic parcellation [AAL2 Rolls et al. (47)] is relatively coarse-grained with a number of 80 cortical regions. As further validation, a replication of the analyses provided here using different, more ﬁne-grained anatomic parcellations is warranted. Previous work on the effects of changes to the glutamatergic and GABAergic system has demonstrated profound alterations on the cortical microcircuit level. For example, numerous computational studies have shown that ScZ-associated changes on the microcircuit level can lead to substantial reductions in gamma power in auditory steady-state response tasks [Metzner et al. (58); Metzner and Steuber (59); Metzner et al. (60); Vierling-Claassen et al. (61)]. Since local gamma oscillations have been hypothesized to at least partially determine the large-scale functional connectivity and temporal dynamics of resting-state activity Cabral et al. (62, 63), it seems surprising that changes to either of the systems did not produce changes in global brain connectivity in our model. 4.3 Limitations Patients in the sample used in this analysis were on typical and atypical antipsychotic medication with a mean dosage of 396.26 (CPZ-equivalent dosage). Antipsychotic medication, however, is known to affect functional connectivity. For example, risperidone treatment has been found to lead to abnormal functional and structural connectivity in striatal areas, prefrontal cortex, and limbic system components Hu et al. (67, 68). Furthermore, Wang et al. (69) found increased FC in the default mode network and decreased FC in the salience network after antipsychotic treatment. Therefore, we cannot rule out that the FC alterations identiﬁed in our analysis are not a result of ScZ pathophysiology but rather an effect of chronic treatment with antipsychotic medication. 4.1 Global changes in connectivity and temporal dynamics Importantly, these regional differences in dynamics covary with expression proﬁles for markers of glutamatergic and GABAergic neurotransmission and E-I balance [Burt et al. (66); Demirtaş et al. (64)]. Therefore, a more detailed, heterogeneous model might be able to shed more light on the effect of E-I balance changes associated with ScZ on large-scale functional networks. Our analysis of the temporal dynamics of the activity, i.e. synchrony and metastability, revealed a decrease in synchrony but no change in metastability. Our ﬁnding of unchanged metastability is in line with previous ﬁndings of Lee et al. (55) on the same dataset but in contrast to very recent work from Hancock et al. (56), proposing metastability as a candidate biomarker for schizophrenia. However, we have to note that Hancock et al. (56) introduced a new measure of metastability with increased sensitivity to detect the differences between healthy controls and ScZ patients. This new measure of metastability did not rely on predeﬁned brain parcellations but rather ﬂexibly deﬁned recurring spatio-temporal modes, so-called ‘communities’ where single brain regions may be grouped into more than one community. As this approach was not applicable to our computational network model we did not employ it in our analysis. Overall, several different metastability measures have been proposed and have been applied in different contexts in neuroscience [Hancock et al. (57)]. 4.1 Global changes in connectivity and temporal dynamics Evidence for large-scale dysconnectivity in functional networks has been accumulated over the last years in ScZ [Liang et al. (7); Bluhm et al. (8); Bassett et al. (11); Liu et al. (12); Bullmore and Sporns (13)]. However, it is still unclear, how these changes relate to changes on the microscopic level. To address this gap, we analysed resting-state fMRI data from healthy participants and patients with chronic ScZ. We identiﬁed a global reduction in functional connectivity that affected both sensory and association areas Our ﬁndings of reduced global brain connectivity are in line with previous research. For example, Lynall et al. (14) and Bassett et al. (54) both found signiﬁcantly reduced global integration in patients with schizophrenia. However, we did not ﬁnd stronger TABLE 5 ScZ-associated changes of noise parameters. TABLE 5 ScZ-associated changes of noise parameters. TABLE 5 ScZ-associated changes of noise parameters. 105% 110% 115% 120% Avg. global GBC -0.078 [-0.98] -0.129 [-1.60] -0.199 [-2.56] -0.260 [-3.56] Avg. GBC sen. -0.082 [-0.98] -0.139 [-1.64] -0.215 [-2.59] -0.285 [-3.64] Avg. GBC ass. -0.078 [-0.95] -0.133 [-1.58] -0.204 [-2.51] -0.268 [-3.57] Synchrony -0.050 [-0.67] -0.080 [-1.05] -0.121 [-1.83] -0.168 [-2.42] Metastability 0.008 [0.26] 0.006 [0.21] 0.010 [0.37] 0.014 [0.52] 125% 130% 135% 140% Avg. global GBC -0.313 [-4.58] -0.362 [-5.71] -0.383 [-6.07] -0.394 [-6.08] Avg. GBC sen. -0.345 [-4.86] -0.399 [-6.01] -0.424 [-6.39] -0.433 [-6.30] Avg. GBC ass. -0.323 [-4.56] -0.373 [-5.62] -0.396 [-6.15] -0.408 [-6.22] Synchrony -0.202 [-2.97] -0.230 [-3.62] -0.244 [-3.95] -0.261 [-4.17] Metastability 0.018 [0.70] 0.019 [0.75] 0.026 [1.03] 0.027 [1.02] Comparison of average global GBC, average GBC in sensory areas, average GBC in association areas, average synchrony, and average metastability with increased noise (from 105% to 140% in steps of 5%). Shown are the mean differences, i.e. the mean of the default condition minus the respective increased noise condition and in brackets the effect size (Hedge’s g). The mean in each condition is calculated over the 40 virtual subjects. Signiﬁcant differences, i.e. a permutation p value of< 0.001, are highlighted in bold. Permutation tests were performed using 5,000 permutations of labels. 09 Frontiers in Psychiatry frontiersin.org Metzner et al. 10.3389/fpsyt.2024.1352641 connectivity disturbances in association areas compared to sensory areas, as previously reported [Yang et al. (20)]. demonstrated that heterogeneous models of cortex, which explicitly incorporate regional differences in dynamics, match experimental resting-state functional connectivity more accurately [Demirtaş et al. (64); Kong et al. (65)]. Frontiers in Psychiatry frontiersin.org 5 Conclusion The current study provides further evidence of large-scale changes in connectivity and temporal dynamics in ScZ through the analysis of resting-state fMRI. Furthermore, through computational modelling, it provides novel evidence that these changes might be explained solely by global reductions in coupling or increases noise levels, although we cannot rule out that local effects also contribute signiﬁcantly. These ﬁndings emphasize the importance of global alterations in ScZ and might have possible implications for the development of treatments. Funding The author(s) declare ﬁnancial support was received for the research, authorship, and/or publication of this article. CM and PU were supported through the Einstein Stiftung Berlin (A-2020-613). Data collection and sharing for this project was funded by NIMH cooperative agreement 1U01 MH097435. Data was downloaded from the COllaborative Informatics and Neuroimaging Suite Data Exchange tool (COINS; http://coins.mrn.org/dx) and data collection was performed at the Mind Research Network, and funded by a Center of Biomedical Research Excellence (COBRE) grant 5P20RR021938/P20GM103472 from the NIH to Dr. Vince Calhoun Author contributions The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpsyt.2024.1352641/ full#supplementary-material CM: Conceptualization, Methodology, Visualization, Writing – original draft, Writing – review & editing. CD: Methodology, Writing - review & editing. FK: Methodology, Writing – review & 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. 4.2 Mechanistic explanations of global changes in ScZ One reason for the lack of impact of the changes might be that we applied them homogeneously. In the work presented here, we only varied glutamatergic or GABAergic strength globally, i.e. without any spatial heterogeneity. Therefore, it seems plausible that these changes disturbed the local, regional nodes all in a similar fashion and thereby did not substantially alter their interrelation, thus not changing global brain connectivity. Indeed, several studies have The ALN model that was used to simulate regional activity has been demonstrated to approximate cortical resting-state activity [Cakan and Obermayer (39); Cakan et al. (43)]. However, it is restricted to the cortex. Including subcortical regions such as the thalamus into whole-brain models is still in its infancy and rarely goes beyond coupling a single cortical and thalamic region [e.g. Jajcay et al. (72), but see Grifﬁths et al. (73)]. The ALN model also presents a simpliﬁcation of the regional circuitry as it approximates and neglects both the variability of cell types, especially the diversity of inhibitory interneurons, and the laminar structure of the cortex. Therefore, the inclusion of more detailed models of regional activity, both in terms of cell type diversity and of laminar structure and connectivity, seems likely to 10 frontiersin.org Metzner et al. 10.3389/fpsyt.2024.1352641 10.3389/fpsyt.2024.1352641 further our understanding of ScZ dysconnectivity and its underlying mechanisms. editing. SF: Methodology, Writing – review & editing. PU: Funding acquisition, Supervision, Writing – review & editing. KO: Conceptualization, Funding acquisition, Supervision, Writing – review & editing. Lastly, the regional ALN model we used had the same parameters regardless of the cortical region it represented, i.e. we implemented a homogeneous model in that respect. As already discussed above, cortical regions are known to differ in various important aspects, whose incorporation are likely to provide additional insight into the pathophysiology of schizophrenia. Ethics statement The studies involving humans were approved by Institutional Review Board of the University of New Mexico. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Conﬂict of interest The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://github.com/ChristophMetzner/ FrontiersPsychiatry2023. Publicly available datasets were analyzed in this study. This data was downloaded from the COllaborative Informatics and Neuroimaging Suite Data Exchange tool (COINS; http://coins.mrn.org/dx) via schizconnect.org. 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. 5. Bullmore ET, Frangou S, Murray R. The dysplastic net hypothesis: an integration of developmental and dysconnectivity theories of schizophrenia. Schizophr Res (1997) 28:143–56. doi: 10.1016/S0920-9964(97)00114-X 6. Pettersson-Yeo W, Allen P, Benetti S, McGuire P, Mechelli A. Dysconnectivity in schizophrenia: where are we now? Neurosci Biobehav Rev (2011) 35:1110–24. doi: 10.1016/j.neubiorev.2010.11.004 7. Liang M, Zhou Y, Jiang T, Liu Z, Tian L, Liu H, et al. Widespread functional disconnectivity in schizophrenia with resting-state functional magnetic resonance imaging. NeuroReport (2006) 17:209–13. doi: 10.1097/01.wnr.0000198434.06518.b8 Frontiers in Psychiatry 1. Lopez AD, Murray CC. The global burden of disease 1990–2020. Nat Med (1998) 4:1241–3. doi: 10.1038/3218 2. Charlson FJ, Ferrari AJ, Santomauro DF, Diminic S, Stockings E, Scott JG, et al. Global epidemiology and burden of schizophrenia: ﬁndings from the global burden of disease study 2016. Schizophr Bull (2018) 44:1195–203. doi: 10.1093/schbul/sby058 3. Friston KJ, and Frith CD. Schizophrenia: a disconnection syndrome. Clin Neurosci (1995) 3:89–97. 4. Friston KJ. Schizophrenia and the disconnection hypothesis. Acta Psychiatrica Scandinavica (1999) 99:68–79. doi: 10.1111/j.1600-0447.1999.tb05985.x 3. Friston KJ, and Frith CD. Schizophrenia: a disconnection syndrome. Clin Neurosci (1995) 3:89–97. 7. Liang M, Zhou Y, Jiang T, Liu Z, Tian L, Liu H, et al. Widespread functional disconnectivity in schizophrenia with resting-state functional magnetic resonance imaging. NeuroReport (2006) 17:209–13. doi: 10.1097/01.wnr.0000198434.06518.b8 2. Charlson FJ, Ferrari AJ, Santomauro DF, Diminic S, Stockings E, Scott JG, et al. Global epidemiology and burden of schizophrenia: ﬁndings from the global burden of disease study 2016. Schizophr Bull (2018) 44:1195–203. doi: 10.1093/schbul/sby058 4. Friston KJ. Schizophrenia and the disconnection hypothesis. Acta Psychiatrica Scandinavica (1999) 99:68–79. doi: 10.1111/j.1600-0447.1999.tb05985.x 1. Lopez AD, Murray CC. The global burden of disease 1990–2020. Nat Med (1998) 4:1241–3. doi: 10.1038/3218 5. Bullmore ET, Frangou S, Murray R. The dysplastic net hypothesis: an integration of developmental and dysconnectivity theories of schizophrenia. Schizophr Res (1997) 28:143–56. doi: 10.1016/S0920-9964(97)00114-X 6. Pettersson-Yeo W, Allen P, Benetti S, McGuire P, Mechelli A. Dysconnectivity in schizophrenia: where are we now? Neurosci Biobehav Rev (2011) 35:1110–24. doi: 10.1016/j.neubiorev.2010.11.004 References Low-dimensional spike rate models derived from networks of adaptive integrate-and-ﬁre neurons: comparison and implementation. PloS Comput Biol (2017) 13:e1005545. doi: 10.1371/ journal.pcbi.1005545 14. Lynall M-E, Bassett DS, Kerwin R, McKenna PJ, Kitzbichler M, Muller U, et al. Functional connectivity and brain networks in schizophrenia. J Neurosci (2010) 30:9477–87. doi: 10.1523/JNEUROSCI.0333-10.2010 15. Chung DW, Fish KN, Lewis DA. Pathological basis for deﬁcient excitatory drive to cortical parvalbumin interneurons in schizophrenia. Am J Psychiatry (2016) 173:1131–9. doi: 10.1176/appi.ajp.2016.16010025 39. Cakan C, Obermayer K. Biophysically grounded mean-ﬁeld models of neural populations under electrical stimulation. PloS Comput Biol (2020) 16:e1007822. doi: 10.1371/journal.pcbi.1007822 16. Chung DW, Geramita MA, Lewis DA. Synaptic variability and cortical gamma oscillation power in schizophrenia. Am J Psychiatry (2022) 179:277–87. doi: 10.1176/ appi.ajp.2021.21080798 40. Brunel N. Dynamics of sparsely connected networks of excitatory and inhibitory spiking neurons. J Comput Neurosci (2000) 8:183–208. doi: 10.1023/A:1008925309027 41. Fourcaud-Trocmé N, Hansel D, Van Vreeswijk C, Brunel N. How spike generation mechanisms determine the neuronal response to ﬂuctuating inputs. J Neurosci (2003) 2311628–11640. doi: 10.1523/JNEUROSCI.23-37-11628.2003 17. Hashimoto T, Volk DW, Eggan SM, Mirnics K, Pierri JN, Sun Z, et al. Gene expression deﬁcits in a subclass of gaba neurons in the prefrontal cortex of subjects with schizophrenia. J Neurosci (2003) 23:6315–26. doi: 10.1523/JNEUROSCI.23-15- 06315.2003 42. Ostojic S, Brunel N. From spiking neuron models to linear-nonlinear models. PloS Comput Biol (2011) 7:e1001056. doi: 10.1371/journal.pcbi.1001056 18. Morris HM, Hashimoto T, Lewis DA. Alterations in somatostatin mrna expression in the dorsolateral prefrontal cortex of subjects with schizophrenia or schizoaffective disorder. Cereb Cortex (2008) 18:1575–87. doi: 10.1093/cercor/bhm186 43. Cakan C, Dimulescu C, Khakimova L, Obst D, Flöel A, Obermayer K. Spatiotemporal patterns of adaptation-induced slow oscillations in a whole-brain model of slow-wave sleep. Front Comput Neurosci (2022) 15:800101. doi: 10.3389/ fncom.2021.800101 19. Moyer CE, Delevich KM, Fish KN, Asafu-Adjei JK, Sampson AR, Dorph- Petersen K-A, et al. Reduced glutamate decarboxylase 65 protein within primary auditory cortex inhibitory boutons in schizophrenia. Biol Psychiatry (2012) 72:734–43. doi: 10.1016/j.biopsych.2012.04.010 44. Friston KJ, Mechelli A, Turner R, Price CJ. Nonlinear responses in fmri: the balloon model, volterra kernels, and other hemodynamics. NeuroImage (2000) 12:466– 77. doi: 10.1006/nimg.2000.0630 20. Yang GJ, Murray JD, Wang X-J, Glahn DC, Pearlson GD, Repovs G, et al. Functional hierarchy underlies preferential connectivity disturbances in schizophrenia. Proc Natl Acad Sci (2016) 113:E219–28. doi: 10.1073/pnas.1508436113 45. Deco G, Ponce-Alvarez A, Mantini D, Romani GL, Hagmann P, Corbetta M. References Restingstate functional connectivity emerges from structurally and dynamically shaped slow linear ﬂuctuations. J Neurosci (2013) 33:11239–52. doi: 10.1523/ JNEUROSCI.1091-13.2013 21. Cabral J, Fernandes HM, Van Hartevelt TJ, James AC, Kringelbach ML, Deco G. Structural connectivity in schizophrenia and its impact on the dynamics of spontaneous functional networks. Chaos: Interdiscip J Nonlinear Sci (2013) 23:57–64. doi: 10.1063/1.4851117 46. Friston KJ, Harrison L, Penny W. Dynamic causal modelling. Neuroimage (2003) 19:1273–302. doi: 10.1016/S1053-8119(03)00202-7 47. Rolls ET, Joliot M, Tzourio-Mazoyer N. Implementation of a new parcellation of the orbitofrontal cortex in the automated anatomical labeling atlas. Neuroimage (2015) 122:1–5. doi: 10.1016/j.neuroimage.2015.07.075 22. Yang GJ, Murray JD, Repovs G, Cole MW, Savic A, Glasser MF, et al. Altered global brain signal in schizophrenia. Proc Natl Acad Sci (2014) 111:7438–43. doi: 10.1073/pnas.1405289111 48. Behrens TE, Berg HJ, Jbabdi S, Rushworth MF, Woolrich MW. Probabilistic diffusion tractography with multiple ﬁbre orientations: What can we gain? neuroimage (2007) 34:144–55. doi: 10.1016/j.neuroimage.2006.09.018 23. Cabral J, Kringelbach M, Deco G. Functional graph alterations in schizophrenia: a result from a global anatomic decoupling? Pharmacopsychiatry (2012) 45:S57–64. doi: 10.1055/s-0032-1309001 49. Winterer G, Ziller M, Dorn H, Frick K, Mulert C, Wuebben Y, et al. Schizophrenia: reduced signal-to-noise ratio and impaired phase-locking during information processing. Clin Neurophysiol (2000) 111:837–49. doi: 10.1016/S1388- 2457(99)00322-3 24. Cabral J, Hugues E, Kringelbach ML, Deco G. Modeling the outcome of structural disconnection on resting-state functional connectivity. Neuroimage (2012) 62:1342–53. doi: 10.1016/j.neuroimage.2012.06.007 25. Dimulescu C, Gareayaghi S, Kamp F, Fromm S, Obermayer K, Metzner C. Structural differences between healthy subjects and patients with schizophrenia or schizoaffective disorder: A graph and control theoretical perspective. Front Psychiatry (2021) 991. doi: 10.3389/fpsyt.2021.669783 50. Winterer G, Weinberger DR. Genes, dopamine and cortical signal-to-noise ratio in schizophrenia. Trends Neurosci (2004) 27:683–90. doi: 10.1016/j.tins.2004.08.002 51. Winterer G, Coppola R, Goldberg TE, Egan MF, Jones DW, Sanchez CE, et al. Prefrontal broadband noise, working memory, and genetic risk for schizophrenia. Am J Psychiatry (2004) 161:490–500. doi: 10.1176/appi.ajp.161.3.490 26. Kay SR, Opler LA, Lindenmayer J-P. The positive and negative syndrome scale (panss): rationale and standardisation. Br J Psychiatry (1989) 155:59–65. doi: 10.1192/ S0007125000291514 52. Cakan C, Jajcay N, Obermayer K. neurolib: a simulation framework for whole- brain neural mass modeling. Cogn Comput (2021) 1–21:1132–52. doi: 10.1101/ 2021.02.18.431886 27. Cetin MS, Christensen F, Abbott CC, Stephen JM, Mayer AR, Cañive JM, et al. References 3. Friston KJ, and Frith CD. Schizophrenia: a disconnection syndrome. Clin Neurosci (1995) 3:89–97. 4. Friston KJ. Schizophrenia and the disconnection hypothesis. Acta Psychiatrica Scandinavica (1999) 99:68–79. doi: 10.1111/j.1600-0447.1999.tb05985.x 11 Frontiers in Psychiatry frontiersin.org Metzner et al. 10.3389/fpsyt.2024.1352641 Metzner et al. 32. Salimi-Khorshidi G, Douaud G, Beckmann CF, Glasser MF, Griffanti L, Smith SM. Automatic denoising of functional mri data: combining independent component analysis and hierarchical fusion of classiﬁers. Neuroimage (2014) 90:449–68. doi: 10.1016/j.neuroimage.2013.11.046 8. Bluhm RL, Miller J, Lanius RA, Osuch EA, Boksman K, Neufeld R, et al. Spontaneous low-frequency ﬂuctuations in the bold signal in schizophrenic patients: anomalies in the default network. Schizophr Bull (2007) 33:1004–12. doi: 10.1093/ schbul/sbm052 9. Zhou Y, Liang M, Tian L, Wang K, Hao Y, Liu H, et al. Functional disintegration in paranoid schizophrenia using resting-state fmri. Schizophr Res (2007) 97:194–205. doi: 10.1016/j.schres.2007.05.029 33. Glasser MF, Sotiropoulos SN, Wilson JA, Coalson TS, Fischl B, Andersson JL, et al. The minimal preprocessing pipelines for the human connectome project. Neuroimage (2013) 80:105–24. doi: 10.1016/j.neuroimage.2013.04.127 g j g 34. Cole MW, Pathak S, Schneider W. Identifying the brain’s most globally connected regions. Neuroimage (2010) 49:3132–48. doi: 10.1016/ j.neuroimage.2009.11.001 10. Anticevic A, Corlett PR, Cole MW, Savic A, Gancsos M, Tang Y, et al. N-methyl- daspartate receptor antagonist effects on prefrontal cortical connectivity better model early than chronic schizophrenia. Biol Psychiatry (2015) 77:569–80. doi: 10.1016/ j.biopsych.2014.07.022 35. Cole MW, Anticevic A, Repovs G, Barch D. Variable global dysconnectivity and individual differences in schizophrenia. Biol Psychiatry (2011) 70:43–50. doi: 10.1016/ j.biopsych.2011.02.010 11. Bassett DS, Bullmore E, Verchinski BA, Mattay VS, Weinberger DR, Meyer- Lindenberg A. Hierarchical organization of human cortical networks in health and schizophrenia. J Neurosci (2008) 28:9239–48. doi: 10.1523/JNEUROSCI.1929-08.2008 36. Deco G, Kringelbach ML, Jirsa VK, Ritter P. The dynamics of resting ﬂuctuations in the brain: metastability and its dynamical cortical core. Sci Rep (2017) 7:3095. doi: 10.1038/s41598-017-03073-5 12. Liu Y, Liang M, Zhou Y, He Y, Hao Y, Song M, et al. Disrupted small-world networks in schizophrenia. Brain (2008) 131:945–61. doi: 10.1093/brain/awn018 37. Brette R, Gerstner W. Adaptive exponential integrate-and-ﬁre model as an effective description of neuronal activity. J Neurophysiol (2005) 94:3637–42. doi: 10.1152/jn.00686.2005 13. Bullmore E, Sporns O. Complex brain networks: graph theoretical analysis of structural and functional systems. Nat Rev Neurosci (2009) 10:186–98. doi: 10.1038/ nrn2575 38. Augustin M, Ladenbauer J, Baumann F, Obermayer K. Frontiers in Psychiatry References Shortterm effects of risperidone monotherapy on spontaneous brain activity in ﬁrst-episode treatment-naïve schizophrenia patients: a longitudinal fmri study. Sci Rep (2016) 6:34287. doi: 10.1038/srep34287 67. Hu M-L, Zong X-F, Zheng J-J, Pantazatos SP, Miller JM, Li Z-C, et al. Shortterm effects of risperidone monotherapy on spontaneous brain activity in ﬁrst-episode treatment-naïve schizophrenia patients: a longitudinal fmri study. Sci Rep (2016) 6:34287. doi: 10.1038/srep34287 59. Metzner C, Steuber V. The beta component of gamma-band auditory steady- state responses in patients with schizophrenia. Sci Rep (2021) 11:20387. doi: 10.1038/ s41598-021-99793-w 60. Metzner C, Zurowski B, Steuber V. The role of parvalbumin-positive interneurons in auditory steady-state response deﬁcits in schizophrenia. Sci Rep (2019) 9:18525. doi: 10.1038/s41598-019-53682-5 68. Hu M, Zong X, Zheng J, Mann J, Li Z, Pantazatos S, et al. Risperidone-induced topological alterations of anatomical brain network in ﬁrst-episode drug-naive schizophrenia patients: a longitudinal diffusion tensor imaging study. psychol Med (2016) 46:2549–60. doi: 10.1017/S0033291716001380 61. Vierling-Claassen D, Siekmeier P, Stufﬂebeam S, Kopell N. Modeling gaba alterations in schizophrenia: a link between impaired inhibition and altered gamma and beta range auditory entrainment. J Neurophysiol (2008) 99:2656–71. doi: 10.1152/ jn.00870.2007 69. Wang Y, Tang W, Fan X, Zhang J, Geng D, Jiang K, et al. Resting-state functional connectivity changes within the default mode network and the salience network after antipsychotic treatment in early-phase schizophrenia. Neuropsychiatr Dis Treat (2017) 13:397–406. doi: 10.2147/NDT.S123598 69. Wang Y, Tang W, Fan X, Zhang J, Geng D, Jiang K, et al. Resting-state functional connectivity changes within the default mode network and the salience network after antipsychotic treatment in early-phase schizophrenia. Neuropsychiatr Dis Treat (2017) 13:397–406. doi: 10.2147/NDT.S123598 62. Cabral J, Luckhoo H, Woolrich M, Joensson M, Mohseni H, Baker A, et al. Exploring mechanisms of spontaneous functional connectivity in meg: how delayed network interactions lead to structured amplitude envelopes of band-pass ﬁltered oscillations. Neuroimage (2014) 90:423–35. doi: 10.1016/j.neuroimage.2013.11.047 70. Faul F, Erdfelder E, Lang A-G, Buchner A. G* power 3: A ﬂexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav Res Methods (2007) 39:175–91. doi: 10.3758/BF03193146 63. Cabral J, Castaldo F, Vohryzek J, Litvak V, Bick C, Lambiotte R, et al. Metastable oscillatory modes emerge from synchronization in the brain spacetime connectome. Commun Phys (2022) 5:184. doi: 10.1038/s42005-022-00950-y 71. Faul F, Erdfelder E, Buchner A, Lang A-G. Statistical power analyses using g* power 3.1: Tests for correlation and regression analyses. Behav Res Methods (2009) 41:1149–60. References Thalamus and posterior temporal lobe show greater inter-network connectivity at rest and across sensory paradigms in schizophrenia. Neuroimage (2014) 97:117–26. doi: 10.1016/j.neuroimage.2014.04.009 53. Yeo BT, Krienen FM, Sepulcre J, Sabuncu MR, Lashkari D, Hollinshead M, et al. The organization of the human cerebral cortex estimated by intrinsic functional connectivity. J Neurophysiol (2011) 106:1125–65. doi: 10.1152/jn.00338.2011 28. Aine C, Bockholt HJ, Bustillo JR, Cañive JM, Caprihan A, Gasparovic C, et al. Multimodal neuroimaging in schizophrenia: description and dissemination. Neuroinformatics (2017) 15:343–64. doi: 10.1007/s12021-017-9338-9 54. Bassett DS, Nelson BG, Mueller BA, Camchong J, Lim KO. Altered resting state complexity in schizophrenia. Neuroimage (2012) 59:2196–207. doi: 10.1016/ j.neuroimage.2011.10.002 29. Woolrich MW, Ripley BD, Brady M, Smith SM. Temporal autocorrelation in univariate linear modeling of fmri data. Neuroimage (2001) 14:1370–86. doi: 10.1006/ nimg.2001.0931 55. Lee WH, Doucet GE, Leibu E, Frangou S. Resting-state network connectivity and metastability predict clinical symptoms in schizophrenia. Schizophr Res (2018) 201:208–16. doi: 10.1016/j.schres.2018.04.029 30. Guo S, He N, Liu Z, Linli Z, Tao H, Palaniyappan L. Brain-wide functional dysconnectivity in schizophrenia: parsing diathesis, resilience, and the effects of clinical expression. Can J Psychiatry (2020) 65:21–9. doi: 10.1177/0706743719890174 56. Hancock F, Rosas FE, McCutcheon RA, Cabral J, Dipasquale O, Turkheimer FE. Metastability as a candidate neuromechanistic biomarker of schizophrenia pathology. PloS One (2023) 18:e0282707. doi: 10.1371/journal.pone.0282707 31. Griffanti L, Salimi-Khorshidi G, Beckmann CF, Auerbach EJ, Douaud G, Sexton CE, et al. Ica-based artefact removal and accelerated fmri acquisition for improved resting state network imaging. Neuroimage (2014) 95:232–47. doi: 10.1016/ j.neuroimage.2014.03.034 57. Hancock F, Rosas FE, Zhang M, Mediano PA, Luppi A, Cabral J, et al. Metastability demystiﬁed—the foundational past, the pragmatic present, and the potential future. (Basel Switzerland: MDPI) (2023). 12 frontiersin.org Metzner et al. 10.3389/fpsyt.2024.1352641 10.3389/fpsyt.2024.1352641 Metzner et al. neuroimaging topography. Nat Neurosci (2018) 21:1251–9. doi: 10.1038/s41593-018- 0195-0 neuroimaging topography. Nat Neurosci (2018) 21:1251–9. doi: 10.1038/s41593-018- 0195-0 58. Metzner C, Schweikard A, Zurowski B. Multifactorial modeling of impairment of evoked gamma range oscillations in schizophrenia. Front Comput Neurosci (2016) 10:89. doi: 10.3389/fncom.2016.00089 59. Metzner C, Steuber V. The beta component of gamma-band auditory steady- state responses in patients with schizophrenia. Sci Rep (2021) 11:20387. doi: 10.1038/ s41598-021-99793-w 58. Metzner C, Schweikard A, Zurowski B. Multifactorial modeling of impairment of evoked gamma range oscillations in schizophrenia. Front Comput Neurosci (2016) 10:89. doi: 10.3389/fncom.2016.00089 67. Hu M-L, Zong X-F, Zheng J-J, Pantazatos SP, Miller JM, Li Z-C, et al. References doi: 10.3758/BRM.41.4.1149 64. Demirtaş M, Burt JB, Helmer M, Ji JL, Adkinson BD, Glasser MF, et al. Hierarchical heterogeneity across human cortex shapes large-scale neural dynamics. Neuron (2019) 101:1181–94. doi: 10.1016/j.neuron.2019.01.017 72. Jajcay N, Cakan C, Obermayer K. Cross-frequency slow oscillation–spindle coupling in a biophysically realistic thalamocortical neural mass model. Front Comput Neurosci (2022) 16:769860. doi: 10.3389/fncom.2022.769860 65. Kong X, Kong R, Orban C, Wang P, Zhang S, Anderson K, et al. Sensory-motor cortices shape functional connectivity dynamics in the human brain. Nat Commun (2021) 12:6373. doi: 10.1038/s41467-021-26704-y 73. Grifﬁths JD, McIntosh AR, Lefebvre J. A connectome-based, corticothalamic model of state-and stimulation-dependent modulation of rhythmic neural activity and connectivity. Front Comput Neurosci (2020) 14:575143. doi: 10.3389/ fncom.2020.575143 66. Burt JB, Demirtas¸ M, Eckner WJ, Navejar NM, Ji JL, Martin WJ, et al. Hierarchy of transcriptomic specialization across human cortex captured by structural 13 13 Frontiers in Psychiatry Frontiers in Psychiatry frontiersin.org
https://openalex.org/W2582063428	https://hal-insu.archives-ouvertes.fr/insu-03321824/file/DDR_ESD_Boers17.pdf	English	null	Inverse stochastic–dynamic models for high-resolution Greenland ice core records	Earth system dynamics	2,017	cc-by	14,075	Earth Syst. Dynam., 8, 1171–1190, 2017 https://doi.org/10.5194/esd-8-1171-2017 © Author(s) 2017. This work is distributed under the Creative Commons Attribution 3.0 License. Earth Syst. Dynam., 8, 1171–1190, 2017 https://doi.org/10.5194/esd-8-1171-2017 © Author(s) 2017. This work is distributed under the Creative Commons Attribution 3.0 License. Inverse stochastic–dynamic models for high-resolution Greenland ice core records Anders Svensson , Matthias Bigler , and Michael Ghil 1Geosciences Department and Laboratoire de Météorologie Dynamique (CNRS and IPSL), École Normale Supérieure and PSL Research University, Paris, France 2Department of Atmospheric and Oceanic Sciences and Institute of Geophysics and Planetary Physics, University of California, Los Angeles, USA 3Department of Mathematics, Virginia Polytechnic Institute and State University, Blacksburg, USA 4Institute of Applied Physics of the Russian Academy of Sciences, Nizhny Novgorod, Russia 5Lamont-Doherty Earth Observatory, Columbia University, Palisades, New York, USA 6Centre for Ice and Climate, University of Copenhagen, Copenhagen, Denmark 7Physics Institute and Oeschger Center for Climate Change Research, University of Bern, Bern, Switzerland Correspondence: Niklas Boers (nboers@lmd.ens.fr) Correspondence: Niklas Boers (nboers@lmd.ens.fr) Received: 23 January 2017 – Discussion started: 24 January 2017 Revised: 19 June 2017 – Accepted: 12 November 2017 – Published: 15 December 2017 Abstract. Proxy records from Greenland ice cores have been studied for several decades, yet many open ques- tions remain regarding the climate variability encoded therein. Here, we use a Bayesian framework for inferring inverse, stochastic–dynamic models from δ18O and dust records of unprecedented, subdecadal temporal resolu- tion. The records stem from the North Greenland Ice Core Project (NGRIP), and we focus on the time interval 59–22 ka b2k. Our model reproduces the dynamical characteristics of both the δ18O and dust proxy records, in- cluding the millennial-scale Dansgaard–Oeschger variability, as well as statistical properties such as probability density functions, waiting times and power spectra, with no need for any external forcing. The crucial ingredients for capturing these properties are (i) high-resolution training data, (ii) cubic drift terms, (iii) nonlinear coupling terms between the δ18O and dust time series, and (iv) non-Markovian contributions that represent short-term memory effects. Published by Copernicus Publications on behalf of the European Geosciences Union. N. Boers et al.: Inverse models for the NGRIP records N. Boers et al.: Inverse models for the NGRIP records 1172 here xi is the empirical observations, 1xi = xi+1 −xi, and 1ti = ti+1 −ti denotes the time spans from one observation to the next. The speciﬁc functional form of F may use some a priori knowledge of the system under study, while the pa- rameters of the proposed model are always inferred by train- ing it on a given set of time series produced by the system. In this sense, the approach is semiempirical, rather than be- ing entirely hypothesis-free. Most existing methodologies for empirical-model derivation are based on least-squares ﬁtting to determine optimal parameters for F. closed system. The term η(t) accounts for the stochastic forc- ing that is now uncorrelated with x, as guaranteed by the MZ formalism. The noise term, however, is not necessarily white in time or space; see Kondrashov et al. (2015) for a detailed derivation of the GLE approach and Appendix A for a sketch of the main ideas and more technical details. The temporal evolution of both the δ18O and dust time series indicates that there exist two alternative, relatively steady states for the underlying dynamical system, namely the colder stadials and the warmer interstadials (Dansgaard et al., 1993; Rasmussen et al., 2014). Transitions from the stadials to the interstadials occur very abruptly, within several decades, during the so-called Dansgaard–Oeschger (DO) events (Johnsen et al., 1992; Dansgaard et al., 1993; Ditlevsen et al., 2005), while transitions in the opposite di- rection are characterized by a comparably slow relaxation process that may last centuries to millennia, depending on the speciﬁc event. In the present study, we are speciﬁcally interested in ﬁt- ting low-dimensional stochastic–dynamic models to high- resolution time series of two paleoclimatic proxy records, namely the δ18O ratios and dust concentrations obtained by the North Greenland Ice Core Project (NGRIP; Ruth et al., 2003; Andersen et al., 2004; Gkinis et al., 2014); see Fig. 1. Low-dimensional conceptual models have a long history in paleoclimate (e.g., Källén et al., 1979; Le Treut and Ghil, 1983; Saltzman and Maasch, 1990; Ghil, 1994; Tziperman et al., 2006; De Saedeleer et al., 2013). Such models typi- cally incorporate a few global or regional climate variables, such as global temperature and ice-sheet volume, nonlinear interactions among these, and astronomical forcing subject to possible stochastic ﬂuctuations (Saltzman and Maasch, 1991; Cruciﬁx and Rougier, 2009). N. Boers et al.: Inverse models for the NGRIP records This bistability suggests using a system of two coupled SDEs with a double-well potential as a model of the pro- cesses generating the two time series of δ18O ratios and dust (Ditlevsen, 1999; Ditlevsen and Ditlevsen, 2009). In addi- tion, we will take into account here possible memory ef- fects in the climate system (Bhattacharya et al., 1982; Ghil et al., 2015) by including explicit non-Markovian terms in the model. In particular, these memory terms are included in order to reproduce the temporal asymmetry of the observed time series: the sharp transitions from the stadials to the in- terstadials, followed by rather smooth transitions from the interstadials back to the stadials. For these data, we thus pro- pose a two-dimensional stochastic delay differential equation as an approximation of the GLE (2): In contrast, we choose here a data-driven, stochastic– dynamic approach: we intend to ﬁnd a system of stochas- tic differential equations (SDEs) to simulate time series that reproduce the statistical and dynamical properties of the ob- served δ18O and dust time series and do so without taking into account exogenous astronomical forcing. The main is- sue with the naive approach of Eq. (1) is that the noise term η, which represents the unobserved variables, will typically be correlated with the state vector x. To overcome this problem, we adapt the recently developed non-Markovian data-driven closure models introduced by Kondrashov et al. (2015) as follows. dx = ( A + d X s=0 Bsx(t −sτ) + C(x,x) + D(x,x,x) ) dt + QdW(t). (3) (3) Here x is the two-dimensional time series of δ18O and dust, which is sampled at time steps ti in the NGRIP ice core. The model has a cubic drift term and retarded, non- Markovian arguments in the linear terms. The matrix Q de- notes the Cholesky decomposition of the covariance matrix of the noise, and W(t) denotes a multidimensional Wiener process. Model parameters will be inferred using maximum likelihood estimation (MLE) and, for comparison, ordinary least-squares ﬁtting. It will turn out that both approaches are in fact equivalent for the speciﬁc optimization problem pro- posed here; see Sect. 2.2 for further details and the explicit version of this SDE that we use in practice. 1 Introduction der consideration, EMR–MSM models have also been shown to be well suited for predictive purposes; e.g., they have con- siderable skill in predicting certain key variables associated with the El Niño–Southern Oscillation (Barnston et al., 2012; Chekroun et al., 2011) and the Madden–Julian Oscillation (Kondrashov et al., 2013). Data-driven stochastic difference equation models have re- cently been successfully applied to a wide range of cli- matic phenomena (Kondrashov et al., 2005, 2006; Kravtsov et al., 2005, 2009). The striking success of this empirical- model reduction (EMR) approach in reproducing dynamical and statistical properties of the underlying dynamical sys- tems has been explained by embedding EMR models in the larger class of multilayer stochastic models (MSMs); the lat- ter models, in turn, were shown to be solidly grounded in the Mori–Zwanzig (MZ; Zwanzig, 1964; Mori, 1965) formalism of statistical physics (Kondrashov et al., 2015). In addition to enhancing our understanding of the geophysical systems un- In general terms, stochastic–dynamic models are derived by approximating the discrete-time divided differences of ob- served time series by a deterministic function F plus residual noise (e.g., Hasselmann, 1976): 1xi 1ti ≈F(xi) + ηi ; (1) 1xi 1ti ≈F(xi) + ηi ; (1) Published by Copernicus Publications on behalf of the European Geosciences Union. www.earth-syst-dynam.net/8/1171/2017/ N. Boers et al.: Inverse models for the NGRIP records Given a multivariate, low-dimensional time series x(t) of partial observations of a much higher-dimensional system, the MZ formalism yields the abstract generalized Langevin equation (GLE): dx dt = F(x) + tZ 0 G(t,s;x(s))ds + η(t). (2) (2) (2) In our application, x is the two-vector of δ18O and log(dust), while the much larger system is the climate system. The ﬁrst term F(x) is Markovian and it accounts for the non- linear self-interactions among the observed variables, while the non-Markovian integral term accounts for the cross- interactions between the observed and unobserved variables; in this formulation, the latter are not present in F(x). This non-Markovian integral term involves the past of the ob- served variables and thus introduces memory effects into the Time series simulated by our empirical model will be com- pared to the original time series in terms of statistical proper- ties, such as the probability density functions (PDFs) of the time series, their power spectra and the average waiting time between sharp transitions from stadials to interstadials. Fur- thermore, we will test the relevance of the different model www.earth-syst-dynam.net/8/1171/2017/ Earth Syst. Dynam., 8, 1171–1190, 2017 N. Boers et al.: Inverse models for the NGRIP records 1173 no uncertainties in the relative timing of the δ18O and dust, since they are obtained from the same ice core. ingredients, such as the nonlinear terms, the memory and the coupling terms, using Bayesian model selection criteria. The general potential of Bayesian parameter inference such as MLE has recently been discussed for stochastic– dynamic climate models from incomplete data (Peavoy et al., 2015). A Bayesian framework to compare different types of models has also recently been used for the speciﬁc case of the NGRIP δ18O record, including a double-well potential model, a relaxation oscillator, and two versions of a mixture of locally linear stochastic models. Based on the Bayesian information criterion, it was concluded there that the two lo- cally linear mixture models are best supported by the ob- servations if three local models are used in each mixture (Kwasniok, 2013). Furthermore, a comprehensive Bayesian approach was employed to infer parameters for a double-well potential model from the NGRIP δ18O record (Krumscheid et al., 2015). Most recently, a double-well potential model was compared to a relaxation oscillator model with different external forcings using comparative Bayesian statistics (Mit- sui and Cruciﬁx, 2017). 2 Data and methods It has recently been shown in general terms how to approx- imate the GLE (2) by a set of SDEs that is relatively easy to derive from the observables x (Kondrashov et al., 2015). Their MSMs both generalize EMR models and provide the correct time-continuous limit of such models. Based on their work, we directly derive here an approximation of Eq. (2) in terms of Eq. (3). N. Boers et al.: Inverse models for the NGRIP records There, the conclusion was that the oscillator model is the more likely model candidate given the data and in particular that external forcing in terms of the ice volume signiﬁcantly improves the statistical model. The lat- ter three studies, however, used a lower-resolution version of the record, and did not consider either memory terms or cou- pling between the δ18O record and the dust record, as will be done here. The δ18O ratios are interpreted as proxies for surface air temperature variability, with algebraically higher ratios cor- responding to warmer temperatures (Johnsen et al., 1992, 2001a; Dansgaard et al., 1993; Andersen et al., 2004). The dust concentrations have been proposed as proxies of large- scale atmospheric circulation changes, with higher particle counts being associated with stronger winds and thus with larger equator-to-pole temperature differences (Fischer et al., 2007; Steffensen et al., 2008). Although the two proxies thus represent very distinct climatic features, they exhibit a high degree of co-variability for the time interval 59–22 ka b2k, as previously reported, e.g., by Johnsen et al. (1997), Ruth et al. (2003) and Rasmussen et al. (2014). This co-variability is best seen when considering the negative natural logarithm −log(dust) of the data along with the δ18O data (Fig. 1c). The raw time series (blue curves in Fig. 1a and b) are pre- processed as follows: ﬁrst, both time series are interpolated to an equidistant time axis with 5 a intervals. Second, gaps of varying length present in the dust time series and totaling about 6% of the data points are ﬁlled by next-neighbor inter- polation. Third, the multimillennial trend is removed from both time series using a 40 ka running mean. Fourth, the noise level of the δ18O record is reduced by applying a But- terworth low-pass ﬁlter of order 4, with a cutoff frequency of 0.02, corresponding to a period of 50 a. 2.1 High-resolution NGRIP data We employ proxy records of δ18O ratios (Andersen et al., 2004; Gkinis et al., 2014) and dust concentrations (Ruth et al., 2003) from the same core at the NGRIP drilling site. The δ18O ratios were regularly sampled every 5 cm, while the dust concentrations were sampled at a resolution of 1 mm (Ruth et al., 2002, 2003). The dust record was resampled here to the lower 5 cm resolution for consistency with the δ18O record. The proxy time series for the two variables have a common chronology, referred to as GICC05 (Svens- son et al., 2008), for the time interval starting at approxi- mately 59 ka b2k. Here, 1 ka equals 1000 a and “b2k” refers to “before AD 2000.” The GICC05 chronology is based on counting annual layers, which are distinguishable due to sea- sonal variations in the ice (Andersen et al., 2006), and the sampling intervals along the core range from 1 to 7 a. Compared to the GLE (2), the non-Markovian term with general kernel G is discretized and replaced by a sequence of Dirac kernels to obtain the second term on the right-hand side of Eq. (3), while the cubic term D(x,x,x) guarantees the stability of the solutions. Essentially, the proposed model is thus a dynamical system with a two-dimensional double-well potential that accounts for two alternative stable states. The additional memory term modulates the transitions between the two wells, which are stochastically forced by Gaussian white noise. In practical applications, the SDE (3) is approximated by the following system of k coupled, discrete difference equa- tions with delays: Dating uncertainties were reported for each measurement of the raw data, and they accumulate toward the more re- mote past, as a consequence of the layer-counting proce- dure, which starts from the top of the core (Svensson et al., 2008; Rasmussen et al., 2014). The dating uncertainties are reported to be roughly 5% and reach a maximum counting error of 2573 a at an age of 59420 ab2k. Note that there are Earth Syst. Dynam., 8, 1171–1190, 2017 www.earth-syst-dynam.net/8/1171/2017/ 1174 N. 2.1 High-resolution NGRIP data The two records are visually almost indistinguishable; the blue is visible only where it exceeds the red. (c) Low-pass ﬁltered δ18O (blue) and −log(dust) (red), together with their 40 ka running means (dashed lines). Note the strong co-variability between the two preprocessed time series during the interval 59–22 ka b2k; the Pearson correlation coefﬁcient on this interval equals rP = 0.84. For details on the preprocessing of the records see Sect. 2.1. xn+1 −xn = ( A + d X s=0 Bsxn−sτ + C(xn,xn) + D(xn,xn,xn) ) δtn + Q(δtn)1/2 χn . (4) The explicit coupled SDE system governing our stochastic–dynamic model is hence given by xn+1 −xn = A1 + 2 X s=0 Bs 11xn−sτ + Bs 12yn−sτ + C11x2 n + C12xnyn + C13y2 n + D11x3 n + D12x2 nyn + D13xny2 n + D14y3 n δtn + Q11ξ1 n + Q12ξ2 n δtn1/2 , (5a) yn+1 −yn = A2 + 2 X s=0 Bs 21xn−kτ + Bs 22yn−kτ + C21x2 n + C22xnyn + C23y2 n + D21x3 n + D22x2 nyn + D23xny2 n + D24y3 n δtn + Q21ξ1 n + Q22ξ2 n δtn1/2 , (5b) (4) Here xn ∈Rk denotes the k-component observed variable x(t) at time t = tn, with 1 ≤n ≤N, and the χn ∈Rk denote k-dimensional, independent white-noise increments. Note that for modeling the NGRIP records, we have k = 2 vari- ables given by the δ18O and dust measurements. (5a) In contrast to all other model parameters, the values for d and τ used in the linear memory part in Eq. (4) are not varied in the parameter optimization: in accordance with the MZ formalism, choosing d = 2 sufﬁces to obtain residuals that are sufﬁciently uncorrelated with the observations x. Opti- mal values of the memory step width τ are chosen in an outer loop so as to have the averaged PDFs of the model- simulated time series, along with the average waiting time between subsequent transitions, as close as possible to those of the observed ones. A choice of τ = 12 time steps, cor- responding to 60 a, will provide optimal approximations of these statistical characteristics; see Fig. 5 below. + C21x2 n + C22xnyn + C23y2 n (5b) where xn = (xn,yn) and τ = 60 a. 2.1 High-resolution NGRIP data Boers et al.: Inverse models for the NGRIP records (a) (c) (b) 15 20 25 30 35 40 45 50 55 60 −50 −45 −40 −35 −30 δ18O [per mill] δ18O raw δ18O preprocessed 15 20 25 30 35 40 45 50 55 60 Age [ka b2k] 0.0 0.5 1.0 1.5 2.0 2.5 dust [⇥106 ml−1] Dust raw Dust preprocessed 15 20 25 30 35 40 45 50 55 60 Age [ka b2k] −48 −45 −42 −39 −36 −33 −30 δ18O [per mill] δ18O preprocessed δ18O 40 ka running mean −15.0 −13.5 −12.0 −10.5 −9.0 −7.5 −6.0 −log(dust) −log(dust) preprocessed −log(dust) 40 ka running mean Figure 1. Time series of δ18O and dust from the NGRIP record for the time interval 15–59 ka b2k; following paleo-record and geochemical use, the time axis points from the present (at left) towards the past (at right). (a) Raw (blue) and preprocessed (red) δ18O time series. (b) Raw (blue) and preprocessed (red) dust time series. The two records are visually almost indistinguishable; the blue is visible only where it exceeds the red. (c) Low-pass ﬁltered δ18O (blue) and −log(dust) (red), together with their 40 ka running means (dashed lines). Note the strong co-variability between the two preprocessed time series during the interval 59–22 ka b2k; the Pearson correlation coefﬁcient on this interval equals rP = 0.84. For details on the preprocessing of the records see Sect. 2.1. (a) (c) (b) 15 20 25 30 35 40 45 50 55 60 −50 −45 −40 −35 −30 δ18O [per mill] δ18O raw δ18O preprocessed 15 20 25 30 35 40 45 50 55 60 Age [ka b2k] 0.0 0.5 1.0 1.5 2.0 2.5 dust [⇥106 ml−1] Dust raw Dust preprocessed 15 20 25 30 35 40 45 50 55 60 Age [ka b2k] −48 −45 −42 −39 −36 −33 −30 δ18O [per mill] δ18O preprocessed δ18O 40 ka running mean −15.0 −13.5 −12.0 −10.5 −9.0 −7.5 −6.0 −log(dust) −log(dust) preprocessed −log(dust) 40 ka running mean (b) (c) Figure 1. Time series of δ18O and dust from the NGRIP record for the time interval 15–59 ka b2k; following paleo-record and geochemical use, the time axis points from the present (at left) towards the past (at right). (a) Raw (blue) and preprocessed (red) δ18O time series. (b) Raw (blue) and preprocessed (red) dust time series. www.earth-syst-dynam.net/8/1171/2017/ 3 Results As a training set for the parameter optimization of our stochastic–dynamic model in Eq. (5), we choose the time interval 59–22 ka b2k; this interval roughly coincides with Marine Isotope Stage 3 (approximately 60–28 ka b2k). Our choice results in N = 7529 data points for each time series. The reason for this choice is that the layer-counted chronol- ogy has only been carried out until 59 ka b2k and that the co-variability between δ18O and log(dust) is substantially re- duced for the more recent part of the record, as already noted by Ruth et al. (2002, 2003) and apparent in Fig. 1c here. LD(5) = Y n 1 2π\|6\|1/2 × exp −1 2 δxn δtn −F5(xn,...,xn−sτ) T 6−1 × δxn δtn −F5(xn,...,xn−sτ) ; (7) (7) We use the natural logarithm of the dust time series (i) be- cause of the large range of dust concentration values and (ii) because it has high co-variability with δ18O (cf. Fig. 1c); it also guarantees that the simulated dust values are positive. This logarithmic scale requires, however, high accuracy in the modeling of the dust concentrations to resolve the multi- plicity of abrupt variations that span several orders of magni- tude; cf. Fig. 1b. here 6 is the covariance matrix of the noise, estimated from the residuals of the least-squares optimization. Note that the functional form of the likelihood function in Eq. (7) assumes that the residuals are normally and inde- pendently distributed. The MZ formalism only ensures that there exists a GLE with noise forcing that is uncorrelated with x. The additional assumption that the noise is white in time is not theoretically guaranteed. Therefore, one has to check empirically how well Gaussian white noise approxi- mates the residual. Given that the stochastic difference equa- tion (Eq. 4) only approximates the theoretical GLE provided by the MZ formalism, one also needs to validate empirically that the residuals are uncorrelated with the observations x (Kondrashov et al., 2015). Our results indicate that for d = 2 memory steps, the resid- uals of the least-squares optimization Eq. (6) are indeed uncorrelated with the observations x – the correlations are less than 10−8 – and they are approximately Gaussian dis- tributed (Fig. A1), while their autocorrelations decay very fast (not shown). These tests empirically support our choice of a Gaussian likelihood function of the form Eq. (7) for the MLE. 2.1 High-resolution NGRIP data In total, there are thus 31 parameters to be estimated. Note that the total set of model parameters includes the standard deviations of the noise residuals, as well as their correlation. Earth Syst. Dynam., 8, 1171–1190, 2017 www.earth-syst-dynam.net/8/1171/2017/ 1175 2.3 Maximum likelihood estimation On the other hand, the MLE is asymptotically optimal. In particular, it is efﬁcient in the sense that the variance of the parameter estimates achieves the so-called Cramér– Rao lower bound, which is optimal, as the number of sam- ples tends to inﬁnity (Andersen, 1970). Srivastava (1965) ex- tended the Gauss–Markov theorem to the multivariate case, where correlations among the variables lead to correlations between the error terms of the distinct variables and thus re- sult in cross-terms in the exponent of Eq. (7). Although both optimization approaches are thus equivalent in the case at hand, the MLE approach has the advantage that the parame- ter estimates can be interpreted as the most likely ones, given the observed data. Recently, Chorin and Lu (2015) introduced a general methodological framework for discrete stochastic parameter- izations. In principle, an optimal parameter set 5∗for the forms A, B, C and D of order 0–3 can be determined by regressing the right-hand side of Eq. (4) onto the observed increments δxn = xn+1 −xn, e.g., by ordinary least-squares (LSQ) optimization: 5∗= argmin 5 δxn δtn −F5(xn,...,xn−sτ) 2 , (6) (6) where F5 denotes the operator corresponding to the right- hand side of Eq. (4), dropping the noise term and using a pa- rameter combination 5. Following, e.g., Kwasniok (2013), Chorin and Lu (2015), Krumscheid et al. (2015), Mitsui and Cruciﬁx (2017), we rely here on Bayesian parameter inference for reduced stochastic models. For the present modeling task, we propose the Gaus- sian likelihood function Earth Syst. Dynam., 8, 1171–1190, 2017 3 Results Furthermore, these results allow us to integrate the stochastic–dynamic model given in Eq. (5) with an Euler– Maruyama scheme. We remark that the approximation of the divided differ- ences δx/δt by the function F5 is in fact a multivariate mul- tiple linear regression, with regressors chosen to be the poly- nomials in Eq. (5). It can easily be seen that MLE and LSQ are equivalent for the case of univariate data and Gaussian errors, since the same term δxn/δtn −F5 is minimized in Eqs. (6) and (7). In fact, subject to the assumption of un- correlated – but not necessarily Gaussian – errors with zero mean and identical variance, the Gauss–Markov theorem en- sures that parameters obtained from LSQ, i.e., from Eq. (6), are the best linear unbiased estimators for such a regression. These estimators are best in the sense that they have the low- est variance. The speciﬁc values of the coefﬁcients of Eq. (5), as de- rived via MLE, are given in Table A1. Note, in particular, the crucial nonlinear and lagged cross-interaction terms. For the reasons explained above, the parameters that minimize the least-squares problem Eq. (6) are identical to the most likely parameter values as determined via MLE up to numer- ical precision, i.e., with a relative error less than 10−5. In order to simulate optimal sample time series for the δ18O and log(dust) variables, the parameter combination 5∗ that maximizes the likelihood function (Eq. 7) is used. The www.earth-syst-dynam.net/8/1171/2017/ Earth Syst. Dynam., 8, 1171–1190, 2017 1176 N. Boers et al.: Inverse models for the NGRIP records (a) (c) (b) 25 30 35 40 45 50 55 60 −50 −45 −40 −35 −30 [per mill] δ18O simulated 25 30 35 40 45 50 55 60 0.0 0.5 1.0 1.5 2.0 2.5 [ ⇥106 ml−1] Dust simulated 25 30 35 40 45 50 55 60 Age [ka b2k] −48 −46 −44 −42 −40 −38 −36 −34 Simulatedδ18O [per mill] δ18O simulated −15 −14 −13 −12 −11 −10 −9 −8 Simulated −log(dust) −log(dust) simulated Figure 2. Simulated δ18O and dust time series. (a) Simulated δ18O time series (red). Gaussian white-noise approximation of the residual removed during the low-pass ﬁltering of the original time series is added back in to obtain the full time series (blue). (b) Simulated dust time series, obtained as the exponential of the simulated log(dust). 3 Results (c) Simulated δ18O time series (blue, same as in A) together with −log(dust) (red). Note that the strong co-variability between the two variables is captured very well by the model: Pearson’s correlation coefﬁcient, averaged over 1000 simulated time series, is rP = 0.85. (a) (c) (b) 25 30 35 40 45 50 55 60 −50 −45 −40 −35 −30 [per mill] δ18O simulated 25 30 35 40 45 50 55 60 0.0 0.5 1.0 1.5 2.0 2.5 [ ⇥106 ml−1] Dust simulated 25 30 35 40 45 50 55 60 Age [ka b2k] −48 −46 −44 −42 −40 −38 −36 −34 Simulatedδ18O [per mill] δ18O simulated −15 −14 −13 −12 −11 −10 −9 −8 Simulated −log(dust) −log(dust) simulated (a) (b) (c) Figure 2. Simulated δ18O and dust time series. (a) Simulated δ18O time series (red). Gaussian white-noise approximation of the residual removed during the low-pass ﬁltering of the original time series is added back in to obtain the full time series (blue). (b) Simulated dust time series, obtained as the exponential of the simulated log(dust). (c) Simulated δ18O time series (blue, same as in A) together with −log(dust) (red). Note that the strong co-variability between the two variables is captured very well by the model: Pearson’s correlation coefﬁcient, averaged over 1000 simulated time series, is rP = 0.85. model is then integrated by the Euler–Maruyama scheme with a uniform step size of δt = 10−5. Compared to the observational data, this step size corresponds to the sam- pling size of 5 a. Essentially, we have thus rescaled the time unit in order to guarantee a stable numerical integration. The stochastic forcing is given by two-dimensional Gaussian white-noise increments multiplied by the Cholesky matrix Q. The residuals of the δ18O and log(dust) are correlated with a Pearson’s correlation coefﬁcient rP = −0.13, resulting in a non-diagonal covariance matrix 6 = QQT. ulations, these values are computed as averages over 1000 simulated sample time series, obtained using the parameter combinations that maximize the likelihood function Eq. (7). The simulated time series (Fig. 2) exhibit abrupt changes that resemble the so-called Dansgaard–Oeschger events, which mark the sharp transitions from colder stadials to warmer interstadials (Dansgaard et al., 1993; Johnsen et al., 2001b; Rasmussen et al., 2014) in the original time series (Fig. 1). Given the more gradual temperature changes from interstadials to stadials, the red curve in Fig. www.earth-syst-dynam.net/8/1171/2017/ 3 Results 2a has a dom- inant sawtooth-shape pattern. Recall that time here, as in Fig. 1, runs from right to left, as it does in Fig. 3a and c of (Krumscheid et al., 2015), which also display a high qual- itative resemblance between their model-simulated and ob- served δ18O time series. Illustrative time series of δ18O and dust, simulated using the most likely model parameter combinations, are shown in Fig. 2. In Fig. 3a and b, the dashed lines show averages and uncertainties in PDFs of 1000 time series simulated using the most likely parameter combinations. Figure 3c and d show the average spectral densities of the latter time series. In this case, error bars would not be visible and are therefore omit- ted. The observed time series are, due to the sawtooth-shaped transitions between stadials and interstadials, not symmet- ric under time reversal. A quantitative measure of the time- reversal asymmetry is provided by the third-order moment (Kwasniok, 2013): The means and standard deviations of the observed time series are reproduced well by the simulations: for the pre- processed δ18O, the mean and standard deviation equal −41.79 ± 1.72 compared to −41.82 ± 1.73 for the simula- tions; the corresponding preprocessed and simulated values for log(dust) are 11.98±0.97 and 12.00±0.92. For the sim- M(θ) = ⟨x(t)x2(t + θ) −x2(t)x(t + θ)⟩t . Figure 4b shows that, for values of θ up to roughly 1000 a, M(θ) computed from dust simulations does exhibit a some- Earth Syst. Dynam., 8, 1171–1190, 2017 www.earth-syst-dynam.net/8/1171/2017/ N. 3 Results Boers et al.: Inverse models for the NGRIP records 1177 10−5 10−4 10−3 10−2 10−1 10−4 10−3 10−2 10−1 100 101 102 103 104 105 PSD δ18O δ18O interpolated δ18O preprocessed δ18O simulated 10−5 10−4 10−3 10−2 10−1 Frequency [1/a] 10−4 10−3 10−2 10−1 100 101 102 103 104 105 PSD dust log(dust) preprocessed log(dust) simulated −50 −48 −46 −44 −42 −40 −38 −36 −34 δ18O [per mill] −0.05 0.00 0.05 0.10 0.15 0.20 0.25 0.30 PDF δ18O δ18O raw δ18O preprocessed δ18O simulated 9 10 11 12 13 14 15 log(dust) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 PDF log(dust) log(dust) raw log(dust) preprocessed log(dust) simulated (a) (c) (b) (d) 10−5 10−4 10−3 10−2 10−1 10−4 10−3 10−2 10−1 100 101 102 103 104 105 PSD δ18O δ18O interpolated δ18O preprocessed δ18O simulated 10−5 10−4 10−3 10−2 10−1 Frequency [1/a] 10−4 10−3 10−2 10−1 100 101 102 103 104 105 PSD dust log(dust) preprocessed log(dust) simulated −50 −48 −46 −44 −42 −40 −38 −36 −34 δ18O [per mill] −0.05 0.00 0.05 0.10 0.15 0.20 0.25 0.30 PDF δ18O δ18O raw δ18O preprocessed δ18O simulated 9 10 11 12 13 14 15 log(dust) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 PDF log(dust) log(dust) raw log(dust) preprocessed log(dust) simulated (a) (c) (b) (d) tatistical properties of the observed and simulated δ18O and dust time series. (a) PDFs for the raw, preprocessed he bimodality, corresponding to the transitions between stadials and interstadials, only arises after applying a ies, leading to the preprocessed time series. (b) PDFs for the raw, preprocessed, and simulated log(dust) time averages over 1000 simulated time series, each obtained from the most likely model parameters (solid red). E e of uncertainties derived from theses 1000 sample time series. (c) Power spectral densities for the interpolated, p and smoothed) and simulated δ18O. (d) Power spectral densities (PSDs) for the preprocessed and simulated l me series, Gaussian white noise with the same standard deviation as the residual of the LSQ (Eq. 6) was added b he PSDs are estimated using the multi-taper method (Vautard et al., 1992), with a total of seven tapers. The PS is an average over 1000 simulated time series and is therefore strongly smoothed. For the spectra, the uncert le and are therefore omitted. 3 Results −50 −48 −46 −44 −42 −40 −38 −36 −34 δ18O [per mill] −0.05 0.00 0.05 0.10 0.15 0.20 0.25 0.30 PDF δ18O δ18O raw δ18O preprocessed δ18O simulated 9 10 11 12 13 14 15 log(dust) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 PDF log(dust) log(dust) raw log(dust) preprocessed log(dust) simulated (a) (b) 10−5 10−4 10−3 10−2 10−1 10−4 10−3 10−2 10−1 100 101 102 103 104 105 PSD δ18O δ18O interpolated δ18O preprocessed δ18O simulated 10−5 10−4 10−3 10−2 10−1 Frequency [1/a] 10−4 10−3 10−2 10−1 100 101 102 103 104 105 PSD dust log(dust) preprocessed log(dust) simulated −50 −48 −46 −44 −42 −40 −38 −36 −34 δ18O [per mill] −0.05 0.00 0.05 0.10 PDF 9 10 11 12 13 14 15 log(dust) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 PDF log(dust) log(dust) raw log(dust) preprocessed log(dust) simulated (c) (b) (d) Figure 3. Statistical properties of the observed and simulated δ18O and dust time series. (a) PDFs for the raw, preprocessed, simulated δ18O. Note how the bimodality, corresponding to the transitions between stadials and interstadials, only arises after applying a low-pass ﬁlter to the time series, leading to the preprocessed time series. (b) PDFs for the raw, preprocessed, and simulated log(dust) time series. PDFs are obtained as averages over 1000 simulated time series, each obtained from the most likely model parameters (solid red). Error bars indicate the 2σ range of uncertainties derived from theses 1000 sample time series. (c) Power spectral densities for the interpolated, preprocessed (i.e., interpolated and smoothed) and simulated δ18O. (d) Power spectral densities (PSDs) for the preprocessed and simulated log(dust). For the simulated time series, Gaussian white noise with the same standard deviation as the residual of the LSQ (Eq. 6) was added before computing the PSD. The PSDs are estimated using the multi-taper method (Vautard et al., 1992), with a total of seven tapers. The PSD shown for the simulations is an average over 1000 simulated time series and is therefore strongly smoothed. For the spectra, the uncertainties would be hardly visible and are therefore omitted. (b) 10−5 10−4 10−3 10−2 10−1 10−4 10−3 10−2 10−1 100 101 102 103 104 105 PSD δ18O δ18O interpolated δ18O preprocessed δ18O simulated 10−5 10−4 10−3 10−2 10−1 Frequency [1/a] 10−4 10−3 10−2 10−1 100 101 102 103 104 105 PSD dust log(dust) preprocessed log(dust) simulated (c) (d) (c) Figure 3. 4 Discussion We studied the δ18O and dust time series obtained from the high-resolution NGRIP record for the 37 000 a interval 59– 22 ka b2k. The results described above show that the statisti- cal properties of these time series – such as their PDFs, spec- tra and average waiting times – can be approximated quite well by the proposed stochastic–dynamic model of Eq. (3). Given the very strong correlations rP between the two vari- ables, with rP ≈0.9 for both observations and simulations, the discrepancies between the resulting M(θ) given δ18O or dust data are rather surprising. These discrepancies sug- gest that M(θ) might not be the best measure for quantifying whether sawtooth-shaped oscillations are present or not. y p p y q The main features of our inverse model are (i) the non- linear terms in the Markovian part, (ii) the inclusion of non- Markovian memory terms and (iii) the coupling terms be- tween the two time series. Cubic terms have previously been used to model the δ18O time series of the NGRIP record (Ditlevsen et al., 2007; Kwasniok, 2013; Krumscheid et al., 2015). Non-Markovian contributions in an SDE model have, so far, only been considered in very few paleoclimatic stud- ies. Pelletier (2003) studied the inﬂuence of delayed ice vol- ume feedbacks on glacial–interglacial variability. In a de- terministic setting, Rial (2004) used a forced logistic delay differential equation model to study DO cycles as well as glacial–interglacial transitions, and Berger (1999) discussed memory contributions in the context of the 100 ka cycle and its association with Milankovitch forcing. We are not aware of modeling efforts that take advantage of the co-variability between the δ18O and log(dust) variables. It is shown in the following that the coupling terms between the two variables are crucial in order to capture the statistical characteristics of the measured NGRIP time series presented above, while the non-Markovian contribution is also signiﬁcant. p p Following Krumscheid et al. (2015), we deﬁne the aver- age waiting time τDO between Dansgaard–Oeschger events as the sum of the average residence times in stadials and interstadials. For this purpose, stadials and interstadials are determined as intervals for which the time series are, respec- tively, below or above the mean of the series. 4 Discussion Due to their comparably high noise level, the high-resolution time series employed here are further smoothed by singular spectrum analysis (SSA) (Vautard et al., 1992; Ghil et al., 2002) us- ing a window size of 500 a and keeping only the ﬁve lead- ing reconstructed components. For the observed δ18O and log(dust), we obtain τDO = 1506 and 1744 a, respectively, compared to τDO = 1370 ± 224 a and 1559 ± 346 a for the simulations, computed as averages over 1000 simulated time series. Note that this deﬁnition of waiting times may not be optimal in view of the high noise level of the time series, leading to different values for δ18O and log(dust). This def- inition is nevertheless employed here for the purpose of fa- cilitating comparison with the results of Krumscheid et al. (2015). The nonlinear terms can be physically motivated by the fact that the observed time series oscillate between two quasi- equilibria, namely the stadials and interstadials. If only linear terms were used, the bimodality of the observed time series, and hence the existence of two quasi-stable states, could not be reproduced (see Figs. A2 and A3). For the dust concentrations (Fig. 2b), there is a striking similarity between the observed and simulated time series in terms of episodes with low dust concentrations of variable durations, as well as the presence of burst episodes of vari- able magnitudes. Over the training interval, the preprocessed time series are correlated at rP = −0.84, which equals the average correlation between the simulated time series. The purely Markovian form of the model approximates the PDFs of the observed time series less well (Figs. 5c and A5). In particular, the bimodality of the PDFs for both δ18O and log(dust) is weaker in this case, indicating that the memory terms do contribute to an appropriate modeling of the persis- tence in the stadials and interstadials, as well as of the tran- sitions between them. In addition, the average waiting times between DO-like transitions are τDO = 1404 a for the δ18O and 1254 a for log(dust), and they are thus too short for the log(dust) time series, in particular. The PDFs of the simulated δ18O and log(dust) (red solid lines), obtained as averages over 1000 time series, are quite similar in shape to those of the preprocessed time series (blue solid lines) of both observed variables; see Fig. N. Boers et al.: Inverse models for the NGRIP records 1178 what similar behavior to the M(θ) computed from the ob- served dust. Quantitatively, the similarity between M(θ) computed for the observed and simulated dust is also sup- ported by Kendall’s τ value (red curve in Fig. 5d). The tem- poral asymmetry is, however, not reproduced by the δ18O simulations (Fig. 4a), as also conﬁrmed by Kendall’s τ (blue curve in Fig. 5d). 3 Results Statistical properties of the observed and simulated δ18O and dust time series. (a) PDFs for the raw, preprocessed, simulated δ18O. Note how the bimodality, corresponding to the transitions between stadials and interstadials, only arises after applying a low-pass ﬁlter to the time series, leading to the preprocessed time series. (b) PDFs for the raw, preprocessed, and simulated log(dust) time series. PDFs are obtained as averages over 1000 simulated time series, each obtained from the most likely model parameters (solid red). Error bars indicate the 2σ range of uncertainties derived from theses 1000 sample time series. (c) Power spectral densities for the interpolated, preprocessed (i.e., 18 Figure 3. Statistical properties of the observed and simulated δ18O and dust time series. (a) PDFs for the raw Figure 3. Statistical properties of the observed and simulated δ18O and dust time series. (a) PDFs for the raw, preprocessed, simulated δ18O. Note how the bimodality, corresponding to the transitions between stadials and interstadials, only arises after applying a low-pass ﬁlter to the time series, leading to the preprocessed time series. (b) PDFs for the raw, preprocessed, and simulated log(dust) time series. PDFs are obtained as averages over 1000 simulated time series, each obtained from the most likely model parameters (solid red). Error bars indicate the 2σ range of uncertainties derived from theses 1000 sample time series. (c) Power spectral densities for the interpolated, preprocessed (i.e., interpolated and smoothed) and simulated δ18O. (d) Power spectral densities (PSDs) for the preprocessed and simulated log(dust). For the simulated time series, Gaussian white noise with the same standard deviation as the residual of the LSQ (Eq. 6) was added before computing the PSD. The PSDs are estimated using the multi-taper method (Vautard et al., 1992), with a total of seven tapers. The PSD shown for the simulations is an average over 1000 simulated time series and is therefore strongly smoothed. For the spectra, the uncertainties would be hardly visible and are therefore omitted. Earth Syst. Dynam., 8, 1171–1190, 2017 www.earth-syst-dynam.net/8/1171/2017/ N. Boers et al.: Inverse models for the NGRIP records N. Boers et al.: Inverse models for the NGRIP records 1179 Figure 4. Third-order statistical moment M(θ) = ⟨x(t)x2(t + θ) −x2(t)x(t + θ)⟩t for the observed NGRIP time series (solid blue), the full model including memory terms with step size τ = 60 a (solid red) and the model without memory terms (dashed red). (a) M(θ) for the δ18O time series and (b) M(θ) for the dust time series. For the simulations, the average of M(θ) over 1000 samples is shown. Note that, for increasing delays θ, the values of M(θ) are affected more and more by the nonstationarity of the data and should therefore be interpreted with care. Figure 4. Third-order statistical moment M(θ) = ⟨x(t)x2(t + θ) −x2(t)x(t + θ)⟩t for the observed NGRIP time series (solid blue), the full model including memory terms with step size τ = 60 a (solid red) and the model without memory terms (dashed red). (a) M(θ) for the δ18O time series and (b) M(θ) for the dust time series. For the simulations, the average of M(θ) over 1000 samples is shown. Note that, for increasing delays θ, the values of M(θ) are affected more and more by the nonstationarity of the data and should therefore be interpreted with care. Table 1. Sample-size-corrected Akaike information criteria (AICc) and Bayesian information criteria (BIC) for the different model versions. Note that the model parameters include the standard deviations and correlation that appear in the respective model’s noise term. For the models with memory, we chose d = 2 memory steps with step size τ = 12, corresponding to 60 a. Nonlinear Memory Coupling No. of parameters AICc BIC × × × 31 4501.93 4287.57 × × 17 4701.05 4583.44 × × 23 5089.18 4929.87 × × 14 5228.15 5131.28 × 9 6058.83 5996.52 × × × 31 4287.57 4501.93 × × 17 4583.44 4701.05 × × 23 4929.87 5089.18 × × 14 5131.28 5228.15 × 9 5996.52 6058.83 and sample-size-corrected Akaike (AICc) information crite- ria; these are deﬁned as AICc = 2pn/(n−p−1)−2log(L∗) and BIC = plog(n)−2log(L∗). Here, p denotes the number of model parameters, n = N −1 = 7528 the total number of data points (a total number of data points N leads to N−1 in- crements to be approximated) and L∗the maximum value of the likelihood function Eq. (7). The lower the value of AICc or BIC for a given model, the higher the relative conﬁdence in that model. 4 Discussion 3a and b. In particular, the bimodality of the PDFs, which reﬂects the rel- ative persistence of stadials and interstadials, is reproduced by our inverse model. Uncertainties in the PDFs are derived on the basis of ensembles of 1000 simulated time series, pro- duced by sampling from the most likely parameter combina- tion. Error bars on the PDFs in Fig. 3a and b reﬂect the 2σ range of these ensembles. When removing all coupling terms between the two vari- ables from the inverse-model Eq. (4), sawtooth-shaped tran- sitions are completely absent from the simulated time se- ries (Fig. A6). Furthermore, the bimodality of the PDFs is missed when excluding the couplings (Fig. A7), and the av- erage waiting times are much too short, namely τDO = 1208 a for the δ18O record and 867 a for the log(dust) record. The spectra of both the δ18O and the log(dust) are well reproduced by our model for the entire frequency range, in- cluding sub-centennial periodicities; see the black and red curves in Fig. 3c. Following previous authors (Kwasniok, 2013; Krumscheid et al., 2015; Mitsui and Cruciﬁx, 2017), we also compare the different model versions in terms of the Bayesian (BIC) Earth Syst. Dynam., 8, 1171–1190, 2017 www.earth-syst-dynam.net/8/1171/2017/ N. Boers et al.: Inverse models for the NGRIP records N. Boers et al.: Inverse models for the NGRIP records 1180 Figure 5. Summary statistics for different memory step sizes τ. (a) Log-likelihood and AICc values for different memory step sizes τ. The AICc is computed as AICc = 2pn/(n−p−1)−2logL∗. (b) Difference between observed and simulated standard deviations. (c) L2 and L∞ distances between observed and simulated PDFs. (d) Kendall’s τ statistic between the third-order moments M(θ), computed for observed and simulated time series. (e) Difference between observations and simulations in terms of average waiting times between subsequent transitions from stadials to interstadials In (b e) statistics for simulated time series are obtained as averages over 1000 simulations using the full model Figure 5. Summary statistics for different memory step sizes τ. (a) Log-likelihood and AICc values for different memory step sizes τ. The AICc is computed as AICc = 2pn/(n−p−1)−2logL∗. (b) Difference between observed and simulated standard deviations. (c) L2 and L∞ distances between observed and simulated PDFs. (d) Kendall’s τ statistic between the third-order moments M(θ), computed for observed and simulated time series. (e) Difference between observations and simulations in terms of average waiting times between subsequent transitions from stadials to interstadials. In (b–e), statistics for simulated time series are obtained as averages over 1000 simulations using the full model. Figure 5. Summary statistics for different memory step sizes τ. (a) Log-likelihood and AICc values for different memory step sizes τ. The AICc is computed as AICc = 2pn/(n−p−1)−2logL∗. (b) Difference between observed and simulated standard deviations. (c) L2 and L∞ distances between observed and simulated PDFs. (d) Kendall’s τ statistic between the third-order moments M(θ), computed for observed and simulated time series. (e) Difference between observations and simulations in terms of average waiting times between subsequent transitions from stadials to interstadials. In (b–e), statistics for simulated time series are obtained as averages over 1000 simulations using the full model. propose higher conﬁdence in the linear model than in the model without memory terms. This would suggest that the memory terms are more important than the higher-order pa- rameters that correspond to the double-well shape of the po- tential. However, the PDFs of the cubic model without mem- ory terms are considerably closer to the observed PDFs than the PDFs obtained from the linear model including memory terms. In particular, by construction, the latter model cannot reproduce the bimodality of the observed PDFs. N. Boers et al.: Inverse models for the NGRIP records with memory terms, the nonlinear coupled model without memory terms, the nonlinear model with memory but with- out coupling terms, and ﬁnally the linear model without memory terms (Table 1). Note that the AICc penalizes higher numbers p of model parameters less strongly than the BIC. Although the AICc was found, under certain conditions, to be optimal in select- ing model candidates (e.g., Burnham and Anderson, 2002; Yang, 2005), notable counterexamples are known (e.g., Pen- land et al., 1991). For the case at hand, both AICc and BIC consistently favor the full model, which includes nonlinear, memory and cou- pling terms. This is followed by the linear coupled model We thus suggest interpreting the values in Table 1 with caution. For example, in the case at hand both AICc and BIC www.earth-syst-dynam.net/8/1171/2017/ Earth Syst. Dynam., 8, 1171–1190, 2017 N. Boers et al.: Inverse models for the NGRIP records www.earth-syst-dynam.net/8/1171/2017/ N. Boers et al.: Inverse models for the NGRIP records Therefore, we suggest that a choice of τ = 60 a provides a good tradeoff between the AICc and BIC, on the one hand, and the statistical characteristics of the simulated time series, on the other. In particular, an accurate reproduc- tion of the correct average waiting time between subsequent transitions from stadials to interstadials is only achieved for 55a≲τ≲60a. It should be emphasized, moreover, that both the AICc and BIC criteria consistently favor the models with memory terms over the models without memory terms, re- gardless of the speciﬁc value of τ. 2. Cubic terms need to be included in the Markovian part of the model. This can be physically motivated by the presence of two quasi-equilibria in the observed time series – the stadials and interstadials – that could not be modeled without two such quasi-stable states in the un- derlying dynamical system. Cubic terms have already been considered in previous attempts to model the δ18O time series of the NGRIP record (Ditlevsen et al., 2007; Kwasniok, 2013; Krumscheid et al., 2015); these at- tempts, however, did not include the dust series, used lower-resolution data and did not consider memory ef- fects. g The model results presented here appear only in the high- resolution version of the NGRIP ice core record, which was originally sampled every 5 cm, a depth sampling that yielded temporal step sizes between 1 and 7 a. For example, interpo- lating the raw data to a uniform grid with 1t = 10 a, instead of 5 a, leads to substantially less accurate approximations of the observed statistical properties of both the δ18O and dust time series (not shown). On the other hand, interpolating the raw data with a uniform sampling step of 3 a is problematic because of the original temporal step sizes, and does not fur- ther improve the results (not shown). It would thus appear that the 5 a uniform grid size is nearly optimal, given the ir- regularities in the sampling and the uncertainties in both the dating and the values of the records. 3. Coupling terms between the δ18O and dust variables substantially improve the statistical characteristics of the simulated time series: the reproduction of the bi- modality of the PDFs, as well as of the correct average waiting time between subsequent transitions from sta- dials to interstadials, are substantially improved when coupling terms are included. 4. N. Boers et al.: Inverse models for the NGRIP records We would thus still argue that the nonlinear contributions are more im- portant than the memory terms. Nevertheless, the AICc and BIC values presented herein provide information-theoretic evidence that the inclusion of memory terms does substan- tially improve the model. We emphasize that the full model proposed herein has the highest number of parameters out of the different candidates but is still the one with the lowest BIC and AIC. Therefore, it can be argued that this number of parameters is not too high, Earth Syst. Dynam., 8, 1171–1190, 2017 www.earth-syst-dynam.net/8/1171/2017/ N. Boers et al.: Inverse models for the NGRIP records 1181 and it is not likely that the full model over-ﬁts the observed data. 1. High-resolution time series have to be used as train- ing data, indicating that the high-frequency variability present in the records plays a vital role for the over- all evolution of the climate processes that generated the NGRIP ice core. Interpolation of the raw data, which is sampled at depth intervals of 5 cm in the core, to 5 a intervals in the preprocessed time series was found to be optimal in our inverse-model setup. This ﬁnding is qualitatively consistent with the assertion of Ryp- dal (2016) that an increase in decadal-scale variabil- ity may be a statistical precursor for the abrupt transi- tions from stadials to interstadials during Dansgaard– Oeschger events. Furthermore, it should be noted that the values of AICc and BIC can only be compared on the basis of the same un- derlying data. Since we use a higher-resolution version of the NGRIP data as compared to the previous authors (Kwasniok, 2013; Krumscheid et al., 2015; Mitsui and Cruciﬁx, 2017), the values for AICc and BIC presented here cannot be com- pared to the values reported in those studies. As noted above, we chose for the memory step size τ = 60 a, corresponding to 12 time steps. In fact, the smallest val- ues of both AICc and BIC are obtained at a considerably smaller step size of τ = 1 time step (5 a) (Fig. 5a). How- ever, the approximation of the correct standard deviations, PDFs, third-order moments and average waiting times by the simulations is strongly improved for larger values of τ (Fig. 5b–e). N. Boers et al.: Inverse models for the NGRIP records Non-Markovian terms that account for memory effects are helpful. Their inclusion allows, to some extent, reproducing the time-reversal asymmetry of the dust time series. The main contribution of including mem- ory terms into the model is, however, to improve the average simulated waiting times between subsequent transitions from stadials to interstadials (cf. Fig. 2e) for 55a≲τ≲60a. The memory terms also help improve the PDFs of simulated time series, in particular for the δ18O time series. Furthermore, in general, the AICc and BIC criteria support the model versions that include memory terms. www.earth-syst-dynam.net/8/1171/2017/ Data availability. The high-resolution NGRIP data used in this study are available online at http://www.icecores.dk. Appendix A: Key ideas in deriving the GLE Table A1. The coefﬁcients of the explicit SDE system, obtained from MLE. The approach to data-driven stochastic–dynamic model- ing taken here is rooted in the MZ formalism of statisti- cal mechanics (Zwanzig, 1964; Mori, 1965; Chorin et al., 2002; Chorin and Hald, 2013), which proposes an integro- differential closed form for model inference from partial data. While the derivation of such an MZ model does not allow one to easily simulate its solutions, it is possible, under suitable hypotheses, to obtain a good approximation thereof by a ﬁnite number of coupled SDEs (Chekroun et al., 2011; Kondrashov et al., 2015). δ18O(x) Dust(y) A1 = −7.54 × 106 A2 = 2.63 × 107 xn B0 11 = −3.16 × 105 B0 21 = 9.03 × 105 yn B0 12 = 8.01 × 105 B0 22 = −3.69 × 106 xn−τ B1 11 = 1.26 × 103 B1 21 = 9.03 × 102 yn−τ B1 12 = 5.49 × 103 B1 22 = 1.38 × 104 xn−2τ B2 11 = −3.89 × 101 B2 21 = 2.31 × 103 yn−2τ B2 12 = 9.19 × 102 B2 22 = 6.30 × 103 x2n C11 = −5.09 × 103 C21 = 1.38 × 104 xnyn C12 = 1.68 × 104 C22 = −6.51 × 104 y2n C13 = −3.97 × 104 C23 = 1.92 × 105 x3n D11 = −3.48 × 101 D21 = 1.26 × 102 x2nyn D12 = 4.09 × 101 D22 = 9.07 × 101 xny2n D13 = −6.30 × 102 D23 = 3.09 × 103 y3n D14 = 3.33 × 102 D24 = −1.72 × 103 ξ1n Q11 = 77.61 Q21 = −13.06 ξ2n Q21 = 0 Q22 = 99.97 Assume that z is a high-dimensional state vector, whose temporal evolution is governed by the following system of ordinary differential equations, which is not explicitly known: dz dt = F(z), z ∈Rn, (A1) (A1) where R denotes the set of real numbers. Assume further- more that z can be decomposed into a sum of an observed vector x and an unobserved vector y, i.e., z = x + y. By orthogonally projecting Eq. (A1) onto the subspace spanned by the observed variables x, via P z = x, we obtain dx dt = PF(x + y) , (A2) (A2) Note that Eq. 5 Conclusions We have shown that a coupled, two-dimensional stochastic– dynamic model with cubic drift term and linear delay terms is capable of reproducing the statistical properties of δ18O and dust time series derived from the high-resolution NGRIP record for the interval 59–22 ka b2k, which roughly corre- sponds to Marine Isotope Stage 3. These statistical properties are expressed in terms of the PDFs of the time series, their power spectra and the waiting times between sharp transi- tions from stadials to interstadials. Our results demonstrate that the statistical characteristics of the roughly 40 ka long, high-resolution NGRIP time se- ries of δ18O and dust considered here can be reproduced by a nonlinear inverse model without taking into account exoge- nous forcing, whether astronomical, solar or volcanic. Based on our results alone, there would thus be no reason to assume that the temporal evolution of the δ18O ratios and dust con- centrations – and hence that of the climatic variabilities they Key ingredients for an accurate simulation of the observed time series are as follows. Earth Syst. Dynam., 8, 1171–1190, 2017 www.earth-syst-dynam.net/8/1171/2017/ N. Boers et al.: Inverse models for the NGRIP records 1182 represent, in particular the DO transitions between stadials and interstadials – are externally forced. We note, though, that Mitsui and Cruciﬁx (2017) have re- cently found evidence that including orbital forcing in mod- eling the 20 a averaged NGRIP Ca2+ time series improves their model’s BIC score. This forcing – however important on longer, astronomical timescales – does not appear to be directly relevant for the millennial-scale DO transitions, ac- cording to the present results. The predictive power of the proposed stochastic–dynamic model for the abrupt transitions from stadials to interstadials should be addressed in future work. Data availability. The high-resolution NGRIP data used in this study are available online at http://www.icecores.dk. Earth Syst. Dynam., 8, 1171–1190, 2017 www.earth-syst-dynam.net/8/1171/2017/ N. Boers et al.: Inverse models for the NGRIP records 1183 Appendix A: Key ideas in deriving the GLE www.earth-syst-dynam.net/8/1171/2017/ Appendix A: Key ideas in deriving the GLE (3) can be approximated by a Markovian SDE by using the Galerkin approximation techniques of Chekroun et al. (2016) (their Appendix C and Remark 5.1). In that respect, Eq. (3) can be put within an SDE format con- sistent with, although different from, the MSMs discussed by Kondrashov et al. (2015). which depends on the unobserved variable y. By introducing the averaging P F(x) := Z Y P F(x + y)dµx(y), (A3) (A3) where µx denotes the probability distribution of y condi- tioned on x, we obtain P F(x + y) = P F(x) \| {z } averaged part + P F(x + y) −PF(x) \| {z } ﬂuctuating part . (A4) (A4) averaged part {z ﬂuctuating part The parameterization of the ﬂuctuating part in Eq. (A4) is at the core of any stochastic parameterization method – whether linear (Penland and Sardeshmukh, 1995) or nonlinear (Majda et al., 2001) – as well as of the MZ formalism. Ergodic-type arguments show that the averaged part can in principle be learned from a time series, assuming the ex- istence of a “nice” invariant measure (Chekroun et al., 2011; Kondrashov et al., 2015): argminf ∈E  lim T →∞ 1 T T Z 0 dx dt −f (x(t;y0)) 2 dt  = P F(x), (A5) (A5) (A5) which holds for almost all y0 with respect to the Lebesgue measure; see Lemma 4.1 in Kondrashov et al. (2015) for fur- ther details. Earth Syst. Dynam., 8, 1171–1190, 2017 1184 N. Boers et al.: Inverse models for the NGRIP records −1.5 −1.0 −0.5 0.0 0.5 1.0 1.5 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Res(δ18O) Res(dust) Figure A1. Probability density of the least-squares residuals for δ18O (blue) and log(dust) (red). The residuals for the log(dust) can be approximated very well by a Gaussian, but for the δ18O the approximation is also sufﬁciently good to justify the choice of a Gaussian likelihood function for the maximum likelihood estimation. −1.5 −1.0 −0.5 0.0 0.5 1.0 1.5 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Res(δ18O) Res(dust) 1.0 Figure A1. Probability density of the least-squares residuals for δ18O (blue) and log(dust) (red). The residuals for the log(dust) can be approximated very well by a Gaussian, but for the δ18O the approximation is also sufﬁciently good to justify the choice of a Gaussian likelihood function for the maximum likelihood estimation. Appendix A: Key ideas in deriving the GLE 25 30 35 40 45 50 55 60 −50 −45 −40 −35 −30 [per mill] δ18O simulated 25 30 35 40 45 50 55 60 0.0 0.5 1.0 1.5 2.0 2.5 [ × 106 ml−1] Dust simulated 25 30 35 40 45 50 55 60 Age [ka b2k] −48 −46 −44 −42 −40 −38 −36 −34 Simulatedδ18O [per mill] δ18O simulated −15 −14 −13 −12 −11 −10 −9 −8 Simulated −log(dust) −log(dust) simulated Figure A2. Same as Fig. 2 in the main text but for the model without nonlinear terms. 25 30 35 40 45 50 55 60 −50 −45 −40 −35 −30 [per mill] δ18O simulated 25 30 35 40 45 50 55 60 0.0 0.5 1.0 1.5 2.0 2.5 [ × 106 ml−1] Dust simulated 25 30 35 40 45 50 55 60 Age [ka b2k] −48 −46 −44 −42 −40 −38 −36 −34 Simulatedδ18O [per mill] δ18O simulated −15 −14 −13 −12 −11 −10 −9 −8 Simulated −log(dust) −log(dust) simulated Figure A2. Same as Fig. 2 in the main text but for the model without nonlinear terms. Earth Syst. Dynam., 8, 1171–1190, 2017 www.earth-syst-dynam.net/8/1171/2017/ N. Boers et al.: Inverse models for the NGRIP records 1185 −50 −48 −46 −44 −42 −40 −38 −36 −34 δ18O [per mill] −0.05 0.00 0.05 0.10 0.15 0.20 0.25 0.30 PDF δ18O δ18O raw δ18O preprocessed δ18O simulated 9 10 11 12 13 14 15 log(dust) −0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 PDF log(dust) log(dust) raw log(dust) preprocessed log(dust) simulated Figure A3. Same as Fig. 3a and b in the main text but for the model without nonlinear terms. 25 30 35 40 45 50 55 60 −50 −45 −40 −35 −30 [per mill] δ18O simulated 25 30 35 40 45 50 55 60 0.0 0.5 1.0 1.5 2.0 2.5 [ × 106 ml−1] Dust simulated 25 30 35 40 45 50 55 60 Age [ka b2k] −48 −46 −44 −42 −40 −38 −36 −34 Simulatedδ18O [per mill] δ18O simulated −15 −14 −13 −12 −11 −10 −9 −8 Simulated −log(dust) −log(dust) simulated Figure A4. Same as Fig. 2 in the main text but for the model without memory terms. www.earth-syst-dynam.net/8/1171/2017/ Earth Syst. Appendix A: Key ideas in deriving the GLE 25 30 35 40 45 50 55 60 −50 −45 −40 −35 −30 [per mill] δ18O simulated 25 30 35 40 45 50 55 60 0.0 0.5 1.0 1.5 2.0 2.5 [ × 106 ml−1] Dust simulated 25 30 35 40 45 50 55 60 Age [ka b2k] −48 −46 −44 −42 −40 −38 −36 −34 Simulatedδ18O [per mill] δ18O simulated −15 −14 −13 −12 −11 −10 −9 −8 Simulated −log(dust) −log(dust) simulated Figure A6. Same as Fig. 2 in the main text but for the model without coupling terms. Note that a coevolution of the observed δ18 log(dust) time series can in this case not be expected to be reproduced. Earth Syst Dynam 8 1171–1190 2017 www earth-syst-dynam net/8/1171/2 −50 −48 −46 −44 −42 −40 −38 −36 −34 δ18O [per mill] −0.05 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 PDF δ18O δ18O raw δ18O preprocessed δ18O simulated 9 10 11 12 13 14 15 log(dust) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 PDF log(dust) log(dust) raw log(dust) preprocessed log(dust) simulated Figure A5. Same as Fig. 3a and b in the main text but for the model without memory terms. −50 −48 −46 −44 −42 −40 −38 −36 −34 δ18O [per mill] −0.05 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 PDF δ18O δ18O raw δ18O preprocessed δ18O simulated 9 10 11 12 13 14 15 log(dust) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 PDF log(dust) log(dust) raw log(dust) preprocessed log(dust) simulated Figure A5. Same as Fig. 3a and b in the main text but for the model without memory terms. 25 30 35 40 45 50 55 60 −50 −45 −40 −35 −30 [per mill] δ18O simulated 25 30 35 40 45 50 55 60 0.0 0.5 1.0 1.5 2.0 2.5 [ × 106 ml−1] Dust simulated 25 30 35 40 45 50 55 60 Age [ka b2k] −48 −46 −44 −42 −40 −38 −36 −34 Simulatedδ18O [per mill] δ18O simulated −15 −14 −13 −12 −11 −10 −9 −8 Simulated −log(dust) −log(dust) simulated Figure A6. Same as Fig. 2 in the main text but for the model without coupling terms. Note that a coevolution of the observed δ18O and log(dust) time series can in this case not be expected to be reproduced. Appendix A: Key ideas in deriving the GLE Dynam., 8, 1171–1 −50 −48 −46 −44 −42 −40 −38 −36 −34 δ18O [per mill] −0.05 0.00 0.05 0.10 0.15 0.20 0.25 0.30 PDF δ18O δ18O raw δ18O preprocessed δ18O simulated 9 10 11 12 13 14 15 log(dust) −0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 PDF log(dust) log(dust) raw log(dust) preprocessed log(dust) simulated Figure A3. Same as Fig. 3a and b in the main text but for the model without nonlinear terms. −50 −48 −46 −44 −42 −40 −38 −36 −34 δ18O [per mill] −0.05 0.00 0.05 0.10 0.15 0.20 0.25 0.30 PDF δ18O δ18O raw δ18O preprocessed δ18O simulated 9 10 11 12 13 14 15 log(dust) −0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 PDF log(dust) log(dust) raw log(dust) preprocessed log(dust) simulated PDF δ18O Figure A3. Same as Fig. 3a and b in the main text but for the model without nonlinear terms. 25 30 35 40 45 50 55 60 −50 −45 −40 −35 −30 [per mill] δ18O simulated 25 30 35 40 45 50 55 60 0.0 0.5 1.0 1.5 2.0 2.5 [ × 106 ml−1] Dust simulated 25 30 35 40 45 50 55 60 Age [ka b2k] −48 −46 −44 −42 −40 −38 −36 −34 Simulatedδ18O [per mill] δ18O simulated −15 −14 −13 −12 −11 −10 −9 −8 Simulated −log(dust) −log(dust) simulated Figure A4. Same as Fig. 2 in the main text but for the model without memory terms. Figure A4. Same as Fig. 2 in the main text but for the model without memory terms. Earth Syst. Dynam., 8, 1171–1190, 2017 www.earth-syst-dynam.net/8/1171/2017/ 1186 N. Boers et al.: Inverse models for the NGRIP records −50 −48 −46 −44 −42 −40 −38 −36 −34 δ18O [per mill] −0.05 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 PDF δ18O δ18O raw δ18O preprocessed δ18O simulated 9 10 11 12 13 14 15 log(dust) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 PDF log(dust) log(dust) raw log(dust) preprocessed log(dust) simulated Figure A5. Same as Fig. 3a and b in the main text but for the model without memory terms. N. Boers et al.: Inverse models for the NGRIP records Competing interests. The authors declare that they have no com- peting ﬁnancial interests. Competing interests. The authors declare that they have no com- peting ﬁnancial interests. Berger, W. H.: The 100-kyr ice-age cycle: Internal oscillation or inclinational forcing?, Int. J. Earth Sci., 88, 305–316, https://doi.org/10.1007/s005310050266, 1999. Bhattacharya, K., Ghil, M., and Vulis, I. L.: Internal variabil- ity of an energy-balance model with delayed albedo effects, J. Atmos. Sci., 39, 1747–1773, https://doi.org/10.1175/1520- 0469(1982)039<1747:IVOAEB>2.0.CO;2, 1982. Acknowledgements. This research was initiated by a collab- oration between D.-D. Rousseau and the late Sigfús Johnsen, to whose memory it is dedicated. N. Boers acknowledges funding by the Alexander von Humboldt Foundation and the German Federal Ministry for Education and Research. M. D. Chekroun, M. Ghil, D. Kondrashov and H. Liu acknowledge support by grant N00014-16-1-2073 from the Multidisciplinary University Research Initiative (MURI) of the Ofﬁce of Naval Research and by National Science Foundation grant OCE-1243175. M. D. Chekroun and H. Liu also acknowledge support by National Science Foun- dation grants DMS-1616981 and DMS-1616450, respectively. D. Kondrashov also acknowledges support by the Government of the Russian Federation (agreement no. 14.Z50.31.0033 with the Institute of Applied Physics of RAS). This is LDEO contribution no. 8167. NGRIP is directed and organized by the Ice and Climate research group, Niels Bohr Institute, University of Copenhagen. It is supported by funding agencies in Denmark (FNU), Belgium (FNRS-CFB), France (IPEV and INSU/CNRS), Germany (AWI), Iceland (RannIs), Japan (MEXT), Sweden (SPRS), Switzerland (SNF) and the USA (NSF, Ofﬁce of Polar Programs). Burnham, K. and Anderson, D.: Model Selection and Mul- timodel Inference: A Practical Information-Theoretic Approach, vol. 172, Springer, New York, 2 edn., https://doi.org/10.1016/j.ecolmodel.2003.11.004, 2002. Chekroun, M. D., Kondrashov, D., and Ghil, M.: Predicting stochas- tic systems by noise sampling, and application to the El Niño- Southern Oscillation, P. Natl. Acad. Sci. USA, 108, 11766– 11771, https://doi.org/10.1073/pnas.1015753108, 2011. Chekroun, M. D., Ghil, M., Liu, H., and Wang, S.: Low- dimensional Galerkin approximations of nonlinear delay dif- ferential equations, Discrete Cont. Dyn.-A, 36, 4133–4177, https://doi.org/10.3934/dcds.2016.36.4133, 2016. Chorin, A. J. and Hald, O. H.: Stochastic Tools in Mathematics and Science, Vol. 58 of Texts in Applied Mathematics, Springer, New York, third edn., 2013. Chorin, A. J. and Lu, F.: Discrete approach to stochas- tic parametrization and dimension reduction in nonlinear dynamics., P. Natl. Acad. Sci. USA, 112, 9804–9809, https://doi.org/10.1073/pnas.1512080112, 2015. Edited by: Anders Levermann Reviewed by: Takahito Mitsui and one anonymous referee Edited by: Anders Levermann Reviewed by: Takahito Mitsui and one anonymous referee Chorin, A. Competing interests. The authors declare that they have no com- peting ﬁnancial interests. J., Hald, O. H., and Kupferman, R.: Optimal prediction with memory, Physica D, 166, 239–257, 2002. Cruciﬁx, M. and Rougier, J.: On the use of simple dynamical sys- tems for climate predictions, Eur. Phys. J.-Spec. Top., 174, 11– 31, 2009. References Andersen, E. B.: Asymptotic properties of conditional maximum- likelihood estimators, J. Roy. Stat. Soc. B Met., 32, 283–301, 1970. Dansgaard, W., Johnsen, S. J., Clausen, H. B., Dahl-Jensen, D., Gundestrup, N. S., Hammer, C. U., Hvidberg, C. S., Stef- fensen, J. P., Sveinbjörnsdóttir, Á. E., Jouzel, J., and Bond, G. C.: Evidence for general instability of past cli- mate from a 250-kyr ice-core record, Nature, 364, 218–220, https://doi.org/10.1038/364218a0, 1993. Andersen, K. K., Azuma, N., Barnola, J.-M., Bigler, M., Biscaye, P., Caillon, N., Chappellaz, J., Clausen, H. B., Dahl-Jensen, D., Fis- cher, H., Flückiger, J., Fritzsche, D., Fujii, Y., Goto-Azuma, K., Grønvold, K., Gundestrup, N. S., Hansson, M., Huber, C., Hvid- berg, C. S., Johnsen, S. J., Jonsell, U., Jouzel, J., Kipfstuhl, S., Landais, A., Leuenberger, M., Lorrain, R., Masson-Delmotte, V., Miller, H., Motoyama, H., Narita, H., Popp, T., Rasmussen, S. O., Raynaud, D., Rothlisberger, R., Ruth, U., Samyn, D., Schwan- der, J., Shoji, H., Siggard-Andersen, M.-L., Steffensen, J. P., Stocker, T., Sveinbjörnsdóttir, a. E., Svensson, A., Takata, M., Tison, J.-L., Thorsteinsson, T., Watanabe, O., Wilhelms, F., and White, J. W. C.: High-resolution record of Northern Hemisphere climate extending into the last interglacial period, Nature, 431, 147–151, https://doi.org/10.1038/nature02805, 2004. De Saedeleer, B., Cruciﬁx, M., and Wieczorek, S.: Is the astronom- ical forcing a reliable and unique pacemaker for climate? A con- ceptual model study, Clim. Dynam., 40, 273–294, 2013. Ditlevsen, P. D.: Observation of alpha stable noise induced millenial climate changes from an ice-core record, Geophys. Res. Lett., 26, 1441–1444, 1999. Ditlevsen, P. D. and Ditlevsen, O. D.: On the stochastic nature of the rapid climate shifts during the last ice age, J. Climate, 22, 446–457, https://doi.org/10.1175/2008JCLI2430.1, 2009. Tison, J.-L., Thorsteinsson, T., Watanabe, O., Wilhelms, F., and White, J. W. C.: High-resolution record of Northern Hemisphere climate extending into the last interglacial period, Nature, 431, 147–151, https://doi.org/10.1038/nature02805, 2004. Ditlevsen, P. D., Kristensen, M. S., and Andersen, K. K.: The recurrence time of Dansgaard–Oeschger events and limits on the possible periodic component, J. Climate, 18, 2594–2603, https://doi.org/10.1175/JCLI3437.1, 2005. Andersen, K. K., Svensson, A., Johnsen, S. J., Ras- mussen, S. O., Bigler, M., Rüthlisberger, R., Ruth, U., Siggaard-Andersen, M. L., Peder Steffensen, J., Dahl- Jensen, D., Vinther, B. M., and Clausen, H. B.: The Greenland Ice Core Chronology 2005, 15–42 ka. Part 1: Construct- Ditlevsen, P. D., Andersen, K. N. Boers et al.: Inverse models for the NGRIP records 1188 ing the time scale, Quaternary Sci. Rev., 25, 3246–3257, https://doi.org/10.1016/j.quascirev.2006.08.002, 2006. ing the time scale, Quaternary Sci. Rev., 25, 3246–3257, https://doi.org/10.1016/j.quascirev.2006.08.002, 2006. Author contributions. MB, D-DR and AS provided the data. NB, MDC, MG, DK and HL conceived of the research. NB conducted the numerical analysis and prepared the manuscript. All authors dis- cussed the results, drew conclusions and edited the manuscript. Author contributions. MB, D-DR and AS provided the data. NB, MDC, MG, DK and HL conceived of the research. NB conducted the numerical analysis and prepared the manuscript. All authors dis- cussed the results, drew conclusions and edited the manuscript. Ba Barnston, A. G., Tippett, M. K., L'Heureux, M. L., Li, S., and Dewitt, D. G.: Skill of real-time seasonal ENSO model pre- dictions during 2002–11: is our capability increasing?, B. Am. Meteorol. Soc., 93, 631–651, https://doi.org/10.1175/BAMS-D- 11-00111.1, 2012. Appendix A: Key ideas in deriving the GLE 25 30 35 40 45 50 55 60 −50 −45 −40 −35 −30 [per mill] δ18O simulated 25 30 35 40 45 50 55 60 0.0 0.5 1.0 1.5 2.0 2.5 [ × 106 ml−1] Dust simulated 25 30 35 40 45 50 55 60 Age [ka b2k] −48 −46 −44 −42 −40 −38 −36 −34 Simulatedδ18O [per mill] δ18O simulated −15 −14 −13 −12 −11 −10 −9 −8 Simulated −log(dust) −log(dust) simulated Figure A6. Same as Fig. 2 in the main text but for the model without coupling terms. Note that a coevolution of the observed δ18O and log(dust) time series can in this case not be expected to be reproduced. Earth Syst. Dynam., 8, 1171–1190, 2017 www.earth-syst-dynam.net/8/1171/2017/ 1187 N. Boers et al.: Inverse models for the NGRIP records N. Boers et al.: Inverse models for the NGRIP records −50 −48 −46 −44 −42 −40 −38 −36 −34 δ18O [per mill] −0.05 0.00 0.05 0.10 0.15 0.20 0.25 0.30 PDF δ18O δ18O raw δ18O preprocessed δ18O simulated 9 10 11 12 13 14 15 log(dust) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 PDF log(dust) log(dust) raw log(dust) preprocessed log(dust) simulated Figure A7. Same as Fig. 3a and b in the main text but for the model without coupling terms. −50 −48 −46 −44 −42 −40 −38 −36 −34 δ18O [per mill] −0.05 0.00 0.05 0.10 0.15 0.20 0.25 0.30 PDF δ18O δ18O raw δ18O preprocessed δ18O simulated 9 10 11 12 13 14 15 log(dust) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 PDF log(dust) log(dust) raw log(dust) preprocessed log(dust) simulated Figure A7. Same as Fig. 3a and b in the main text but for the model without coupling terms. Earth Syst. Dynam., 8, 1171–1190, 2017 www.earth-syst-dynam.net/8/1171/2017/ www.earth-syst-dynam.net/8/1171/2017/ N. Boers et al.: Inverse models for the NGRIP records N. Boers et al.: Inverse models for the NGRIP records 1189 Kondrashov, D., Kravtsov, S., and Ghil, M.: Empirical mode re- duction in a model of extratropical low-frequency variability, J. Atmos. Sci., 63, 1859–1877, https://doi.org/10.1175/JAS3719.1, 2006. tion of the claimed 1470 years cycle, Clim. Past, 3, 129–134, https://doi.org/10.5194/cp-3-129-2007, 2007. tion of the claimed 1470 years cycle, Clim. Past, 3, 129–134, https://doi.org/10.5194/cp-3-129-2007, 2007. Fischer, H., Siggaard-Andersen, M., Ruth, U., Röthlis- berger, R., and Wolff, E. W.: Glacial/interglacial changes in mineral dust and sea-salt records in polar ice cores: sources, transport, and deposition, Rev. Geophys., 45, 1–26, https://doi.org/10.1029/2005RG000192, 2007. Kondrashov, D., Chekroun, M. D., Robertson, A. W., and Ghil, M.: Low-order stochastic model and “past-noise forecasting” of the Madden–Julian Oscillation, Geophys. Res. Lett., 40, 5305–5310, https://doi.org/10.1002/grl.50991, 2013. Ghil, M.: Cryothermodynamics: the chaotic dynamics of paleo- climate, Physica D, 77, 130–159, https://doi.org/10.1016/0167- 2789(94)90131-7, 1994. Kondrashov, D., Chekroun, M. D., and Ghil, M.: Data-driven non-Markovian closure models, Physica D, 297, 33–55, https://doi.org/10.1016/j.physd.2014.12.005, 2015. Ghil, M., Allen, M. R., Dettinger, M. D., Ide, K., Kon- drashov, D., Mann, M. E., Robertson, A. W., Saunders, A., Tian, Y., Varadi, F., and Yiou, P.: Advanced spectral meth- ods for climatic time series, Rev. Geophys., 40, 1003, https://doi.org/10.1029/2000RG000092, 2002. Kravtsov, S., Kondrashov, D., and Ghil, M.: Multilevel regres- sion modeling of nonlinear processes: Derivation and appli- cations to climatic variability, J. Climate, 18, 4404–4424, https://doi.org/10.1175/JCLI3544.1, 2005. Ghil, M., Chekroun, M. D., and Stepan, G.: A collection on “Cli- mate Dynamics: multiple scales and memory effects”, Proc. R. Soc. A, 471, 20150097, https://doi.org/10.1098/rspa.2015.0097, 2015. Kravtsov, S., Kondrashov, D., and Ghil, M.: Empirical model re- duction and the modelling hierarchy in climate dynamics, and the geosciences, in: Stochastic Physics and Climate modeling, edited by Palmer, T., and Williams, P., Cambridge University Press, Cambridge, available at: http://www.atmos.ucla.edu/tcd/ PREPRINTS/BookEMR_Text.pdf, 35–72, 2009. Gkinis, V., Simonsen, S. B., Buchardt, S. L., White, J. W. C., and Vinther, B. M.: Water isotope diffusion rates from the North- GRIP ice core for the last 16,000 years – Glaciological and pa- leoclimatic implications, Earth Planet. Sc. Lett., 405, 132–141, https://doi.org/10.1016/j.epsl.2014.08.022, 2014. Krumscheid, S., Pradas, M., Pavliotis, G. A., and Kalliada- sis, S.: Data-driven coarse graining in action: Modelling and prediction of complex systems, Phys. Rev. E, 92, 042139, https://doi.org/10.1103/PhysRevE.92.042139, 2015. Hasselmann, K.: Stochastic climate models Part I. The- ory, Tellus, 28, 473–485, https://doi.org/10.1111/j.2153- 3490.1976.tb00696.x, 1976. Kwasniok, F.: Analysis and modelling of glacial climate transitions using simple dynamical systems, Philos. T. R. Soc. A, 371, 1–22, 2013. N. Boers et al.: Inverse models for the NGRIP records Johnsen, S., Clausen, H., Dansgaard, W., Fuhrer, K., Gun- destrup, N., Hammer, C., Iversen, P., Jouzel, J., Stauf- fer, B., and Steffensen, J.: Irregular glacial interstadials recorded in a new Greenland ice core, Nature, 359, 311–313, https://doi.org/10.1038/359311a0, 1992. Le Treut, H., and Ghil, M.: Orbital forcing, climatic interactions, and glaciation cycles, J. Geophys. Res.-Oceans, 88, 5167–5190, https://doi.org/10.1029/JC088iC09p05167, 1983. Majda, A. J., Timofeyev, I., and Vanden Eijnden, E.: A mathe- matical framework for stochastic climate models, Commun. Pur. Appl. Math., 54, 891–974, 2001. p g , Johnsen, S. J., Clausen, H. B., Dansgaard, W., Gundestrup, N. S., Hammer, C. U., Andersen, U., Andersen, K. K., Hvidberg, C. S., Dahl-Jensen, D., Steffensen, J. P., Shoji, H., Sveinbjornsdot- tir, A. E., White, J., Jouzel, J., and Fisher, D.: The d18O record along the Greenland Ice Core Project deep ice core and the prob- lem of possible Eemian climatic instability, J. Geophys. Res.- Oceans, 102, 26397–26410, https://doi.org/10.1029/97JC00167, 1997. Mitsui, T. and Cruciﬁx, M.: Inﬂuence of external forcings on abrupt millennial-scale climate changes: a statistical modelling study, Clim. Dynam., 48, 2729–2749, https://doi.org/10.1007/s00382- 016-3235-z, 2017. Mori, H.: A continued-fraction representation of the time- correlation functions, Prog. Theor. Phys., 34, 399–416, 1965. Johnsen, S. J., Dahl-Jensen, D., Gundestrup, N., Steffensen, J. P., Clausen, H. B., Miller, H., Masson-Delmotte, V., Sveinbjörns- dottir, A. E., and White, J.: Oxygen isotope and palaeotemper- ature records from six Greenland ice-core stations: Camp Cen- tury, Dye-3, GRIP, GISP2, Renland and NorthGRIP, J. Quater- nary Sci., 16, 299–307, https://doi.org/10.1002/jqs.622, 2001a. Peavoy, D., Franzke, C. L. E., and Roberts, G. O.: Systematic physics constrained parameter estimation of stochastic differen- tial equations, Comput. Stat. Data An., 83, 182–199, 2015. Pelletier, J. D.: Coherence resonance and ice ages, J. Geophys. Res.-Atmos., 108, 1–14, https://doi.org/10.1029/2002JD003120, 2003. Penland, C. and Sardeshmukh, P. D.: The optimal growth of tropi- cal sea surface temperature anomalies, J. Climate, 8, 1999–2024, 1995. Johnsen, S. J., Dahl-Jensen, D., Gundestrup, N., Steffensen, J. P., Clausen, H. B., Miller, H., Masson-Delmotte, V., Sveinbjorns- dottir, A. E., and White, J.: Oxygen isotope and palaeotemper- ature records from six Greenland ice-core stations: Camp Cen- tury, Dye-3, GRIP, GISP2, Renland and NorthGRIP, J. Quater- nary Sci., 16, 299–307, https://doi.org/10.1002/jqs.622, 2001b. Penland, C., Ghil, M., and Weickmann, K.: Adaptive ﬁltering and maximum entropy spectra with application to changes in at- mospheric angular momentum, J. Geophys. Res.-Atmos., 96, 22659–22671, https://doi.org/10.1029/91JD02107, 1991. References K., and Svensson, A.: The DO- climate events are probably noise induced: statistical investiga- Earth Syst. Dynam., 8, 1171–1190, 2017 www.earth-syst-dynam.net/8/1171/2017/ N. Boers et al.: Inverse models for the NGRIP records N. Boers et al.: Inverse models for the NGRIP records 1190 graphic framework for abrupt climatic changes during the Last Glacial period based on three synchronized Green- land ice-core records: Reﬁning and extending the INTI- MATE event stratigraphy, Quaternary Sci. Rev., 106, 14–28, https://doi.org/10.1016/j.quascirev.2014.09.007, 2014. Steffensen, J. P., Andersen, K. K., Bigler, M., Clausen, H. B., Dahl-Jensen, D., Fischer, H., Goto-Azuma, K., Hansson, M., Johnsen, S. J., Jouzel, J., Masson-Delmotte, V., Popp, T., Ras- mussen, S. O., Röthlisberger, R., Ruth, U., Stauffer, B., Siggaard- Andersen, M.-L., Sveinbjörnsdóttir, Á. E., Svensson, A., and White, J. W. C.: High-resolution Greenland ice core data show abrupt climate change happens in few years, Science, 321, 680– 684, https://doi.org/10.1126/science.1157707, 2008. Rial, J. A.: Abrupt climate change: chaos and order at orbital and millennial scales, Global Planet. Change, 41, 95–109, https://doi.org/10.1016/j.gloplacha.2003.10.004, 2004. Ruth, U., Wagenbach, D., Bigler, M., Steffensen, J. P., Röth- lisberger, R., and Miller, H.: High-resolution micropar- ticle proﬁles at NorthGRIP, Greenland: case studies of the calcium-dust relationship, Ann. Glaciol., 35, 237–242, https://doi.org/10.3189/172756402781817347, 2002. Svensson, A., Andersen, K. K., Bigler, M., Clausen, H. B., Dahl- Jensen, D., Davies, S. M., Johnsen, S. J., Muscheler, R., Par- renin, F., Rasmussen, S. O., Röthlisberger, R., Seierstad, I., Steffensen, J. P., and Vinther, B. M.: A 60 000 year Green- land stratigraphic ice core chronology, Clim. Past, 4, 47–57, https://doi.org/10.5194/cp-4-47-2008, 2008. Ruth, U., Wagenbach, D., Steffensen, J. P., and Bigler, M.: Con- tinuous record of microparticle concentration and size dis- tribution in the central Greenland NGRIP ice core during the last glacial period, J. Geophys. Res.-Atmos., 108, 1–12, https://doi.org/10.1029/2002JD002376, 2003. Tziperman, E., Raymo, M. E., Huybers, P., and Wunsch, C.: Con- sequences of pacing the Pleistocene 100 kyr ice ages by non- linear phase locking to Milankovitch forcing, Paleoceanography, 21, PA4206, https://doi.org/10.1029/2005PA001241, 2006. Rypdal, M.: Early-Warning Signals for the onsets of Green- land Interstadials and the Younger Dryas–Preboreal transi- tion, J. Climate, 29, 4047–4056, https://doi.org/10.1175/JCLI-D- 15-0828.1, 2016. Vautard, R., Yiou, P., and Ghil, M.: Singular-spectrum analysis: A toolkit for short, noisy chaotic signals, Physica D, 58, 95–126, https://doi.org/10.1016/0167-2789(92)90103-T, 1992. Yang, Y.: Can the strengths of AIC and BIC be shared? A conﬂict between model indentiﬁcation and regression estimation, Biometrika, 92, 937–950, https://doi.org/10.1093/biomet/92.4.937, 2005. Saltzman, B. and Maasch, K. A.: A ﬁrst-order global model of late Cenozoic climate, T. Roy. Soc. Edin.-Earth, 81, 315–325, 1990. Saltzman, B. and Maasch, K. A.: A ﬁrst-order global model of late Cenozoic climatic change I I. N. Boers et al.: Inverse models for the NGRIP records Källén, E., Crafoord, C., Ghil, M., and Kaumllleacuten, E.: Free oscillations in a climate model with ice-sheet dynam- ics, J. Atmos. Sci., 36, 2292–2303, https://doi.org/10.1175/1520- 0469(1979)036<2292:foiacm>2.0.co;2, 1979. Rasmussen, S. O., Bigler, M., Blockley, S. P., Blunier, T., Buchardt, S. L., Clausen, H. B., Cvijanovic, I., Dahl-Jensen, D., Johnsen, S. J., Fischer, H., Gkinis, V., Guillevic, M., Hoek, W. Z., Lowe, J. J., Pedro, J. B., Popp, T., Seierstad, I. K., Stef- fensen, J. P., Svensson, A. M., Vallelonga, P., Vinther, B. M., Walker, M. J. C., Wheatley, J. J., and Winstrup, M.: A strati- Johnsen, S. J., Fischer, H., Gkinis, V., Guillevic, M., Hoek, W. Z., Kondrashov, D., Kravtsov, S., Robertson, A. W., and Ghil, M.: A hi- erarchy of data-based ENSO models, J. Climate, 18, 4425–4444, https://doi.org/10.1175/JCLI3567.1, 2005. Earth Syst. Dynam., 8, 1171–1190, 2017 www.earth-syst-dynam.net/8/1171/2017/ www.earth-syst-dynam.net/8/1171/2017/ N. Boers et al.: Inverse models for the NGRIP records Further analysis based on a simpli- ﬁcation of CO2 dynamics, Clim. Dynam., 5, 201–210, 1991. Zwanzig, R.: On the identity of three generalized master equations, Physica, 30, 1109–1123, 1964. Srivastava, J. N.: A multivariate extension of the gauss- markov theorem, Ann. I. Stat. Math., 17, 63–66, https://doi.org/10.1007/BF02868153, 1965. Earth Syst. Dynam., 8, 1171–1190, 2017 www.earth-syst-dynam.net/8/1171/2017/
https://openalex.org/W2117166804	https://journals.plos.org/plosmedicine/article/file?id=10.1371/journal.pmed.0010052&type=printable	English	null	Is It Ethical to Use Enhancement Technologies to Make Us Better than Well?	PLoS medicine	2,004	cc-by	3,682	Is It Ethical to Use Enhancement Technologies to Make Us Better than Well? Arthur Caplan, Carl Elliott DOI: 10.1371/journal.pmed.0010052.g001 It is in our nature as humans to strive for self-improvement (Illustration: Margaret Shear) B ackground to the debate: A variety of biomedical technologies are being developed that can be used for purposes other than treating disease. Such “enhancement technologies” can be used to improve our appearance and regulate our emotions, with the goal of feeling “better than well.” While these technologies can help people adapt to their rapidly changing lifestyles, their use raises important ethical issues. Open access, freely available online Open access, freely available online The PLoS Medicine Debate The PLoS Medicine Debate Open access, freely available online Carl Elliott’s Viewpoint: Pharma’s Gain May Be Our Loss Putting the brakes on biologically driven human betterment would have real consequences for science. Some lines of research would be slowed or restricted [3,5,8]. Their application would be declared off-limits or at least tightly regulated [1,2,3,4,5,7,8,9]. Those of us who worry about medical enhancement are usually less worried about the technologies themselves than about the larger social effects of embracing them too enthusiastically. Just as you do not need to object to cars to worry about urban sprawl, you do not need to object to enhancement technologies to question where these technologies may be taking us. It is not just technophobes who wonder whether a society that consumes 90% of the world’s supply of methylphenidate (Ritalin), where the most proﬁ table class of drugs is antidepressants, and where cosmetic surgeons perform liposuction on prime-time television is a society that has somehow lost its way. Why is the drive to improve ourselves so disturbing to the anti-meliorists? Their arguments cluster around three key worries: that the pursuit of perfection by biomedical means is vain, selﬁ sh, and unrewarding [1,2,3,6,7], that improving ourselves is unfair [1,3,4], and that enhancement or improvement violates human nature [2,4,5,7,8,9] and may actually destroy it [2,5,7,9]. It is the last of these arguments that is at the core of anti-meliorist concerns. It cannot simply be the pursuit of improvement that is making anti-meliorists nervous. Many religious traditions and spiritual movements seek perfection [10,11,12,13], but these evoke no negative commentary from the anti-meliorists. Nor do efforts to improve animals and plants set this crowd aﬂ utter. Rather, it is biomedical knowledge being applied to you and me that is the crux of their concern. They fear that in applying new biomedical knowledge to improve human beings, something essential about humanity will be lost. If biomedical tinkering is allowed, we will destroy the very thing that makes us human—our nature. Let’s look at three of the most commercially successful medical enhancements of recent years: selective serotonin reuptake inhibitors, hormone replacement therapy, and the diet drug fenﬂ uramine-phentermine (Fen-Phen). What can we learn from these interventions? First, the manufacturers of enhancement technologies will usually exploit the blurry line between enhancement and treatment in order to sell drugs. Because enhancement technologies must be prescribed by physicians, drug manufacturers typically market the technologies not as enhancements, but as treatments for newly discovered or under-recognized disorders. Carl Elliott’s Viewpoint: Pharma’s Gain May Be Our Loss Selective serotonin reuptake inhibitors were marketed not as personality enhancers, or even only as treatments for clinical depression, but as treatments for questionable illnesses like “premenstrual dysphoric disorder” [16]. Fen-Phen was sold not as a mere diet drug but as a treatment for obesity, which Wyeth, the manufacturer, portrayed as a dangerous public health problem [17]. Estrogen replacement therapy was initially marketed as a risk-free way for women to extend their youthfulness. But when a 1974 study found that estrogen Anti-meliorism rests, however, on a very shaky foundation. To support their position, the anti-meliorists must state what human nature is. They do not. They must also be very clear about why they see human nature as static. They are not. And they must advance an argument about why human nature, which has presumably evolved in response to an enormous array of random forces, tells us anything about what is good or desirable in terms of the traits humans should possess. They cannot. The ﬁ ght over whether there is any such thing as human nature is a long-standing one [14]. But one can concede that we are shaped by a causally powerful set of genetic inﬂ uences and still remain skeptical as to whether these produce a single “nature” that all members of humanity possess. Is there a single trait or ﬁ xed set of traits that deﬁ nes the nature of who we are and have been throughout our entire existence on this planet? Unless they can articulate this Platonic essence, anti- meliorists do not have a foundation for their argument that change, improvement, and betterment are grave threats to humanity. DOI: 10.1371/journal.pmed.0010052.g002 Where is the pursuit of the perfect face, body, and mind taking us? (Illustration: Margaret Shear) Worse still for anti-meliorists, we are clearly creatures who have long tinkered with ourselves, using all manner of technologies from clothing to telescopes to computers to airplanes. Our view of our “nature” is closely linked to the technologies that we have invented and to which we have adapted [15]. We are already technological creatures. Nor is there any normative guidance offered by our evolutionary history that shows why we should not try to improve upon the biological design with which we are endowed. Arthur Caplan’s Viewpoint: Nobody Is Perfect— But Why Not Try to Be Better? Perfection has come in for a lot of bad press recently. A torrent of books and articles has recently appeared [1,2,3,4,5,6,7,8,9], all raising serious ethical questions about the wisdom and morality of trying to use biomedical knowledge to perfect ourselves or our offspring. g p p g Biomedical scientists and physicians might be inclined to ignore this literature as just so much abstract philosophical handwringing. After all, it is almost impossible to ﬁ nd mainstream scientists arrogant enough to proclaim their interest in perfecting anything, much less themselves or their fellow human beings. DOI: 10.1371/journal.pmed.0010052.g001 It is in our nature as humans to strive for self-improvement (Illustration: Margaret Shear) Beating up on the pursuit of perfection is silly. As Salvadore Dali famously pointed out, “Have no fear of perfection— you’ll never reach it.” Critics of those who allegedly seek to perfect human beings know this. While often couching their critiques in language that assails the pursuit of perfection, what they really are attacking is the far more oft-expressed— albeit far less lofty—desire to improve or enhance a particular behavior or trait by the application of emerging biomedical knowledge in genetics, neuroscience, pharmacology, and physiology. Those who might accurately be termed “anti- meliorists” wonder how we will ever resist the obvious temptation to put this knowledge to use to alter ourselves. They are quick to note that we have already given in to such temptation—we augment our breasts, smooth our wrinkles, and pump ourselves full of antidepressants. The PLoS Medicine Debate discusses important but controversial issues in clinical practice, public health policy, or health in general. PLoS Medicine \| www.plosmedicine.org December 2004 \| Volume 1 \| Issue 3 \| e69 \| e52 172 PLoS Medicine \| www.plosmedicine.org Caplan’s Response to Elliott’s Viewpoint replacement therapy was associated with an increased risk of endometrial cancer, the manufacturers added progesterone, renamed the combination “hormone” replacement therapy, and recast it as a treatment for medical problems associated with menopause such as osteoporosis [6]. Elliott professes to be unhappy about enhancement. What arguments does he present to support his unhappiness? Not many, and the arguments that he does offer miss the point completely. Second, an alarming number of supposedly risk-free enhancements have later been associated with unanticipated side effects, some of them deadly. Wyeth has set aside over $16 billion to compensate the thousands of patients who have developed valvular heart disease and pulmonary hypertension after taking Fen-Phen [18]. A 2002 National Institutes of Health study found that hormone replacement therapy was associated with such an elevated risk of heart disease, stroke, pulmonary emboli, and breast cancer that the study was stopped prematurely [19]. Selective serotonin reuptake inhibitors are currently embroiled in controversy over whether they are associated with an elevated risk of suicide [20]. If people want to feel better, sleep less, have fewer hot ﬂ ashes, better vision, or fewer wrinkles, then they may want to use enhancement technologies to achieve these things. Technology in itself isn’t driving us in any particular direction—I believe that we decide where it should go. Elliott, however, gravely warns us that you and I do not really decide a direction when it comes to matters of enhancement. It is—listen carefully for the Darth Vader–esque hissing—drug companies! The rest of Elliott’s viewpoint amounts to what is his increasingly familiar harangue against the pharmaceutical industry. The drug companies sucker us into buying enhancement by getting us hooked on pseudotherapies. The drug companies rob us of our will to fend off their siren-like messages of better living through their chemistry. And the drug companies get us feeling so bad about ourselves that we empty our wallets on their latest overpriced geegaws. Third, the most successful enhancement technologies have been backed by tremendously inﬂ uential public relations campaigns. These campaigns have included ghostwritten journal articles, industry-funded front groups, and lucrative payments to academics, professional societies, and university centers [21]. For example, GlaxoSmithKline marketed paroxetine (Paxil) by promoting the previously obscure diagnosis of “social anxiety disorder” through phony support groups, celebrity spokespeople, a direct-to-consumer illness awareness campaign, and generous payments to key opinion leaders [22]. Caplan’s Response to Elliott’s Viewpoint The pharmaceutical industry can buy politicians to pass industry-friendly legislation; it can buy academic scientists to publish favorable journals articles; it can buy professional societies and patient support groups to spread the word on the newly medicalized disorders that its interventions are developed to treat [24]. It can even buy bioethicists to dispense with any moral concerns [25]. In this kind of political and economic climate, how likely is it that dissenting voices will have any effect before it is too late? Elliott’s Response to Caplan’s Viewpoint The traditional worry about enhancement technologies is that users of the technologies are buying individual well-being at the expense of some larger social good. I may improve my own athletic ability by taking steroids, but I set off a steroid arms race that destroys my sport. I may get cosmetic surgery for my “Asian eyes” or use skin lighteners for my dark skin, but I reinforce the implicitly racist social norms that say that Asian eyes or dark skin are traits to be ashamed of. The worry is that some aspect of the way we live together, collectively, is going to be damaged by actions that we take individually [4]. Caplan does not defend medical enhancement so much as attack its critics. Or rather, he attacks a small group of conservative critics who want to preserve “human nature.” He dispatches those critics with admirable precision, but I am not sure why he believes that group of critics includes me. My worry about enhancement technologies has little to do with human nature. My worry is that we will ignore important human needs at the expense of frivolous human desires; that dominant social norms will crowd out those of the minority; that the self-improvement agenda will be set not by individuals, but by powerful corporate interests; and that in the pursuit of betterment, we will actually make ourselves worse off. A market-driven health-care system brings this worry much closer to home. The pharmaceutical industry is now the most proﬁ table and politically powerful industry in the United States [23]. It also has a huge ﬁ nancial interest in creating a demand for enhancement technologies. The pharmaceutical industry can buy politicians to pass industry-friendly It’s no secret that many Americans are deeply ashamed of their personal shortcomings and inadequacies. Nor is it any secret that these shortcomings and inadequacies can be exploited for commercial proﬁ t. But do we really want to submit our health-care system to the same forces that have made millionaires out of motivational speakers and diet book authors? legislation; it can buy academic scientists to publish favorable journals articles; it can buy professional societies and patient support groups to spread the word on the newly medicalized disorders that its interventions are developed to treat [24]. It can even buy bioethicists to dispense with any moral concerns [25]. Carl Elliott’s Viewpoint: Pharma’s Gain May Be Our Loss Augmenting breasts or prolonging erections may be vain and even a waste of scarce resources, but seeking to use our knowledge to enhance our vision, memory, learning skills, immunity, or metabolism is not obviously either. Ultimately, anti-meliorism posits a static vision of human nature to which the anti-meliorists mandate we reconcile ourselves. If anything is clear about human nature, it is that this is not an accurate view of who we have been or what we are now, or a view that should determine what we become. Where is the pursuit of the perfect face, body, and mind taking us? (Illustration: Margaret Shear) December 2004 \| Volume 1 \| Issue 3 \| e52 173 PLoS Medicine \| www.plosmedicine.org Caplan’s Response to Elliott’s Viewpoint The manufacturers of estrogen replacement therapy marketed the hormone in the 1960s by funding a “research foundation” for Robert Wilson, the gynecologist and author of the best-selling book Feminine Forever [6]. Wyeth marketed Fen-Phen by funding obesity research centers, launching public ﬁ tness campaigns, contracting with a medical education company to produce a series of ghostwritten journal articles, and making generous payments to academic physicians who then published extensively and testiﬁ ed for the drug’s safety to the Food and Drug Administration [17]. Pharmaceutical companies may be evil incarnate. And we may be putty in their pecuniary little hands. But that has nothing at all to do with the question of whether there is anything wrong with pursuing enhancement. When Elliott eagerly dons his hair shirt to bemoan Big Pharma, he ﬁ nds so much sin to revel in that he forgets to give a reason, any reason, why enhancement is, in itself, immoral. At most he presents an argument for keeping the pharmaceutical industry out of enhancement. Okay, so let’s take Big Pharma out of the picture. If we left the encouragement of enhancement to the government, the military, schools, foundations, doctors, or parents, would this now be morally acceptable? I think sometimes it would be. And nothing that Elliott says provides any reason to think otherwise. The traditional worry about enhancement technologies is that users of the technologies are buying individual well-being at the expense of some larger social good. I may improve my own athletic ability by taking steroids, but I set off a steroid arms race that destroys my sport. I may get cosmetic surgery for my “Asian eyes” or use skin lighteners for my dark skin, but I reinforce the implicitly racist social norms that say that Asian eyes or dark skin are traits to be ashamed of. The worry is that some aspect of the way we live together, collectively, is going to be damaged by actions that we take individually [4]. A market-driven health-care system brings this worry much closer to home. The pharmaceutical industry is now the most proﬁ table and politically powerful industry in the United States [23]. It also has a huge ﬁ nancial interest in creating a demand for enhancement technologies. Abbreviation: Fen-Phen, fenﬂ uramine-phentermine Abbreviation: Fen-Phen, fenﬂ uramine-phentermine Abbreviation: Fen-Phen, fenﬂ uramine-phentermine p p 7. Kass LR (2002) Life, liberty, and the defense of dignity: The challenge for bioethics. San Francisco: Encounter Books. 313 p. y g bioethics. San Francisco: Encounter Books. 313 p. Arthur Caplan is chair of the Department of Medical Ethics, University of Pennsylva- nia School of Medicine, Philadelphia, Pennsylvania, United States of America. E-mail: Caplan@mail.med.upenn.edu 8. Kristol W, Cohen E, editors (2002) The future is now: America confronts the new genetics. Lanham (Maryland): Rowman and Littleﬁ eld. 357 p. g y p 9. Sandel S (2004 April) The case against perfection. Atlantic Monthly: 51–62. ( p ) g p y 10. Isaacson W (2003) Benjamin Franklin: An American life. New York: Simon and Schuster. 590 p. Carl Elliott is associate professor at the Center for Bioethics at the University of Minnesota, Minneapolis, Minnesota, United States of America, and the author of Better Than Well: American Medicine Meets the American Dream. E-mail: ellio023@tc. umn.edu 11. Whorton J (1984) Crusaders for ﬁ tness: The history of American health reformers. Princeton (New Jersey): Princeton University Press. 359 p. y y p 12. Caplan AL (2004) Is biomedical research too dangerous to pursue? Science 303: 1142. Competing Interests: AC was a member of Dupont’s biotechnology advisory panel, advising on genetically modiﬁ ed organisms. He previously served on the scientiﬁ c advisory board of Celera genomics. From 1997–1999 he served as a consultant to Pﬁ zer on the launch of sildenaﬁ l (Viagra) as part of the company’s scientiﬁ c/eth- ics advisory board. Subsequently Pﬁ zer sponsored a course on research ethics presented by the Center for Bioethics at Pﬁ zer headquarters in which he was one of the lecturers. CE declares that he has no competing interests. 13. Saint Teresa of Avila (1964) The way of perfection. Peers EA, translator. Garden City, New York: Image Books. 320 p. Available: www.ccel.org/t/ teresa/way/main.html. Accessed 16 October 2004. teresa/way/main.html. Accessed 16 October 2004. y 14. Pinker S (2002) The blank slate: The modern denial of human nature. New York: Viking. 509 p. g p 15. Tenner E (2003) Our own devices: The past and future of body technology New York: Alfred A. Knopf. 314 p. 15. Tenner E (2003) Our own devices: The past and future of body technology. New York: Alfred A. Knopf. 314 p. 16. Elliott’s Response to Caplan’s Viewpoint In this kind of political and economic climate, how likely is it that dissenting voices will have any effect before it is too late? Skepticism about enhancement technologies is not equivalent to a wish to set back medical research and December 2004 \| Volume 1 \| Issue 3 \| e52 174 17. Mundy A (2001) Dispensing with the truth: The victims, the drug companies, and the dramatic story behind the battle over Fen-Phen. New York: St. Martin’s Press. 402 p. declare some applications off-limits. This is a debate about enhancing human traits, not curing human illness. To say that our medical research agenda will be set back if we restrict enhancement technologies makes no more sense than saying that cancer surgery will be set back if the American Broadcasting Corporation cancels its cosmetic surgery reality TV show Extreme Makeover. p 18. Barrett A (2004 May 24) Wyeth: The class action that wouldn’t quit. Business Week: 88. 19. Writing Group for the Women’s Health Initiative Investigators (2002) Risks and beneﬁ ts of estrogen plus progestin in healthy postmenopausal women: Principal results from the Women’s Health Initiative randomized controlled trial. JAMA 288: 321–333. J 20. Whittington C, Kendall T, Fonagy P, Cottrell D, Cotgrove A, et al. (2004) Selective serotonin reuptake inhibitors in childhood depression: Systematic review of published versus unpublished data. Lancet 363: 1341–1344. We live in a country where 46 million uninsured people cannot get basic medical care, while the rest of us spend a billion dollars a year on baldness remedies. It is not just the inequity here that is so impressive. It is the fact that we have gotten so accustomed to the inequity that we do not see it as obscene. 21. Kassirer J (2004) On the take: How medicine’s complicity with big business can endanger your health. New York: Oxford University Press. 251 p. 22. Moynihan R, Heath I, Henry D (2002) Selling sickness: The pharmaceutical industry and disease mongering. BMJ 324: 886–891. y g g 23. Greider K (2003) The big ﬁ x: How the pharmaceutical industry rips off American consumers. New York: Public Affairs. 189 p. 24. Angell M (2004) The truth about the drug companies: How they deceive us and what to do about it. New York: Random House. 305 p. 24. Angell M (2004) The truth about the drug companies: Ho 1. President’s Council on Bioethics (2003) Beyond therapy: Biotechnology and the pursuit of happiness. New York: Dana Press. 400 p. p pp p 2. McKibben W (2003) Enough: Staying human in an engineered age. New York: Times Books. 271 p. References p 25. Elliott C (2004) Pharma buys a conscience. Am Prospect 12(17): 16–20. p pp p 2. McKibben W (2003) Enough: Staying human in an engineered age. New York: Times Books. 271 p. Citation: Caplan A, Elliott C (2004) Is it ethical to use enhancement technologies to make us better than well? PLoS Med 1(3): e52. 3. Callahan D (2003) What price better health? Hazards of the research imperative. Berkeley: University of California Press. 329 p. 4. Elliott C (2003) Better than well: American medicine meets the American dream. New York: W. W. Norton. 357 p. Copyright: © 2004 Caplan and Elliott. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits un- restricted use, distribution, and reproduction in any medium, provided the original work is properly cited. p 5. Fukuyama F (2003) Our posthuman future: Consequ p 5. Fukuyama F (2003) Our posthuman future: Consequen biotechnology revolution. New York: Picador. 272 p. y p q biotechnology revolution. New York: Picador. 272 p. gy p 6. Rothman S, Rothman D (2003) The pursuit of perfection: The promise and perils of medical enhancement. New York: Pantheon Books. 292 p. 3. Callahan D (2003) What price better health? Hazards of the research imperative. Berkeley: University of California Press. 329 p. Abbreviation: Fen-Phen, fenﬂ uramine-phentermine Moynihan R (2002) Drug ﬁ rms hype disease as sales ploy, industry chief claims. BMJ 324: 867. 16. Moynihan R (2002) Drug ﬁ rms hype disease as sales ploy, industry chief claims. BMJ 324: 867. DOI: 10.1371/journal.pmed.0010052 December 2004 \| Volume 1 \| Issue 3 \| e52 175 PLoS Medicine \| www.plosmedicine.org
https://openalex.org/W4367181923	https://nottingham-repository.worktribe.com/OutputFile/20280187	English	null	Differentiated Neurons Are More Vulnerable to Organophosphate and Carbamate Neurotoxicity than Undifferentiated Neurons Due to the Induction of Redox Stress and Accumulate Oxidatively-Damaged Proteins	Brain sciences	2,023	cc-by	16,900	Citation: Mudyanselage, A.W.; Wijamunige, B.C.; Kocon, A.; Carter, W.G. Differentiated Neurons Are More Vulnerable to Organophosphate and Carbamate Neurotoxicity than Undifferentiated Neurons Due to the Induction of Redox Stress and Accumulate Oxidatively-Damaged Proteins. Brain Sci. 2023, 13, 728. https://doi.org/10.3390/ brainsci13050728 Keywords: aldicarb; azamethiphos; chlorpyrifos; cholinergic toxicity; developmental neurotoxicity; non-cholinergic mechanisms; pesticides Academic Editor: James O’Callaghan Received: 5 April 2023 Revised: 21 April 2023 Accepted: 25 April 2023 Published: 26 April 2023 brain sciences brain sciences brain sciences Differentiated Neurons Are More Vulnerable to Organophosphate and Carbamate Neurotoxicity than Undifferentiated Neurons Due to the Induction of Redox Stress and Accumulate Oxidatively-Damaged Proteins e 1,2 , Buddhika C. Wijamunige 1,2 , Artur Kocon 1 and Wayne G. Carter 1,* Anusha W. Mudyanselage 1,2 , Buddhika C. Wijamunige 1,2 , Artur Kocon 1 and Wayne G. C 1 School of Medicine, University of Nottingham, Royal Derby Hospital Centre, Uttoxeter Road, Derby DE22 3DT, UK; wijesekara@agri.sab.ac.lk (A.W.M.); buddhikawijamunige@agri.sab.ac.lk (B.C.W.); artek.1993@googlemail.com (A.K.) 2 Faculty of Agricultural Sciences, Sabaragamuwa University of Sri Lanka, Belihuloya 70140, Sri Lanka * Correspondence: wayne.carter@nottingham.ac.uk; Tel.: +44-(0)1332-724738 1 School of Medicine, University of Nottingham, Royal Derby Hospital Centre, Uttoxeter Road, Derby DE22 3DT, UK; wijesekara@agri.sab.ac.lk (A.W.M.); buddhikawijamunige@agri.sab.ac.lk (B.C.W.); artek.1993@googlemail.com (A.K.) 2 Faculty of Agricultural Sciences, Sabaragamuwa University of Sri Lanka, Belihuloya 70140, Sri Lanka * Correspondence: wayne.carter@nottingham.ac.uk; Tel.: +44-(0)1332-724738 Abstract: Organophosphate (OP) and carbamate pesticides are toxic to pests through targeted inhibi- tion of acetylcholinesterase (AChE). However, OPs and carbamates may be harmful to non-target species including humans and could induce developmental neurotoxicity if differentiated or dif- ferentiating neurons are particularly vulnerable to neurotoxicant exposures. Hence, this study compared the neurotoxicity of OPs, chlorpyrifos-oxon (CPO), and azamethiphos (AZO) and the carbamate pesticide, aldicarb, to undifferentiated versus differentiated SH-SY5Y neuroblastoma cells. OP and carbamate concentration-response curves for cell viability were undertaken using 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays and cellular bioenergetic capacity assessed via quantitation of cellular ATP levels. Concentration-response curves for inhibition of cellular AChE activity were also generated and the production of reactive oxygen species (ROS) was monitored using a 2′,7′-dichloroﬂuorescein diacetate (DCFDA) assay. The OPs and aldicarb reduced cell viability, cellular ATP levels, and neurite out- growth in a concentration-dependent fashion, from a threshold concentration of ≥10 µM. Neurotoxic potency was in the order AZO > CPO > aldicarb for undifferentiated cells but CPO > AZO > aldicarb for differentiated cells and this toxic potency of CPO reﬂected its more extensive induction of reactive oxygen species (ROS) and generation of carbonylated proteins that were characterized by western blotting. Hence, the relative neurotoxicity of the OPs and aldicarb in part reﬂects non-cholinergic mechanisms that are likely to contribute to developmental neurotoxicity. 1. Introduction Pesticides are utilized for the control and management of pests in agricultural, indus- trial, and domestic settings. Commercial pesticide usage improves the economic viability of crops and increases food production, but pesticides could impact the health of non-target species [1]. Organophosphate (OP) pesticides are widely used insecticides due to their broad-spectrum activity and relatively low cost [2]. However, the somewhat indiscriminate use of OPs over several decades has produced conditions conducive to the development of insects with pesticide resistance, potential environmental and ecological damage, and the possibility of detrimental effects on human health from both acute and chronic pesticide exposures [1–3]. Copyright: © 2023 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/). Chlorpyrifos (CPF) has been in use since 1965 and is one of the most extensively employed OP insecticides utilized for crop protection, landscaping pest control, and in https://www.mdpi.com/journal/brainsci Brain Sci. 2023, 13, 728. https://doi.org/10.3390/brainsci13050728 Brain Sci. 2023, 13, 728 2 of 20 domestic settings, due to its potent and broad biocidal nature [4,5]. Metabolic biotransfor- mation of CPF results in desulphuration and the production of chlorpyrifos-oxon (CPO), a metabolite of CPF that is a potent acetylcholinesterase (AChE) inhibitor [2,4–7]. Inhibition of AChE and the induction of cholinergic toxicity is the recognized mode of action of CPF or CPO, although non-cholinergic mechanisms of toxicity, including induction of redox stress and the adduction of non-cholinesterase targets, have also been documented [1,8–12]. Furthermore, a link has been proposed between subclinical and chronic low-dose exposures to OPs including CPF, particularly prenatally, and subsequent neurodevelopmental and neurobehavioural deﬁcits in children and adults [13–16]. Azamethiphos is also an OP pesticide (organothiophosphate) but azamethiphos is an oxon and therefore, unlike CPF, does not require hepatic biotransformation. AZO is a widely used synthetic OP insecticide that is bioactive as an AChE inhibitor [17,18]. AZO is also used for the control of ectoparasites in aquacultures including salmon and trout [17,19]. Azamethiphos is also an OP pesticide (organothiophosphate) but azamethiphos is an oxon and therefore, unlike CPF, does not require hepatic biotransformation. AZO is a widely used synthetic OP insecticide that is bioactive as an AChE inhibitor [17,18]. AZO is also used for the control of ectoparasites in aquacultures including salmon and trout [17,19]. Carbamate insecticides are derivatives of N-methyl carbamic acid. They are widely used in agricultural production, as well as for the protection of human and animal health from insect vector-mediated diseases [20]. Global carbamate pesticide usage is second only to that of OP pesticides as insecticides of choice due to their broad-spectrum biocidal activity. Aldicarb is an oxime methylcarbamate insecticide often soil-applied and used to control insects and nematodes and employed in animal husbandry as an acaricide [21–23]. Like OP pesticides, the intended mechanism of action for carbamate insecticides, such as aldicarb, is via inhibition of AChE [20,23–25]. Carbamate insecticides are derivatives of N-methyl carbamic acid. They are widely used in agricultural production, as well as for the protection of human and animal health from insect vector-mediated diseases [20]. Global carbamate pesticide usage is second only to that of OP pesticides as insecticides of choice due to their broad-spectrum biocidal activity. Aldicarb is an oxime methylcarbamate insecticide often soil-applied and used to control insects and nematodes and employed in animal husbandry as an acaricide [21–23]. Like OP pesticides, the intended mechanism of action for carbamate insecticides, such as aldicarb, is via inhibition of AChE [20,23–25]. However, although AChE inhibition is central to the neurotoxicity of OP and car- bamate pesticides, this is unlikely to represent the sole mechanism responsible for the symptomology and disorders that arise from exposure to these chemicals and their metabo- lites. Their high reactivity accounts for the binding and/or adduction of cellular targets other than cholinesterase enzymes, including neuropathy target esterase (NTE), that can trigger organophosphate-induced delayed neurotoxicity (OPIDN) [1,6,8–11,18,26,27]. In addition, pesticides can induce cellular redox stress through the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) [12,26,28]. Pesticide exposure can also deplete the activity of the cellular antioxidant enzymes catalase, superoxide dismutase, and glutathione peroxidase, as well as the levels of glutathione, the major cellular thiol and antioxidant [12,26,28]. ROS are produced in healthy cells under normal metabolic conditions but are present at relatively low levels and scavenged by the antioxidant system; however, sufﬁcient pesticide induction of ROS can potentially overwhelm the antioxidant system, triggering cell death [26,28]. OPs and carbamates may induce neurotoxicity that results in the degeneration of neu- rons [29–31], and for some OPs, such as chlorpyrifos, neurotoxic effects may be particularly damaging if encountered prenatally [13–16]. An increased vulnerability to developmen- tal neurotoxicity could arise if differentiating and/or recently differentiated neurons are more susceptible to pesticide neurotoxicity. Hence, this study aimed to directly compare the neurotoxicity of the bioactive forms of chlorpyrifos, azamethiphos, and aldicarb to undifferentiated versus differentiated neurons and consider the mechanisms of toxicity. 2.2. Cell Culture SH-SY5Y cells were grown in a culture medium composed of 43.5% Eagle’s minimum essential medium (EMEM) (M4655, Sigma-Aldrich, Poole, UK) supplemented with 43.5% Ham’s F12 nut mix (217665-029, Gibco, Waltham, MA, USA), 10% heat-inactivated foetal bovine serum (FBS) (F9665, Sigma-Aldrich, Poole, UK), 1% non-essential Amino Acid Solution (M7145, Sigma-Aldrich, Poole, UK), 2 mM glutamine (01077 Life Technologies, Paisley, UK) and 1% penicillin-streptomycin solution containing 10,000 IU penicillium and 10 mg/mL streptomycin (Sigma-Aldrich, Poole, UK) in ﬂasks (T25, 130189, ThermoFisher Scientiﬁc, Rochester, UK) at 37 ◦C with an atmosphere of 5% CO2 and 95% humidity, and passaged as required. p g q SH-SY5Y cells were differentiated as described by Encinas et al. (2000) [34] after seeding onto either poly-D-Lysine hydrobromide (PDL) (50 µg/mL) (P6407, Sigma-Aldrich, Poole, UK) -coated cell cultureware or in 96-well microtitre plates (6005649, Perkin Elmer, Groningen, The Netherlands) with 10% FBS media and after settling, cells were grown to 60% conﬂuency. The following day, the cells were treated with differentiation media (10 µM all-trans retinoic acid (RA) (R26625, Sigma-Aldrich, Poole, UK) in low-serum medium (1% FBS) for 6 days and then treated with 20 ng/mL brain-derived neurotrophic factor (BDNF) (B3795, Sigma-Aldrich, Poole, UK) with low-serum media containing RA for a further 2 days, at which time the cells displayed a fully-differentiated morphology according to Shipley et al. (2016) [35]. All experiments were conducted using passage 13 to prevent any morphological outgrowth of the culture and the potential for genetic drift associated with multiple passaging. 2.1. Chemicals and Reagents SH-SY5Y human neuroblastoma cells were obtained from the European Collection of Authenticated Cell Culture (ECACC) (ECACC-94030304). Chlorpyrifos-oxon (CPO) di- ethyl (3,5,6-trichloropyridin-2-yl) phosphate (C9H11Cl3NO4P, MW = 344.5 g/mol, purity 97.2–99.1%) and Azamethiphos (AZO) (6-chloro-3-(dimethoxyphosphorylsulfanylmethyl)- [1,3]oxazolo[4,5-b]pyridin-2-one), (C9H10ClN2O5PS, MW = 324.7 g/mol, purity 95–99.5%) were purchased from Greyhound Chromatography, Birkenhead, UK. Aldicarb ([(E)-(2-methyl- 2-methylsulfanylpropylidene)amino] N-methylcarbamate), (C7H14N2O2S, MW = 190.27 g/mol, purity 99.5%) was from Chem Service Inc. (West Chester, PA, USA) as supplied by Grey- hound Chromatography, Birkenhead, UK. Pesticide stock solutions were prepared at 50 mM Brain Sci. 2023, 13, 728 3 of 20 in 99.5% pure ethyl alcohol (product 459844, Sigma-Aldrich, Poole, UK). 3-(4,5 dimethylthiazol- 2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) (product M5655) isopropanol, and dimethyl sulphoxide (DMSO) (product D8418) were purchased from Sigma-Aldrich, Poole, UK and used for MTT cell viability assays. Acetylthiocholine iodide (ATCI) (product A5751) and 5,5′-Dithiobis-(2-Nitrobenzoic Acid) (DTNB) (product D8130) were purchased from Sigma, Poole, UK and were used for a modiﬁed Ellman’s assay adapted for a 96-well plate [32,33]. 2′,7′-dichloroﬂuorescein diacetate (DCFDA) (D6883, Sigma-Aldrich, Poole, UK), and 30% H2O2 in H2O (H1009, Sigma-Aldrich, Poole, UK) was used as a positive control in the mea- surements of ROS. Radioimmunoprecipitation assay (RIPA, 20-188, Millipore, Burlington, MA, USA) buffer containing protease inhibitors (04693124001, Roche, Munich, Germany) and phosphatase inhibitor cocktail (P0044, Sigma-Aldrich, Poole, UK) were used for cell lysate preparation. 10 mM 2,4-dinitrophenylhydrazine (DNPH) (D199303, Sigma-Aldrich, Poole, UK) prepared in 2N HCL (231-5957, Scientiﬁc Laboratory Suppliers (SLS), Not- tingham, UK), trichloroacetic acid (TCA) (T0699, Sigma-Aldrich, Poole, UK), ethyl acetate (270989, Sigma-Aldrich, Poole, UK), and guanidine hydrochloride (50950, Sigma-Aldrich, Poole, UK) were used for the protein carbonyl content (PCC) assays. 2.3.1. MTT Assay The effect of CPO, AZO, or aldicarb (0–200 µM) on the viability of both undifferentiated and differentiated cells was determined by a Thiazolyl Blue Tetrazolium Bromide (MTT) reduction assay [36]. In brief, 3 × 104 cells/well were grown in laminin-coated 96-well clear- bottom tissue culture plates. Undifferentiated cells were challenged with each agent for 24 h within the concentration ranges speciﬁed by diluting each compound in the cell culture medium. Differentiated cells that were grown on 96-well plates were treated with each compound diluted in cell culture medium. After incubation with the compounds, spent media was removed and then replaced with the corresponding media for differentiated or undifferentiated cells containing 10% of 5 mg/mL MTT and incubated for 4 h. Wells that only received 10% MTT and respective growth media served as background controls. The formazan crystals generated were suspended in 1:1 DMSO and isopropanol solution and the absorbance of wells read at 570 nm using a spectrophotometer (Multiskan Spectrum, Brain Sci. 2023, 13, 728 4 of 20 4 of 20 Thermo Electron Corporation, Vantaa, Finland). Experiments were performed in triplicates from which an average was taken, and blank (negative control) values were subtracted. Cell viability was expressed as a percentage of survival relative to treated cells from at least ﬁve repeated experiments. The concentration of agent producing a 50% inhibition of cell viability (IC50 values) was obtained from the concentration-response curves and expressed as means ± standard error of the mean (SEM). Thermo Electron Corporation, Vantaa, Finland). Experiments were performed in triplicates from which an average was taken, and blank (negative control) values were subtracted. Cell viability was expressed as a percentage of survival relative to treated cells from at least ﬁve repeated experiments. The concentration of agent producing a 50% inhibition of cell viability (IC50 values) was obtained from the concentration-response curves and expressed as means ± standard error of the mean (SEM). 2.3.2. Lactate Dehydrogenase (LDH) Assay The production of active extracellular LDH in response to pesticide exposures was measured using an LDH assay kit (ab65393, Abcam, Cambridge, UK) according to the manufacturer’s instructions. After agent treatment (1–200 µM, or vehicle control), 50 µL of spent media was removed and LDH activity was measured spectrophotometrically at 450 nm (Multiskan Spectrum, Thermo Electron Corporation, Finland). Assays were performed in triplicates, with blank values from a negative control subtracted from test values. IC50 values were obtained from the concentration-response curves and expressed as means ± standard error of the mean (SEM). Experiments were performed with an n-number of at least ﬁve. 2.4. Measurements of Neurite Extension in Differentiated Cells SH-SY5Y cells were seeded at 5 × 105 cells/well in 12-well poly-D-Lysine coated plates and grown for 24 h to ensure cell adhesion. Cells were then treated with pesticides in differentiation media for 24 h. Cell images were captured using a phase contrast microscope (Olympus, DP70, London, UK). Cells were considered differentiated if each neuronal cell contained at least one process that was longer than its cell body. The neurite length from 100 randomly chosen cells were measured in ﬁve selected regions of each well using the neurite tracer tool in Image J (Image J 1.49k, National Institute of Health, Bethesda, MD, USA). Results are expressed as mean percentage neurite length (±SEM) relative to vehicle-control treated cells. 2.3.3. Measurement of Intracellular ATP Levels Undifferentiated and differentiated cells that were grown in 6-well plates were treated with MTT cell viability inhibition concentrations that produced 10, 20, 50, and an 80% loss of cell viability. Intracellular ATP levels were quantiﬁed using an ATP luminescence assay kit (ATP Bioluminescence Assay Kit CLS II (product 11 699 695 001, Roche, Germany), according to the manufacturer’s protocol. ATP standards were prepared according to the kit protocol across a concentration range of 1 × 10−4 to 1 × 10−10 M and after the addition of luciferase reagent to each well, the luminescence was measured using a luminometric plate reader (Thermo Fisher Scientiﬁc, Fluoroskan Ascent FC, Vantaa, Finland) using an integration time of 1 s. The ATP content in control and pesticide-treated samples were interpolated from the ATP standard curve. Experiments were performed in triplicates and ﬁve individual experiments were undertaken from which a mean was calculated, with blank values subtracted. 2.4. Measurements of Neurite Extension in Differentiated Cells 2.8. Protein Quantitation The quantitation of the protein concentration in cell homogenates was performed using a modiﬁed Lowry assay [38]. Bovine serum albumin (BSA) protein standards of 1.25, 2.5, 5, 7.5, and 10 µg (5000206, Bio-Rad, Hertfordshire, UK) were used to generate a standard curve. For a volume of 40 µL of cell lysates or protein standards, 20 µL of Reagent A (500-0113, Bio-Rad, Hertfordshire, UK) was added and then 160 µL of Reagent B (500-0114, Bio-Rad, Hertfordshire, UK) and the samples mixed. After 15 min, spectrophoto- metric measurements were taken at 740 nm using a SpectraMax plate reader (Multiskan Spectrum, Thermo Electron Corporation, Finland). Protein amounts of unknowns were interpolated from the BSA standard curve. 2.6. Measurements of Reactive Oxygen Species The generation of reactive oxygen species (ROS) were quantiﬁed using a 2′,7′-dichloro- ﬂuorescein diacetate (DCFDA) assay. Undifferentiated or differentiated cells were seeded in clear-bottom black 96-well plates. Cells were treated with agents at concentrations that reduced cell viability by 10, 20, 50, and 80% (by MTT assay) or treated with vehicle control for 6 h and 24 h. DCFDA at 50 µM was added to each well 30 min before the end of the experiment to allow for cellular incorporation. After the treatments, cells were washed twice with ice-cold PBS and then ﬂuorescence quantiﬁed with a 485 nm excitation and 535 nm emission (Thermo Fisher Scientiﬁc, Fluoroskan Ascent FC device, ThermoFisher, Finland). Undifferentiated or differentiated cells (with their respective media) were treated for 30 min with 0.5 mM H2O2 together with 50 µM DCFDA as a positive control for ROS generation, with the values generated set at 100% ﬂuorescence. Six replicate assays were performed for each data point, from which an average was calculated after subtracting blank values generated from media alone with DCFDA. 2.5. Measurements of Acetylcholinesterase (AChE) Activity Inhibition of AChE was assessed using an Ellman’s assay modiﬁed for harvested cells [32,33]. Cells that had been treated with neurotoxicant for 24 h were harvested into ice-cold potassium phosphate buffer pH 8.0 and centrifuged at 13,000× g for 5 min at 4 ◦C. The supernatant was discarded, and the resultant cell pellet was retained and resuspended in 1 mL of potassium phosphate buffer pH 8.0. A volume of 100 µL was then assayed as a 1:1 mixture with ATCI and DTNB. Absorbance of the lysate was immediately measured at 412 nm using a spectrophotometer (Multiskan Spectrum, Thermo Electron Corporation, Finland) in kinetic mode, with readings taken every minute at 37 ◦C, protected from light, for a total of 10 min. Since the absorbance background increased with time, the associated absorbance of buffer blanks was subtracted at each time point for each data point. Corrected Brain Sci. 2023, 13, 728 5 of 20 5 of 20 absorbance readings for each treatment were normalized to the means of the vehicle control and AChE activity presented as a percentage relative to the vehicle control. absorbance readings for each treatment were normalized to the means of the vehicle control and AChE activity presented as a percentage relative to the vehicle control. 2.7. Cell Lysis and Fractionation After cell treatments, cells were scraped into 0.5 mL of radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors. The cell suspension was vortexed thoroughly in the RIPA buffer and then passed through a 28 g needle 25 times to ensure homogenization. Homogenates were stored at −20 ◦C until required. Thawed homogenates were fractionated by differential centrifugation. Firstly, low-speed centrifuga- tion at 500× g for 10 min at 4 ◦C was performed to pellet the cell debris and nuclear fraction. The supernatant produced was then centrifuged at 23,100× g for 40 min at 4 ◦C to generate a crude cytosolic extract, leaving a pellet that was a membrane-enriched fraction [37]. 2.11. Statistical Analysis Results for cell viability and ATP assays are presented as means ± standard error of the mean (SEM), with statistical analysis performed using GraphPad Prism 9.2.0 (GraphPad Prism, San Diego, CA, USA). Concentration-response curves were generated to interpolate the inhibition concentrations in each experiment by using a non-linear regression curve ﬁt model. Curves were plotted using Prism as lines of best ﬁt. Comparison between control and treatment groups was performed using either one-way analysis of variance (ANOVA) or two-way ANOVA with Dennett’s multiple comparison test and Tukey’s multiple comparisons, respectively. A p value of <0.05 or lower was considered statistically signiﬁcant, with asterisks used to indicate levels of signiﬁcance: * p < 0.05; p < 0.01; * p < 0.001; ** p < 0.0001. 2.9. Quantitation of Protein Carbonyl Content Quantitation of the levels of cellular protein carbonyl content (PCC) was performed based on published methods [39,40]. Cytosolic extracts from undifferentiated or differ- entiated cells were challenged with neurotoxicants and extracts prepared as described in Section 2.7. To 500 µg of protein, an equivalent volume of 10 mM 2,4-dinitrophenylhydrazine (DNPH) (prepared in 2N HCl) was added, samples were vortexed and then left in the dark for 1 h at room temperature, with vortex mixing every 10 min. An equivalent volume of ice-cold 20% (w/v) trichloroacetic acid (TCA) was added and samples were incubated for 15 min on ice. Samples were then spun at 10,000× g for 5 min at 4 ◦C, the supernatant was discarded, and the pellets were washed with 1:1 ethanol:ethyl acetate (v/v) and vortex mixing. Samples were spun at 10,000× g for 5 min at 4 ◦C and the supernatant discarded. Washing with 20% ice-cold TCA and then 1:1 ethanol:ethyl acetate was repeated and then samples were air-dried for 5 min to allow complete evaporation of solvents. Protein pellets were resuspended in an equal volume of 6 M guanidine hydrochloride in 50 mM phosphate buffer, pH 2.3, with incubation at 37 ◦C for 30 min and vortex mixing. PCC of test samples Brain Sci. 2023, 13, 728 6 of 20 was determined via a spectrophotometric reading at 366 nm (Multiskan Spectrum, Thermo Electron Corporation, Finland) using a molar absorption coefﬁcient of 22,000 M−1 cm−1 after subtraction of blanks. Assays were performed in triplicate from which an average was calculated. A minimum n-number of ﬁve was performed for each data point, from which an average was determined. 2.10. Characterisation of Carbonylated Proteins Using Oxyblots 2.10. Characterisation of Carbonylated Proteins Using Oxyblots Carbonylated proteins produced in response to neurotoxicant treatments were char- acterized after gel electrophoresis via the use of an OxyBlot Protein Oxidation Detection Kit (S71590, Millipore, Temecula, CA, USA). Cytosolic proteins from pesticide-treated cells were prepared to a concentration of 2 mg/mL and 20 µg derivatized with 2,4- dinitrophenylhydrazine (DNPH) after denaturation with 12% sodium dodecyl sulphate (SDS), according to the manufacturer’s protocol. Samples were neutralized after 15 min and 0.05% of β-mercaptoethanol was added. Proteins were separated by gel electrophoresis using XCell SureLock Mini-Cell Electrophoresis System (E10001, ThermoFisher Scientiﬁc, Rochester, UK) and then electroblotted onto polyvinylidene diﬂuoride (PVDF) membranes (88518, ThermoFisher Scientiﬁc, Rochester, UK) as previously described [41]. PVDF mem- branes were dried overnight to ﬁx the proteins. Membranes were rewetted in 10% (v/v) acetic acid, 50% methanol (v/v) and 40% (v/v) ultrapure water for 5 min and then equili- brated with phosphate-buffered saline (PBS) containing 0.05% Tween-20 (PBS-T) washing buffer, blocked for one hour at room temperature with 1% BSA in PBS-T, and then incubated overnight (≈16 h) at 4 ◦C with a rabbit anti-DNP primary antibody at a 1:150 dilution. Target primary antibody bound to carbonylated proteins was detected using a goat anti- rabbit IgG (HRP-conjugated) secondary antibody at a 1:300 dilution. Detection of immune complexes was accomplished by application of Clarity Western ECL Substrate (BioRad, Hertfordshire, UK), with light captured using a ChemiDoc MP imager (BioRad, Hertford- shire, UK), set for auto-exposure readings to ensure linearity of signal, with representative blots included in Figures. 3. Results 3.1. Chlorpyrifos-Oxon, Azamethiphos and Aldicarb Are More Toxic to Differentiated than Undifferentiated Neurons and Reduce Neurite Outgrowth 3.1. Chlorpyrifos-Oxon, Azamethiphos and Aldicarb Are More Toxic to Differentiated than Undifferentiated Neurons and Reduce Neurite Outgrowth Undifferentiated and differentiated neuroblastoma SH-SY5Y cells were incubated with chlorpyrifos-oxon (CPO), azamethiphos (AZO), or aldicarb (Figure 1) across a broad concentration range of 1–200 µM for 24 h and cell metabolic activity and viability quantiﬁed using an MTT assay. CPO was applied directly to neuronal cells to represent the biologically active metabolite of chlorpyrifos (CPF) that acts as a potent AChE inhibitor. 7 of 20 7 of 20 7 of 20 7 of 20 Brain Sci. 2023, 13, 728 ai i , , Figure 1. Chemical structures of neurotoxicants investigated in this study. Figure 1. Chemical structures of neurotoxicants investigated in this study. Figure 1. Chemical structures of neurotoxicants investigated in this study. Figure 1. Chemical structures of neurotoxicants investigated in this study. Figure 1. Chemical structures of neurotoxicants investigated in this study. Figure 1. Chemical structures of neurotoxicants investigated in this study. Cell viability of SH-SY5Y cells was reduced after OP or aldicarb exposure in a concen- tration-dependent manner, with differentiated neurons more sensitive to exposures with reduced cell viability at lower concentrations (refer to Figure 2A,B). The concentrations that reduced cell viability by 50% (IC50 values) were calculated by non-linear regression and val- ues have been included in Table 1. The threshold for a signiﬁcant reduction of cell viability for both cell phenotypes was a neurotoxicant concentration of ≥10 µM. Cell viability of SH-SY5Y cells was reduced after OP or aldicarb exposure in a concentration-dependent manner, with differentiated neurons more sensitive to exposures with reduced cell viability at lower concentrations (refer to Figure 2A,B). The concentrations that reduced cell viability by 50% (IC50 values) were calculated by non-linear regression and values have been included in Table 1. The threshold for a signiﬁcant reduction of cell viability for both cell phenotypes was a neurotoxicant concentration of ≥10 µM. Cell viability of SH-SY5Y cells was reduced after OP or aldicarb exposure in a concen- tration-dependent manner, with differentiated neurons more sensitive to exposures with reduced cell viability at lower concentrations (refer to Figure 2A,B). The concentrations that reduced cell viability by 50% (IC50 values) were calculated by non-linear regression and val- ues have been included in Table 1. 3. Results The threshold for a signiﬁcant reduction of cell viability for both cell phenotypes was a neurotoxicant concentration of ≥10 µM. (A) (B) Figure 2. Toxicity of CPO, AZO, and aldicarb to undifferentiated and differentiated SHSY-5Y cells meas- ured using an MTT assay. Undifferentiated (A) or differentiated (B) SHSY-5Y cells were treated with CPO, AZO, or aldicarb and cell metabolic activity was quantified using an MTT assay. Absorbance read- ings were normalised to the viability of vehicle controls, providing cell viability as a percentage relative to the vehicle control. Readings were taken from five individual experiments with three replicates in each treatment concentration. Results were analysed using one-way ANOVA with Dunnett’s multiple com- parison tests and expressed as mean ± standard error of the mean (SEM). For significance, p < 0.0001. (A) (B) Figure 2. Toxicity of CPO, AZO, and aldicarb to undifferentiated and differentiated SHSY-5Y cells meas- ured using an MTT assay. Undifferentiated (A) or differentiated (B) SHSY-5Y cells were treated with CPO, AZO, or aldicarb and cell metabolic activity was quantified using an MTT assay. Absorbance read- ings were normalised to the viability of vehicle controls, providing cell viability as a percentage relative to the vehicle control. Readings were taken from five individual experiments with three replicates in each treatment concentration. Results were analysed using one-way ANOVA with Dunnett’s multiple com- parison tests and expressed as mean ± standard error of the mean (SEM). For significance, p < 0.0001. Figure 2. Toxicity of CPO, AZO, and aldicarb to undifferentiated and differentiated SHSY-5Y cells measured using an MTT assay. Undifferentiated (A) or differentiated (B) SHSY-5Y cells were treated with CPO, AZO, or aldicarb and cell metabolic activity was quantiﬁed using an MTT assay. Ab- sorbance readings were normalised to the viability of vehicle controls, providing cell viability as a percentage relative to the vehicle control. Readings were taken from ﬁve individual experiments with three replicates in each treatment concentration. Results were analysed using one-way ANOVA with Dunnett’s multiple comparison tests and expressed as mean ± standard error of the mean (SEM). For signiﬁcance, p < 0.0001. (B) (B) (A) (A) (A) (A) (B) (B) Figure 2. Toxicity of CPO, AZO, and aldicarb to undifferentiated and differentiated SHSY-5Y cells meas- ured using an MTT assay. 3. Results R2 values were calculated after graph plotting in Prism to provide a numerical quantitation of how the curve ﬁts the expected non-linear model. AChE, acetylcholinesterase; ATP, adenosine triphosphate; LDH, lactate dehydrogenase; MTT, (4,5-dime- thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; N/A, not applicable. Table 1. Toxicity of CPO, AZO, and aldicarb to undifferentiated and differentiated SH-SY5Y cells. The concentrations (µM) of the neurotoxicants that produced 50% assay inhibition (IC50) are displayed, inter- polated from concentration-response curves, and expressed as means ± 95% conﬁdence intervals from ﬁve independent experiments. R2 values were calculated after graph plotting in Prism to provide a numerical quantitation of how the curve ﬁts the expected non-linear model. AChE, acetylcholinesterase; ATP, adenosine triphosphate; LDH, lactate dehydrogenase; MTT, (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; N/A, not applicable. The concentrations (µM) of the neurotoxicants that produced 50% assay inhibition (IC50) are dis- played, interpolated from concentration-response curves, and expressed as means ±95% conﬁdence intervals from ﬁve independent experiments. R2 values were calculated after graph plotting in Prism to provide a numerical quantitation of how the curve ﬁts the expected non-linear model. AChE, acetylcholinesterase; ATP, adenosine triphosphate; LDH, lactate dehydrogenase; MTT, (4,5-dime- thylthiazol-2-yl)-2 5-diphenyltetrazolium bromide; N/A not applicable As an alternative measure of cell viability, the production of active, extracellular LDH was quantiﬁed after 24-h exposure to the neurotoxicants. Undifferentiated or differentiated cells displayed a concentration-dependent loss of cell viability (Figure 3A,B), with a sig- niﬁcant reduction of viability from agent concentrations of ≥10 µM. Similar to the MTT data, the OPs (CPO and AZO) were more toxic than aldicarb to both cell phenotypes (lower IC50 values) and the potency of toxicity decreased in the order AZO > CPO > aldicarb in undifferentiated cells but CPO > AZO > aldicarb for differentiated cells. Furthermore, all compounds were more neurotoxic to differentiated cells than undifferentiated cells, with lower IC50 values (refer to Table 1). As an alternative measure of cell viability, the production of active, extracellular LDH was quantified after 24-h exposure to the neurotoxicants. Undifferentiated or differentiated cells displayed a concentration-dependent loss of cell viability (Figure 3A,B), with a signifi- cant reduction of viability from agent concentrations of ≥10 µM. Similar to the MTT data, the OPs (CPO and AZO) were more toxic than aldicarb to both cell phenotypes (lower IC50 values) and the potency of toxicity decreased in the order AZO > CPO > aldicarb in undif- ferentiated cells but CPO > AZO > aldicarb for differentiated cells. 3. Results Furthermore, all com- pounds were more neurotoxic to diﬀerentiated cells than undiﬀerentiated cells, with lower IC50 values (refer to Table 1). (A) (B) Figure 3. Toxicity of CPO, AZO, and aldicarb to undiﬀerentiated and diﬀerentiated SHSY-5Y cells measured using a LDH assay. Undiﬀerentiated (A) or diﬀerentiated (B) SHSY-5Y cells were treated with CPO, AZO, or aldicarb and extracellular LDH production was quantiﬁed using an LDH activ- ity assay. Absorbance readings were corrected by subtracting the values of blanks, then the resultant values were normalised to the LDH production from a vehicle control, providing LDH production as a percentage value relative to the vehicle control. Readings were taken from ﬁve independent experiments with three replicates for each treatment. Results were analysed using one-way ANOVA with Dunnett’s multiple comparison tests and expressed as means ± standard error of the mean (SEM). For signiﬁcance, * p < 0.001, ** p < 0.0001. We next investigated the eﬀects of neurotoxicants on cellular bioenergetics via the Figure 3. Toxicity of CPO, AZO, and aldicarb to undifferentiated and differentiated SHSY-5Y cells measured using a LDH assay. Undifferentiated (A) or differentiated (B) SHSY-5Y cells were treated with CPO, AZO, or aldicarb and extracellular LDH production was quantiﬁed using an LDH activity assay. Absorbance readings were corrected by subtracting the values of blanks, then the resultant values were normalised to the LDH production from a vehicle control, providing LDH production as a percentage value relative to the vehicle control. Readings were taken from ﬁve independent experiments with three replicates for each treatment. Results were analysed using one-way ANOVA with Dunnett’s multiple comparison tests and expressed as means ± standard error of the mean (SEM). For signiﬁcance, * p < 0.001, ** p < 0.0001. (A) (B) (B) (A) (A) (B) Figure 3. Toxicity of CPO, AZO, and aldicarb to undiﬀerentiated and diﬀerentiated SHSY-5Y cells measured using a LDH assay. Undiﬀerentiated (A) or diﬀerentiated (B) SHSY-5Y cells were treated with CPO, AZO, or aldicarb and extracellular LDH production was quantiﬁed using an LDH activ- ity assay. Absorbance readings were corrected by subtracting the values of blanks, then the resultant values were normalised to the LDH production from a vehicle control, providing LDH production as a percentage value relative to the vehicle control. Readings were taken from ﬁve independent experiments with three replicates for each treatment. 3. Results Absorbance readings were corrected by subtracting the values of blanks, then the resultant values were normalised to the LDH production from a vehicle control, providing LDH production as a percentage value relative to the vehicle control. Readings were taken from ﬁve independent experiments with three replicates for each treatment. Results were analysed using one-way ANOVA with Dunnett’s multiple comparison tests and expressed as means ± standard error of the mean (SEM). For signiﬁcance, * p < 0.001, ** p < 0.0001. We next investigated the eﬀects of neurotoxicants on cellular bioenergetics via the quantitation of intracellular ATP levels. CPO, AZO, and aldicarb signiﬁcantly decreased ATP levels in proportion to their applied concentrations in both SHSY-5Y cell phenotypes Figure 3. Toxicity of CPO, AZO, and aldicarb to undifferentiated and differentiated SHSY-5Y cells measured using a LDH assay. Undifferentiated (A) or differentiated (B) SHSY-5Y cells were treated with CPO, AZO, or aldicarb and extracellular LDH production was quantiﬁed using an LDH activity assay. Absorbance readings were corrected by subtracting the values of blanks, then the resultant values were normalised to the LDH production from a vehicle control, providing LDH production as a percentage value relative to the vehicle control. Readings were taken from ﬁve independent experiments with three replicates for each treatment. Results were analysed using one-way ANOVA with Dunnett’s multiple comparison tests and expressed as means ± standard error of the mean (SEM). For signiﬁcance, * p < 0.001, ** p < 0.0001. We next investigated the effects of neurotoxicants on cellular bioenergetics via the i i f i ll l ATP l l CPO AZO d ldi b i iﬁ l d d Table 1. Toxicity of CPO, AZO, and aldicarb to undifferentiated and differentiated SH-SY5Y cells. 3. Results Cell type Agent MTT LDH ATP Neurite Retraction AChE Inhibition IC50 R2 IC50 R2 IC50 R2 IC50 R2 IC50 R2 Undifferentiated CPO 29.4 ± 2.1 0.970 27.8 ± 2.1 0.971 29.6 ± 2.1 0.956 N/A - 0.28 ± 0.0 0.996 Differentiated 17.3 ± 0.9 0.987 17.2 ± 1.1 0.967 18.8 ± 0.1 0.999 10.8 ± 0.1 0.914 0.19 ± 0.0 0.987 Undifferentiated AZO 26.9 ± 1.1 0.987 26.5 ± 0.9 0.992 26.0 ± 0.6 0.995 N/A - 0.30 ± 0.0 0.992 Differentiated 19.6 ± 0.5 0.996 20.5 ± 1.6 0.954 20.7 ± 0.1 0.999 16.5 ± 1.3 0.894 0.22 ± 0.0 0.986 Undifferentiated Aldicarb 39.6 ± 3.6 0.951 35.4 ± 1.4 0.990 40.2 ± 2.9 0.945 N/A - 0.62 ± 0.0 0.967 Differentiated 31.6 ± 2.2 0.973 29.9 ± 1.6 0.981 32.4 ± 1.2 0.987 30.2 ± 4.7 0.703 0.38 ± 0.0 0.970 The concentrations (µM) of the neurotoxicants that produced 50% assay inhibition (IC50) are displayed, inter- polated from concentration-response curves, and expressed as means ± 95% conﬁdence intervals from ﬁve independent experiments. R2 values were calculated after graph plotting in Prism to provide a numerical quantitation of how the curve ﬁts the expected non-linear model. AChE, acetylcholinesterase; ATP, adenosine triphosphate; LDH, lactate dehydrogenase; MTT, (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; N/A, not applicable. Table 1. Toxicity of CPO, AZO, and aldicarb to undiﬀerentiated and diﬀerentiated SH-SY5Y cells. Cell type Agent MTT LDH ATP Neurite Retraction AChE Inhibition IC50 R2 IC50 R2 IC50 R2 IC50 R2 IC50 R2 Undifferentiated CPO 29.4 ± 2.1 0.970 27.8 ± 2.1 0.971 29.6 ± 2.1 0.956 N/A - 0.28 ± 0.0 0.996 Differentiated 17.3 ± 0.9 0.987 17.2 ± 1.1 0.967 18.8 ± 0.1 0.999 10.8 ± 0.1 0.914 0.19 ± 0.0 0.987 Undifferentiated AZO 26.9 ± 1.1 0.987 26.5 ± 0.9 0.992 26.0 ± 0.6 0.995 N/A - 0.30 ± 0.0 0.992 Differentiated 19.6 ± 0.5 0.996 20.5 ± 1.6 0.954 20.7 ± 0.1 0.999 16.5 ± 1.3 0.894 0.22 ± 0.0 0.986 Undifferentiated Aldicarb 39.6 ± 3.6 0.951 35.4 ± 1.4 0.990 40.2 ± 2.9 0.945 N/A - 0.62 ± 0.0 0.967 Differentiated 31.6 ± 2.2 0.973 29.9 ± 1.6 0.981 32.4 ± 1.2 0.987 30.2 ± 4.7 0.703 0.38 ± 0.0 0.970 The concentrations (µM) of the neurotoxicants that produced 50% assay inhibition (IC50) are dis- played, interpolated from concentration-response curves, and expressed as means ±95% conﬁdence intervals from ﬁve independent experiments. 3. Results Undifferentiated (A) or differentiated (B) SHSY-5Y cells were treated with CPO, AZO, or aldicarb and cell metabolic activity was quantified using an MTT assay. Absorbance read- ings were normalised to the viability of vehicle controls, providing cell viability as a percentage relative to the vehicle control. Readings were taken from five individual experiments with three replicates in each treatment concentration. Results were analysed using one-way ANOVA with Dunnett’s multiple com- parison tests and expressed as mean ± standard error of the mean (SEM). For significance, p < 0.0001. Figure 2. Toxicity of CPO, AZO, and aldicarb to undifferentiated and differentiated SHSY-5Y cells meas- ured using an MTT assay. Undifferentiated (A) or differentiated (B) SHSY-5Y cells were treated with CPO, AZO, or aldicarb and cell metabolic activity was quantified using an MTT assay. Absorbance read- ings were normalised to the viability of vehicle controls, providing cell viability as a percentage relative to the vehicle control. Readings were taken from five individual experiments with three replicates in each treatment concentration. Results were analysed using one-way ANOVA with Dunnett’s multiple com- parison tests and expressed as mean ± standard error of the mean (SEM). For significance, p < 0.0001. Figure 2. Toxicity of CPO, AZO, and aldicarb to undifferentiated and differentiated SHSY-5Y cells measured using an MTT assay. Undifferentiated (A) or differentiated (B) SHSY-5Y cells were treated with CPO, AZO, or aldicarb and cell metabolic activity was quantiﬁed using an MTT assay. Ab- sorbance readings were normalised to the viability of vehicle controls, providing cell viability as a percentage relative to the vehicle control. Readings were taken from ﬁve individual experiments with three replicates in each treatment concentration. Results were analysed using one-way ANOVA with Dunnett’s multiple comparison tests and expressed as mean ± standard error of the mean (SEM). For signiﬁcance, p < 0.0001. Brain Sci. 2023, 13, 728 8 of 20 Table 1. Toxicity of CPO, AZO, and aldicarb to undifferentiated and differentiated SH-SY5Y cells. 3. Results Cell type Agent MTT LDH ATP Neurite Retraction AChE Inhibition IC50 R2 IC50 R2 IC50 R2 IC50 R2 IC50 R2 Undifferentiated CPO 29.4 ± 2.1 0.970 27.8 ± 2.1 0.971 29.6 ± 2.1 0.956 N/A - 0.28 ± 0.0 0.996 Differentiated 17.3 ± 0.9 0.987 17.2 ± 1.1 0.967 18.8 ± 0.1 0.999 10.8 ± 0.1 0.914 0.19 ± 0.0 0.987 Undifferentiated AZO 26.9 ± 1.1 0.987 26.5 ± 0.9 0.992 26.0 ± 0.6 0.995 N/A - 0.30 ± 0.0 0.992 Differentiated 19.6 ± 0.5 0.996 20.5 ± 1.6 0.954 20.7 ± 0.1 0.999 16.5 ± 1.3 0.894 0.22 ± 0.0 0.986 Undifferentiated Aldicarb 39.6 ± 3.6 0.951 35.4 ± 1.4 0.990 40.2 ± 2.9 0.945 N/A - 0.62 ± 0.0 0.967 Differentiated 31.6 ± 2.2 0.973 29.9 ± 1.6 0.981 32.4 ± 1.2 0.987 30.2 ± 4.7 0.703 0.38 ± 0.0 0.970 The concentrations (µM) of the neurotoxicants that produced 50% assay inhibition (IC50) are displayed, inter- polated from concentration-response curves, and expressed as means ± 95% conﬁdence intervals from ﬁve independent experiments. R2 values were calculated after graph plotting in Prism to provide a numerical quantitation of how the curve ﬁts the expected non-linear model. AChE, acetylcholinesterase; ATP, adenosine triphosphate; LDH, lactate dehydrogenase; MTT, (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; N/A, not applicable. As an alternative measure of cell viability, the production of active, extracellular LDH was quantiﬁed after 24-h exposure to the neurotoxicants. Undifferentiated or differentiated cells displayed a concentration-dependent loss of cell viability (Figure 3A,B), with a sig- niﬁcant reduction of viability from agent concentrations of ≥10 µM. Similar to the MTT data, the OPs (CPO and AZO) were more toxic than aldicarb to both cell phenotypes (lower IC50 values) and the potency of toxicity decreased in the order AZO > CPO > aldicarb in undifferentiated cells but CPO > AZO > aldicarb for differentiated cells. Furthermore, all compounds were more neurotoxic to differentiated cells than undifferentiated cells, with lower IC50 values (refer to Table 1). Brain Sci. 2023, 13, x 8 of 20 Table 1. Toxicity of CPO, AZO, and aldicarb to undiﬀerentiated and diﬀerentiated SH-SY5Y cells. 3. Results Cell type Agent MTT LDH ATP Neurite Retraction AChE Inhibition IC50 R2 IC50 R2 IC50 R2 IC50 R2 IC50 R2 Undifferentiated CPO 29.4 ± 2.1 0.970 27.8 ± 2.1 0.971 29.6 ± 2.1 0.956 N/A - 0.28 ± 0.0 0.996 Differentiated 17.3 ± 0.9 0.987 17.2 ± 1.1 0.967 18.8 ± 0.1 0.999 10.8 ± 0.1 0.914 0.19 ± 0.0 0.987 Undifferentiated AZO 26.9 ± 1.1 0.987 26.5 ± 0.9 0.992 26.0 ± 0.6 0.995 N/A - 0.30 ± 0.0 0.992 Differentiated 19.6 ± 0.5 0.996 20.5 ± 1.6 0.954 20.7 ± 0.1 0.999 16.5 ± 1.3 0.894 0.22 ± 0.0 0.986 Undifferentiated Aldicarb 39.6 ± 3.6 0.951 35.4 ± 1.4 0.990 40.2 ± 2.9 0.945 N/A - 0.62 ± 0.0 0.967 Differentiated 31.6 ± 2.2 0.973 29.9 ± 1.6 0.981 32.4 ± 1.2 0.987 30.2 ± 4.7 0.703 0.38 ± 0.0 0.970 The concentrations (µM) of the neurotoxicants that produced 50% assay inhibition (IC50) are dis- played, interpolated from concentration-response curves, and expressed as means ±95% conﬁdence intervals from ﬁve independent experiments. R2 values were calculated after graph plotting in Prism to provide a numerical quantitation of how the curve ﬁts the expected non-linear model. AChE, acetylcholinesterase; ATP, adenosine triphosphate; LDH, lactate dehydrogenase; MTT, (4,5-dime- thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; N/A, not applicable. As an alternative measure of cell viability, the production of active, extracellular LDH was quantified after 24-h exposure to the neurotoxicants. Undifferentiated or differentiated cells displayed a concentration-dependent loss of cell viability (Figure 3A,B), with a signifi- cant reduction of viability from agent concentrations of ≥10 µM. Similar to the MTT data, the OPs (CPO and AZO) were more toxic than aldicarb to both cell phenotypes (lower IC50 values) and the potency of toxicity decreased in the order AZO > CPO > aldicarb in undif- ferentiated cells but CPO > AZO > aldicarb for differentiated cells. Furthermore, all com- pounds were more neurotoxic to diﬀerentiated cells than undiﬀerentiated cells, with lower IC50 values (refer to Table 1). (A) (B) Figure 3. Toxicity of CPO, AZO, and aldicarb to undiﬀerentiated and diﬀerentiated SHSY-5Y cells measured using a LDH assay. Undiﬀerentiated (A) or diﬀerentiated (B) SHSY-5Y cells were treated with CPO, AZO, or aldicarb and extracellular LDH production was quantiﬁed using an LDH activ- ity assay. 3. Results Results were analysed using one-way ANOVA with Dunnett’s multiple comparison tests and expressed as means ± standard error of the mean (SEM). For signiﬁcance, * p < 0.001, ** p < 0.0001. We next investigated the eﬀects of neurotoxicants on cellular bioenergetics via the Figure 3. Toxicity of CPO, AZO, and aldicarb to undifferentiated and differentiated SHSY-5Y cells measured using a LDH assay. Undifferentiated (A) or differentiated (B) SHSY-5Y cells were treated with CPO, AZO, or aldicarb and extracellular LDH production was quantiﬁed using an LDH activity assay. Absorbance readings were corrected by subtracting the values of blanks, then the resultant values were normalised to the LDH production from a vehicle control, providing LDH production as a percentage value relative to the vehicle control. Readings were taken from ﬁve independent experiments with three replicates for each treatment. Results were analysed using one-way ANOVA with Dunnett’s multiple comparison tests and expressed as means ± standard error of the mean (SEM). For signiﬁcance, * p < 0.001, ** p < 0.0001. quantitation of intracellular ATP levels. CPO, AZO, and aldicarb signiﬁcantly decreased ATP levels in proportion to their applied concentrations in both SHSY-5Y cell phenotypes We next investigated the effects of neurotoxicants on cellular bioenergetics via the quantitation of intracellular ATP levels. CPO, AZO, and aldicarb signiﬁcantly decreased ATP levels in proportion to their applied concentrations in both SHSY-5Y cell phenotypes Brain Sci. 2023, 13, 728 Brain Sci. 2023, 13, 9 of 20 0 (Figure 4A,B). The induced reductions of ATP levels generated IC50 values similar to those from MTT and LDH assays (refer to Table 1). (Figure 4A,B). The induced reductions of ATP levels generated IC50 values similar to those from MTT and LDH assays (refer to Table 1). 9 of 20 (Figure 4A,B). The induced reductions of ATP levels generated IC50 values similar to those from MTT and LDH assays (refer to Table 1). (Figure 4A,B). The induced reductions of ATP levels generated IC50 values similar to those from MTT and LDH assays (refer to Table 1). 9 of 20 (A) (B) Figure 4. Toxicity of CPO, AZO, and aldicarb to undifferentiated and differentiated SHSY-5Y cells meas- ured using an ATP assay. Undifferentiated (A) or differentiated (B) SHSY-5Y cells were treated with CPO, AZO, or aldicarb and intracellular ATP levels were quantified using an ATP assay. 3. Results Inhibition concentra- tions were interpolated from MTT assays with IC10, IC20, IC50, and IC80 concentrations selected for ATP assays. Absorbance readings were corrected by subtracting blank values with the resultant values nor- malised to the ATP level of vehicle controls, providing ATP levels as a percentage value relative to the vehicle control. Readings were taken from five individual experiments with three replicates quantified for each treatment. Results were analysed using one-way ANOVA with Dunnett’s multiple comparison tests and expressed as means ± standard error of the mean (SEM). For significance, p < 0. 0001. The effect of the neurotoxicants on cell number and cell morphology was further eval- uated by bright-field, phase-contrast microscopy. Cell numbers declined in proportion to increasing agent concentration and neurotoxicants also reduced the levels of neurite out- Figure 4. Toxicity of CPO, AZO, and aldicarb to undifferentiated and differentiated SHSY-5Y cells measured using an ATP assay. Undifferentiated (A) or differentiated (B) SHSY-5Y cells were treated with CPO, AZO, or aldicarb and intracellular ATP levels were quantiﬁed using an ATP assay. Inhibi- tion concentrations were interpolated from MTT assays with IC10, IC20, IC50, and IC80 concentrations selected for ATP assays. Absorbance readings were corrected by subtracting blank values with the resultant values normalised to the ATP level of vehicle controls, providing ATP levels as a percentage value relative to the vehicle control. Readings were taken from ﬁve individual experiments with three replicates quantiﬁed for each treatment. Results were analysed using one-way ANOVA with Dunnett’s multiple comparison tests and expressed as means ± standard error of the mean (SEM). For signiﬁcance, p < 0. 0001. (Figure 4A,B). The induced reductions of ATP levels generated IC50 values similar to those from MTT and LDH assays (refer to Table 1). (A) (B) Figure 4. Toxicity of CPO, AZO, and aldicarb to undifferentiated and differentiated SHSY-5Y cells meas- ured using an ATP assay. Undifferentiated (A) or differentiated (B) SHSY-5Y cells were treated with CPO, AZO, or aldicarb and intracellular ATP levels were quantified using an ATP assay. Inhibition concentra- tions were interpolated from MTT assays with IC10, IC20, IC50, and IC80 concentrations selected for ATP assays. Absorbance readings were corrected by subtracting blank values with the resultant values nor- malised to the ATP level of vehicle controls, providing ATP levels as a percentage value relative to the vehicle control. 3. Results Readings were taken from five individual experiments with three replicates quantified for each treatment. Results were analysed using one-way ANOVA with Dunnett’s multiple comparison tests and expressed as means ± standard error of the mean (SEM) For significance p < 0 0001 (A) (Figure 4A,B). The induced redu from MTT and LDH assays (refe (B) ions of ATP levels generated IC50 values similar to those to Table 1). (A) (B) Figure 4. Toxicity of CPO, AZO, and aldicarb to undifferentiated and differentiated SHSY-5Y cells meas- ured using an ATP assay. Undifferentiated (A) or differentiated (B) SHSY-5Y cells were treated with CPO, AZO, or aldicarb and intracellular ATP levels were quantified using an ATP assay. Inhibition concentra- tions were interpolated from MTT assays with IC10, IC20, IC50, and IC80 concentrations selected for ATP assays. Absorbance readings were corrected by subtracting blank values with the resultant values nor- malised to the ATP level of vehicle controls, providing ATP levels as a percentage value relative to the vehicle control. Readings were taken from five individual experiments with three replicates quantified for each treatment. Results were analysed using one-way ANOVA with Dunnett’s multiple comparison tests and expressed as means ± standard error of the mean (SEM). For significance, p < 0. 0001. The effect of the neurotoxicants on cell number and cell morphology was further eval- uated by bright-field, phase-contrast microscopy. Cell numbers declined in proportion to increasing agent concentration and neurotoxicants also reduced the levels of neurite out- Figure 4. Toxicity of CPO, AZO, and aldicarb to undifferentiated and differentiated SHSY-5Y cells measured using an ATP assay. Undifferentiated (A) or differentiated (B) SHSY-5Y cells were treated with CPO, AZO, or aldicarb and intracellular ATP levels were quantiﬁed using an ATP assay. Inhibi- tion concentrations were interpolated from MTT assays with IC10, IC20, IC50, and IC80 concentrations selected for ATP assays. Absorbance readings were corrected by subtracting blank values with the resultant values normalised to the ATP level of vehicle controls, providing ATP levels as a percentage value relative to the vehicle control. Readings were taken from ﬁve individual experiments with three replicates quantiﬁed for each treatment. Results were analysed using one-way ANOVA with Dunnett’s multiple comparison tests and expressed as means ± standard error of the mean (SEM). For signiﬁcance, p < 0. 0001. A) (B) Figure 4. 3. Results Toxicity of CPO, AZO, and aldicarb to undifferentiated and differentiated SHSY-5Y cells meas- ured using an ATP assay. Undifferentiated (A) or differentiated (B) SHSY-5Y cells were treated with CPO, AZO, or aldicarb and intracellular ATP levels were quantified using an ATP assay. Inhibition concentra- tions were interpolated from MTT assays with IC10, IC20, IC50, and IC80 concentrations selected for ATP assays. Absorbance readings were corrected by subtracting blank values with the resultant values nor- malised to the ATP level of vehicle controls, providing ATP levels as a percentage value relative to the vehicle control. Readings were taken from five individual experiments with three replicates quantified for each treatment. Results were analysed using one-way ANOVA with Dunnett’s multiple comparison tests and expressed as means ± standard error of the mean (SEM) For significance, p < 0 0001 growth in pre-differentiated cells in a concentration-dependent manner (Figure 5A–D). At the higher agent concentrations, cells adopted a rounded morphology, particularly after CPO or AZO treatments, but some cells survived and were resistant to cell death even at the highest pesticide concentrations examined (200 µM). CPO was the most potent inhibitor of neurite retraction followed by AZO and then aldicarb (refer to Table 1). The effect of the neurotoxicants on cell number and cell morphology was further evaluated by bright-ﬁeld, phase-contrast microscopy. Cell numbers declined in proportion to increasing agent concentration and neurotoxicants also reduced the levels of neurite outgrowth in pre-differentiated cells in a concentration-dependent manner (Figure 5A–D). At the higher agent concentrations, cells adopted a rounded morphology, particularly after CPO or AZO treatments, but some cells survived and were resistant to cell death even at the highest pesticide concentrations examined (200 µM). CPO was the most potent inhibitor of neurite retraction followed by AZO and then aldicarb (refer to Table 1). The effect of the neurotoxicants on cell number and cell morphology was further eval- uated by bright-field, phase-contrast microscopy. Cell numbers declined in proportion to increasing agent concentration and neurotoxicants also reduced the levels of neurite out- growth in pre-differentiated cells in a concentration-dependent manner (Figure 5A–D). At the higher agent concentrations, cells adopted a rounded morphology, particularly after CPO or AZO treatments, but some cells survived and were resistant to cell death even at the highest pesticide concentrations examined (200 µM). 3. Results Assay absorbance readings were normalised to vehicle control after subtracting blank values and expressed as a percentage of vehicle controls. Readings were obtained from ﬁve individual experiments with three replicates measured for every data point. Results were analysed using one-way ANOVA with Dunnett’s multiple comparison tests and expressed as means ± standard error of the mean (SEM). For signiﬁcance, p < 0.0001. 3.2. Chlorpyrifos-Oxon, Azamethiphos and Aldicarb Induced Production of Reactive Oxygen Species and Oxidatively-Damaged Proteins (B) (A) (B) (A) (B) Figure 6. Toxicity of CPO, AZO, and aldicarb to undifferentiated and differentiated SHSY-5Y cells measured via inhibition of AChE. Undifferentiated (A) or differentiated (B) SHSY-5Y cells were treated with CPO, AZO, or aldicarb over the concentration range of 0–3 µM for 24 h and the inhibition of AChE quantified using a modified Ellman’s assay. Assay absorbance readings were normalised to ve- hicle control after subtracting blank values and expressed as a percentage of vehicle controls. Readings were obtained from five individual experiments with three replicates measured for every data point Results were analysed using one-way ANOVA with Dunnett’s multiple comparison tests and ex- pressed as means ± standard error of the mean (SEM). For significance, p < 0.0001. 3.2. Chlorpyrifos-Oxon, Azamethiphos and Aldicarb Induced Production of Reactive Oxygen Species and Oxidatively-Damaged Proteins Figure 6. Toxicity of CPO, AZO, and aldicarb to undifferentiated and differentiated SHSY-5Y cells measured via inhibition of AChE. Undifferentiated (A) or differentiated (B) SHSY-5Y cells were treated with CPO, AZO, or aldicarb over the concentration range of 0–3 µM for 24 h and the inhibition of AChE quantiﬁed using a modiﬁed Ellman’s assay. Assay absorbance readings were normalised to vehicle control after subtracting blank values and expressed as a percentage of vehicle controls. Readings were obtained from ﬁve individual experiments with three replicates measured for every data point. Results were analysed using one-way ANOVA with Dunnett’s multiple comparison tests and expressed as means ± standard error of the mean (SEM). For signiﬁcance, p < 0.0001. 3.2. Chlorpyrifos-Oxon, Azamethiphos and Aldicarb Induced Production of Reactive Oxygen S i d O id ti l D d P t i 3.2. Chlorpyrifos-Oxon, Azamethiphos and Aldicarb Induced Production of Reactive Oxygen Species and Oxidatively-Damaged Proteins Th i d i f i i (ROS ) i f SHSY 3.2. 3. Results CPO was the most potent inhibitor of neurite retraction followed by AZO and then aldicarb (refer to Table 1). (A) (A) Figure 5. Cont. (A) (A) Figure 5. Cont. Brain Sci. 2023, 13, 728 Brain Sci. 2023, 13, x 10 of 20 10 of 20 (B) (C) (D) Figure 5. Toxicity of CPO, AZO, and aldicarb to differentiated SHSY-5Y cells assessed using microscopy. SH-SY5Y cells were induced to differentiate for 24 h and then treated with CPO (A), AZO (B), or aldicarb Figure 5. Toxicity of CPO, AZO, and aldicarb to differentiated SHSY-5Y cells assessed using croscopy. SH-SY5Y cells were induced to differentiate for 24 h and then treated with CPO (A), A (B), or aldicarb (C) over a concentration range of 0–200 µM and morphological changes were capt using phase-contrast microscopy. Neurite lengths were quantiﬁed using the neurite tracer to ImageJ (National Institute of Health, USA) from ﬁve independent experiments with three replic (D). Results were analysed using one-way ANOVA with Dunnett’s multiple comparison tests expressed as means ± standard error of the mean (SEM). For signiﬁcance, p < 0. 0001. (B) (C) (B) (B) (B) (C) (C) (C) (D) (D) Figure 5. Toxicity of CPO, AZO, and aldicarb to differentiated SHSY-5Y cells assessed using microscopy. SH-SY5Y cells were induced to differentiate for 24 h and then treated with CPO (A), AZO (B), or aldicarb Figure 5. Toxicity of CPO, AZO, and aldicarb to differentiated SHSY-5Y cells assessed using mi- croscopy. SH-SY5Y cells were induced to differentiate for 24 h and then treated with CPO (A), AZO (B), or aldicarb (C) over a concentration range of 0–200 µM and morphological changes were captured using phase-contrast microscopy. Neurite lengths were quantiﬁed using the neurite tracer tool in ImageJ (National Institute of Health, USA) from ﬁve independent experiments with three replicates (D). Results were analysed using one-way ANOVA with Dunnett’s multiple comparison tests and expressed as means ± standard error of the mean (SEM). For signiﬁcance, p < 0. 0001. Brain Sci. 2023, 13, 728 11 of 20 as means 11 of 20 as means Cholinergic toxicity to SHSY-5Y cells was considered through analysis of the inhibi- tion of endogenous AChE. 3. Results Undifferentiated and differentiated cells were incubated with the neurotoxicants for 24 h over a concentration range of 0–3 µM (range determined by preliminary experiments; results not included) and the inhibition of AChE quantiﬁed using a modiﬁed Ellman’s assay [32,33] (Figure 6A,B). The concentration of agent that produced 50% inhibition of AChE (IC50) was calculated by non-linear regression and values have been included in Table 1. The neurotoxicants were more potent inhibitors of AChE in differentiated than undifferentiated cells, with lower IC50 values. Cholinergic toxicity to SHSY-5Y cells was considered through analysis of the inhibition of endogenous AChE. Undifferentiated and differentiated cells were incubated with the neurotoxicants for 24 h over a concentration range of 0–3 µM (range determined by prelim- inary experiments; results not included) and the inhibition of AChE quantified using a mod- ified Ellman’s assay [32,33] (Figure 6A,B). The concentration of agent that produced 50% inhibition of AChE (IC50) was calculated by non-linear regression and values have been in- cluded in Table 1. The neurotoxicants were more potent inhibitors of AChE in differentiated than undiﬀerentiated cells, with lower IC50 values. (A) (B) Figure 6. Toxicity of CPO, AZO, and aldicarb to undifferentiated and differentiated SHSY-5Y cells measured via inhibition of AChE. Undifferentiated (A) or differentiated (B) SHSY-5Y cells were treated with CPO, AZO, or aldicarb over the concentration range of 0–3 µM for 24 h and the inhibition of AChE quantified using a modified Ellman’s assay. Assay absorbance readings were normalised to ve- hicle control after subtracting blank values and expressed as a percentage of vehicle controls. Readings were obtained from five individual experiments with three replicates measured for every data point. Results were analysed using one-way ANOVA with Dunnett’s multiple comparison tests and ex- pressed as means ± standard error of the mean (SEM). For significance, p < 0.0001. 3.2. Chlorpyrifos-Oxon, Azamethiphos and Aldicarb Induced Production of Reactive Oxygen Species and Oxidatively-Damaged Proteins The i du tio of ea ti e o y e pe ie (ROS ) i e po e to t eat e t of SHSY 5Y Figure 6. Toxicity of CPO, AZO, and aldicarb to undifferentiated and differentiated SHSY-5Y cells measured via inhibition of AChE. Undifferentiated (A) or differentiated (B) SHSY-5Y cells were treated with CPO, AZO, or aldicarb over the concentration range of 0–3 µM for 24 h and the inhibition of AChE quantiﬁed using a modiﬁed Ellman’s assay. 3. Results Chlorpyrifos-Oxon, Azamethiphos and Aldicarb Induced Production of Reactive Oxygen Species and Oxidatively-Damaged Proteins The induction of reactive oxygen species (ROSs) in response to treatment of SHSY-5Y cells with CPO, AZO, or aldicarb after 6 and 24 h was tracked using a DCFDA assay. ROS levels were quantified relative to the levels produced by 500 µM H2O2 as a positive control cellular redox stressor. ROS levels within undifferentiated or differentiated cells increased sig- nificantly after 6 h, in accordance with agent concentration, and plateaued at 50–80 µM (Figure 7A,B). Similarly, a 24-h exposure to the neurotoxicants induced ROS levels that increased in proportion to their concentrations and plateaued at 50–80 µM (Figure 7C,D). Collectively, ROS levels were significantly higher in differentiated cells than in undifferentiated cells at high pes- ticide concentrations and were associated with reduced cell viability (refer to Supplementary Data). ROS levels were higher at 6 than 24 h and, for the higher neurotoxicant concentrations, were produced in the order CPO > AZO > Aldicarb (Figure 7A–D). The induction of reactive oxygen species (ROS) in response to treatment of SHSY- 5Y cells with CPO, AZO, or aldicarb after 6 and 24 h was tracked using a DCFDA as- say. ROS levels were quantiﬁed relative to the levels produced by 500 µM H2O2 as a positive control cellular redox stressor. ROS levels within undifferentiated or differenti- ated cells increased signiﬁcantly after 6 h, in accordance with agent concentration, and plateaued at 50–80 µM (Figure 7A,B). Similarly, a 24-h exposure to the neurotoxicants induced ROS levels that increased in proportion to their concentrations and plateaued at 50–80 µM (Figure 7C,D). Collectively, ROS levels were signiﬁcantly higher in differentiated cells than in undifferentiated cells at high pesticide concentrations and were associated with reduced cell viability (refer to Supplementary Data). ROS levels were higher at 6 than 24 h and, for the higher neurotoxicant concentrations, were produced in the order CPO > AZO > Aldicarb (Figure 7A–D). 12 of 20 12 of 20 Brain Sci. 2023, 13, 728 Brain Sci. 2023, 13, x (A) (B) (C) Figure 7. Cont. (A) (B) (A) (A) (B) ( ) ( ) (B) ( ) (C) (C) (C) Figure 7. Cont. Figure 7. Cont. Brain Sci. 2023, 13, 728 ain Sci. 2023, 13, x 13 of 20 13 of 20 (D) Figure 7. 3. Results Undifferentiated (A,C) or differentiated (B,D) SHSY-5Y with CPO, AZO, or aldicarb (MTT IC10, IC20, IC50, and IC80 concentrations) and ROS tiﬁed using a DCFDA assay after 6 or 24 h. Cellular ROS levels were normalised treatments and presented as a percentage of vehicle control. Results were obtaine vidual experiments with three replicates measured for every data point. Results w ing one-way ANOVA with Dunnett’s multiple comparison tests and expressed as error of the mean (SEM) For significance p < 0 0001 Figure 7. Toxicity of CPO, AZO, and aldicarb to undifferentiated and differentiated SHSY-5Y cells measured using a DCFDA assay. Undifferentiated (A,C) or differentiated (B,D) SHSY-5Y cells were treated with CPO, AZO, or aldicarb (MTT IC10, IC20, IC50, and IC80 concentrations) and ROS levels were quantiﬁed using a DCFDA assay after 6 or 24 h. Cellular ROS levels were normalised to vehicle control treatments and presented as a percentage of vehicle control. Results were obtained from ﬁve individual experiments with three replicates measured for every data point. Results were analysed using one-way ANOVA with Dunnett’s multiple comparison tests and expressed as means ± standard error of the mean (SEM). For signiﬁcance, p < 0.0001. with CPO, AZO, or aldicarb (MTT IC10, IC20, IC50, and IC80 concentrations) and ROS leve ﬁed using a DCFDA assay after 6 or 24 h. Cellular ROS levels were normalised to ve reatments and presented as a percentage of vehicle control. Results were obtained fr idual experiments with three replicates measured for every data point. Results were ng one-way ANOVA with Dunnett’s multiple comparison tests and expressed as mean rror of the mean (SEM). For significance, p < 0.0001. The levels of protein carbonyl content (PCC) (oxidatively-damaged pro The levels of protein carbonyl content (PCC) (oxidatively-damaged sponse to a 24-h exposure to CPO, AZO, or aldicarb were quantified. All thr icantly increased PCC in a concentration-dependent manner in both undif differentiated cells (Figure 8A,B). PCC levels mirrored those of ROSs such h d C O A O ld b ( A B) h l l ﬁ The levels of protein carbonyl content (PCC) (oxidatively-damaged proteins) in re- sponse to a 24-h exposure to CPO, AZO, or aldicarb were quantiﬁed. All three agents signiﬁcantly increased PCC in a concentration-dependent manner in both undifferentiated and differentiated cells (Figure 8A,B). 3. Results Toxicity of CPO, AZO, and aldicarb to undifferentiated and differentiated SH ured using a DCFDA assay. Undifferentiated (A,C) or differentiated (B,D) SHSY-5Y with CPO, AZO, or aldicarb (MTT IC10, IC20, IC50, and IC80 concentrations) and ROS tiﬁed using a DCFDA assay after 6 or 24 h. Cellular ROS levels were normalised treatments and presented as a percentage of vehicle control. Results were obtaine vidual experiments with three replicates measured for every data point. Results w ing one-way ANOVA with Dunnett’s multiple comparison tests and expressed as m error of the mean (SEM). For significance, p < 0.0001. Figure 7. Toxicity of CPO, AZO, and aldicarb to undifferentiated and differentiated SHSY-5Y cells measured using a DCFDA assay. Undifferentiated (A,C) or differentiated (B,D) SHSY-5Y cells were treated with CPO, AZO, or aldicarb (MTT IC10, IC20, IC50, and IC80 concentrations) and ROS levels were quantiﬁed using a DCFDA assay after 6 or 24 h. Cellular ROS levels were normalised to vehicle control treatments and presented as a percentage of vehicle control. Results were obtained from ﬁve individual experiments with three replicates measured for every data point. Results were analysed using one-way ANOVA with Dunnett’s multiple comparison tests and expressed as means ± standard error of the mean (SEM). For signiﬁcance, p < 0.0001. (D) Figure 7. Toxicity of CPO, AZO, and aldicarb to undifferentiated and differentiated SHSY-5 ured using a DCFDA assay. Undifferentiated (A,C) or differentiated (B,D) SHSY-5Y cells with CPO, AZO, or aldicarb (MTT IC10, IC20, IC50, and IC80 concentrations) and ROS level iﬁed using a DCFDA assay after 6 or 24 h. Cellular ROS levels were normalised to ve reatments and presented as a percentage of vehicle control. Results were obtained fr vidual experiments with three replicates measured for every data point. Results were ng one-way ANOVA with Dunnett’s multiple comparison tests and expressed as mean error of the mean (SEM). For significance, p < 0.0001. The levels of protein carbonyl content (PCC) (oxidatively-damaged pro (D) (D) Figure 7. Toxicity of CPO, AZO, and aldicarb to undifferentiated and differentiated ured using a DCFDA assay Undifferentiated (A C) or differentiated (B D) SHSY (D) aldica erentia Figure 7. Toxicity of CPO, AZO, and aldicarb to undifferentiated and differentiated SH ured using a DCFDA assay. 3. Results PCC levels mirrored those of ROS such that induction was in the order CPO > AZO > aldicarb (Figure 8A,B), with levels signiﬁcantly higher in differentiated cells compared to undifferentiated cells (refer to Supplementary Data). ponse to a 24-h exposure to CPO, AZO, or aldicarb were quantified. All three ag cantly increased PCC in a concentration-dependent manner in both undiffere differentiated cells (Figure 8A,B). PCC levels mirrored those of ROSs such tha was in the order CPO > AZO > aldicarb (Figure 8A,B), with levels signiﬁcant diﬀerentiated cells compared to undiﬀerentiated cells (refer to Supplementary was in the order CPO > AZO > aldicarb (Figure 8A,B), with levels signiﬁc diﬀerentiated cells compared to undiﬀerentiated cells (refer to Supplemen (A) (A) Figure 8. Cont. er CPO AZO aldicarb cells compared to undiﬀer cells (refer to (A) Figure 8. Cont. 14 of 20 14 of 20 Brain Sci. 2023, 13, 728 Sci. 2023, 13, x (B) Figure 8. CPO, AZO, and aldicarb induction of protein carbonyl content in undifferent ferentiated SHSY-5Y cells. Undifferentiated (A) or differentiated (B) SHSY-5Y cells wer CPO, AZO, or aldicarb and the levels of protein carbonyl content (PCC) were quantif PCC was quantified after DNPH derivatization of the cytosolic fractions of treated cell photometric readings normalised to vehicle control treatments after subtracting the blan assays were performed for each data point and ﬁve individual experiments were un sults were analysed using one-way ANOVA with Dunnett’s multiple comparison pressed as means ± standard error of the mean (SEM) For signiﬁcance p < 0 0001 Figure 8. CPO, AZO, and aldicarb induction of protein carbonyl content in undifferentiated and differentiated SHSY-5Y cells. Undifferentiated (A) or differentiated (B) SHSY-5Y cells were treated with CPO, AZO, or aldicarb and the levels of protein carbonyl content (PCC) were quantiﬁed after 24 h. PCC was quantiﬁed after DNPH derivatization of the cytosolic fractions of treated cells and spectrophotometric readings normalised to vehicle control treatments after subtracting the blanks. Triplicates assays were performed for each data point and ﬁve individual experiments were undertaken. Results were analysed using one-way ANOVA with Dunnett’s multiple comparison tests and expressed as means ± standard error of the mean (SEM). For signiﬁcance, p < 0.0001. 14 of 20 (B) Figure 8. CPO, AZO, and aldicarb induction of protein carbonyl content in undifferentiated and dif- ferentiated SHSY-5Y cells. 3. Results Re- sults were analysed using one-way ANOVA with Dunnett’s multiple comparison tests and ex- pressed as means ± standard error of the mean (SEM). For signiﬁcance, p < 0.0001. p ( ) g p To characterise the oxidatively-damaged proteins, cytosolic proteins from tiated and differentiated SH-SY5Y cells that had been treated with the neuroto resolved by gel electrophoresis and carbonylated proteins localised by immun To characterise the oxidatively-damaged proteins, cytosolic proteins from undiffer- entiated and differentiated SH-SY5Y cells that had been treated with the neurotoxicants were resolved by gel electrophoresis and carbonylated proteins localised by immune (oxy)- blotting (Figure 9). To characterise the oxidatively-damaged proteins, cytosolic proteins from undifferen- tiated and differentiated SH-SY5Y cells that had been treated with the neurotoxicants were resolved by gel electrophoresis and carbonylated proteins localised by immune (oxy)-blot- ting (Figure 9). y g p y p y ting (Figure 9). Figure 9. CPO, AZO, and aldicarb induction of carbonylated proteins in undifferentiated Figure 9. CPO, AZO, and aldicarb induction of carbonylated proteins in undifferentiated and differenti- ated SHSY-5Y cells characterized by oxy-blotting. Undifferentiated or differentiated SHSY-5Y cells were treated with CPO, AZO, or aldicarb at their MTT IC10, IC20, IC50, and IC80 concentrations and carbonylated proteins detected using an oxy-blot. Three independent blotting experiments were performed with each pesticide with representative blots included. The most prominent and reproducible carbonylated proteins were characterized by Figure 9. CPO, AZO, and aldicarb induction of carbonylated proteins in undifferentiated and differentiated SHSY-5Y cells characterized by oxy-blotting. Undifferentiated or differentiated SHSY-5Y cells were treated with CPO, AZO, or aldicarb at their MTT IC10, IC20, IC50, and IC80 concentrations and carbonylated proteins detected using an oxy-blot. Three independent blotting experiments were performed with each pesticide with representative blots included. y p y y g p y p y ting (Figure 9). Figure 9 CPO AZO and aldicarb induction of carbonylated proteins in undifferentiated Figure 9. CPO, AZO, and aldicarb induction of carbonylated proteins in undifferentiated and differenti- ated SHSY-5Y cells characterized by oxy-blotting. Undifferentiated or differentiated SHSY-5Y cells were treated with CPO, AZO, or aldicarb at their MTT IC10, IC20, IC50, and IC80 concentrations and carbonylated proteins detected using an oxy-blot. Three independent blotting experiments were performed with each pesticide with representative blots included. Figure 9. CPO, AZO, and aldicarb induction of carbonylated proteins in undifferentiated and differentiated SHSY-5Y cells characterized by oxy-blotting. 3. Results Undifferentiated (A) or differentiated (B) SHSY-5Y cells were treated with CPO, AZO, or aldicarb and the levels of protein carbonyl content (PCC) were quantified after 24 h. PCC was quantified after DNPH derivatization of the cytosolic fractions of treated cells and spectro- photometric readings normalised to vehicle control treatments after subtracting the blanks. Triplicates assays were performed for each data point and ﬁve individual experiments were undertaken. Re- sults were analysed using one-way ANOVA with Dunnett’s multiple comparison tests and ex- pressed as means ± standard error of the mean (SEM). For signiﬁcance, p < 0.0001. (B) 14 of (B) Figure 8. CPO, AZO, and aldicarb induction of protein carbonyl content in undifferen ferentiated SHSY-5Y cells. Undifferentiated (A) or differentiated (B) SHSY-5Y cells wer CPO, AZO, or aldicarb and the levels of protein carbonyl content (PCC) were quantif PCC was quantified after DNPH derivatization of the cytosolic fractions of treated cel photometric readings normalised to vehicle control treatments after subtracting the blan assays were performed for each data point and ﬁve individual experiments were un sults were analysed using one-way ANOVA with Dunnett’s multiple comparison pressed as means ± standard error of the mean (SEM) For signiﬁcance p < 0 0001 Figure 8. CPO, AZO, and aldicarb induction of protein carbonyl content in undifferentiated and differentiated SHSY-5Y cells. Undifferentiated (A) or differentiated (B) SHSY-5Y cells were treated with CPO, AZO, or aldicarb and the levels of protein carbonyl content (PCC) were quantiﬁed after 24 h. PCC was quantiﬁed after DNPH derivatization of the cytosolic fractions of treated cells and spectrophotometric readings normalised to vehicle control treatments after subtracting the blanks. Triplicates assays were performed for each data point and ﬁve individual experiments were undertaken. Results were analysed using one-way ANOVA with Dunnett’s multiple comparison tests and expressed as means ± standard error of the mean (SEM). For signiﬁcance, ** p < 0.0001. (B) Figure 8. CPO, AZO, and aldicarb induction of protein carbonyl content in undifferentiated and dif- ferentiated SHSY-5Y cells. Undifferentiated (A) or differentiated (B) SHSY-5Y cells were treated with CPO, AZO, or aldicarb and the levels of protein carbonyl content (PCC) were quantified after 24 h. PCC was quantified after DNPH derivatization of the cytosolic fractions of treated cells and spectro- photometric readings normalised to vehicle control treatments after subtracting the blanks. Triplicates assays were performed for each data point and ﬁve individual experiments were undertaken. 3. Results Undifferentiated or differentiated SHSY-5Y cells were treated with CPO, AZO, or aldicarb at their MTT IC10, IC20, IC50, and IC80 concentrations and carbonylated proteins detected using an oxy-blot. Three independent blotting experiments were performed with each pesticide with representative blots included. Figure 9 CPO AZO and aldicarb induction of carbonylated proteins in undifferentiated Figure 9. CPO, AZO, and aldicarb induction of carbonylated proteins in undifferentiated and differenti- ated SHSY-5Y cells characterized by oxy-blotting. Undifferentiated or differentiated SHSY-5Y cells were treated with CPO, AZO, or aldicarb at their MTT IC10, IC20, IC50, and IC80 concentrations and carbonylated proteins detected using an oxy-blot. Three independent blotting experiments were performed with each pesticide with representative blots included. Figure 9. CPO, AZO, and aldicarb induction of carbonylated proteins in undifferentiated and differentiated SHSY-5Y cells characterized by oxy-blotting. Undifferentiated or differentiated SHSY-5Y cells were treated with CPO, AZO, or aldicarb at their MTT IC10, IC20, IC50, and IC80 concentrations and carbonylated proteins detected using an oxy-blot. Three independent blotting experiments were performed with each pesticide with representative blots included. Figure 9. CPO, AZO, and aldicarb induction of carbonylated proteins in undifferentiated ated SHSY-5Y cells characterized by oxy-blotting. Undifferentiated or differentiated SHSY treated with CPO, AZO, or aldicarb at their MTT IC10, IC20, IC50, and IC80 conce The most prominent and reproducible carbonylated proteins were characterized by their denatured molecular weights of approximately 35, 50, 80, and 90 kDa. The accumu- lation of oxidatively-damaged proteins primarily increased in proportion to agent con- The most prominent and reproducible carbonylated proteins were characterized by their denatured molecular weights of approximately 35, 50, 80, and 90 kDa. The accu- Brain Sci. 2023, 13, 728 15 of 20 15 of 20 mulation of oxidatively-damaged proteins primarily increased in proportion to agent concentration, most notably the ≈50 kDa protein. mulation of oxidatively-damaged proteins primarily increased in proportion to agent concentration, most notably the ≈50 kDa protein. y p Collectively, differentiated cells were more vulnerable to the neurotoxic effects of the OPs and aldicarb and this was in part mediated through the induction of redox stress as a non-cholinergic mechanism. 4. Discussion 4.1. Differentiated Neurons Are More Vulnerable to the Neurotoxic Effects of CPO, AZO, and Aldicarb 4.1. Differentiated Neurons Are More Vulnerable to the Neurotoxic Effects of CPO, AZO, and Aldicarb In this manuscript, the neurotoxic properties of two OPs, CPO and AZO, and the carbamate pesticide, aldicarb, were investigated. All three compounds were cytotoxic and reduced neuronal viability and were detrimental to cellular bioenergetics (ATP produc- tion) (Figures 2–4). Neurotoxicity potency was in the order AZO > CPO > aldicarb for undifferentiated cells but CPO > AZO > aldicarb for differentiated cells. Vulnerability to neurotoxicity for differentiated cells was evidenced through suppression of neurite outgrowth (Figure 5), increased inhibition of AChE (Figure 6), and increased production of damaging ROS (Figure 7). CPO induced the highest levels of ROS and corresponding production of oxidatively-damaged proteins, and these were higher in differentiated cells than undifferentiated cells and were characterized by their denatured molecular weights (Figures 8 and 9). g SH-SY5Y cells were used as a homogeneous neuronal cell model, often used for toxicity studies, since they can be speciﬁcally differentiated to undergo a morphological change and generate neuritic projections, with altered neurotransmitter responsiveness consistent with a cholinergic phenotype [34,35,42–44]. Through a direct comparison of undifferentiated with differentiated SH-SY5Y cells, an increased vulnerability of differenti- ated cells to neurotoxicants was established. MTT assays provided a measurement of cell metabolic activity as a surrogate for cell viability, with the neurotoxicant-induced loss of cell viability substantiated using LDH assays and a shutdown of cellular ATP production (Figures 2–4). 4.2. Differentiated Neurons Are More Vulnerable to Cholinergic Toxicity from CPO, AZO, and Aldicarb The recognized acute neurotoxicity of many OPs and carbamates is via the targeted inhibition of AChE and the induction of cholinergic crisis. By challenging cells with the bioactive oxon forms of the OP pesticides (CPO and AZO) and aldicarb, we were able to consider their relative potency as cholinesterase inhibitors. All three compounds were strong inhibitors of AChE with IC50 values of below 1 µM, but the OPs were more potent than aldicarb, with CPO displaying the lowest IC50 concentration (Figure 6 and Table 1). A key difference between the AChE inhibition by the two OPs and aldicarb is the stability of the AChE-OP and the AChE–carbamate complex, since the organophosphylation of AChE is often stable with slow spontaneous hydrolysis (particularly for O,O’-diethyl adducts such as those produced from CPO), whereas carbamylation at the active site serine of AChE is readily reversible and can spontaneously hydrolyse within hours [23]. Furthermore, carbamate binding to AChE does not undergo non-enzymatic dealkylation (ageing) which can further limit hydrolysis of the organophosphorylation such that it becomes essentially irreversible. Thus, if the potential for prolonged inhibition of AChE is solely considered, aldicarb should be considered less toxic than either CPO or AZA. However, the potentially rapid binding of aldicarb to AChE and associated acute toxicity (rat oral LD50 of 0.65 mg/kg) [45] without the need for bioactivation, has resulted in aldicarb (and its formulations) being classiﬁed as extremely hazardous (Class Ia) in contrast to the moderately hazardous listing for chlorpyrifos (CPF) and Azamethiphos (AZO) (Class II) (rat oral LD50 of 82 mg/kg and 1180 mg/kg, respectively) [46–48]. g g g g p y The production of AChE inhibitor response curves for treatments with CPO, AZO, and aldicarb revealed that differentiated neurons were more vulnerable to neurotoxicity, with lower IC50 values (by 27–39%). Collectively, this neurotoxicant inhibition of AChE Brain Sci. 2023, 13, 728 16 of 20 16 of 20 occurs at lower concentrations (IC50 values of 0.19–0.61 µM) than those investigated for ROS production. However, consideration should be given to the combination of both mech- anisms of toxicity since inhibition of AChE may be transient. AZO produces O,O’-dimethyl adducts with AChE which are less stable than the O,O’-diethyl adducts produced from CPO, and can be hydrolyzed after several hours. 4.2. Differentiated Neurons Are More Vulnerable to Cholinergic Toxicity from CPO, AZO, and Aldicarb Nevertheless, irrespective of whether pesticide-AChE O,O’-dimethyl or O,O’-diethyl adducts are formed, AChE will be regener- ated through protein turnover, with an estimated half-life of 3–12 days [49], and this may be even more rapid for an irreversible inhibitor-bound enzyme [49]. Furthermore, AChE has a rapid enzymatic turnover number [50]; hence, even low levels of the enzyme could still potentially cleave ACh efﬁciently, assuming the substrate is not limiting. In addition, relatively high (or repeated) exposures to OP inhibitors are usually needed to surpass the threshold of approximately 50–75% AChE inhibition associated with mild to moderate OP poisoning [51]. By contrast, the structure-activity relationships of other proteins ad- ducted by pesticides may differ from that for AChE [1,6,8–11,18,26,27,52] and some could potentially be long-lasting and contribute to pesticide-induced ill health. Furthermore, as proposed herein, oxidatively-damaged proteins are generated after exposure to neuro- toxicants at a concentration below the threshold required to induce neuronal death, and therefore this form of protein post-translational modiﬁcation could potentially contribute to acute or chronic cellular damage. 4.3. Differentiated Neurons Are More Vulnerable to Non-Cholinergic Toxicity from CPO, AZO, and Aldicarb We also examined the non-cholinergic mechanism of neurotoxicant-generated redox stress through the induction of ROS and the production of carbonylated proteins. After cell treatment, differentiated cells produced signiﬁcantly more ROS than their undifferentiated counterparts and hence, signiﬁcantly higher levels of PCC (refer to Supplementary Data). The induction of PCC was evidenced by the detection and characterization of oxidatively- damaged proteins and herein we provide the ﬁrst characterization of these carbonylated proteins in response to CPO, AZO, and aldicarb. The most prominent detection of carbony- lation was at denatured molecular weights of approximately 35, 50, 80, and 90 kDa. The greater levels of oxidative damage to these proteins, particularly the ≈50 kDa protein band, may represent an increased vulnerability to oxidative damage as a consequence of their higher protein expression in differentiation cells. SH-SY-5Y cell differentiation is characterized by the expression of mature neuronal markers including βIII-tubulin, microtubule-associated protein-2 (MAP-2), and MAP-tau; cytoskeletal or cytoskeletal-associated proteins required for the production of neurite projections [34,35,42–44,53]. The denatured molecular weights of immunoreactive tubulin, MAP-2 and MAP-tau [54] could correspond to the oxidatively-damaged protein bands at approximately 50, 80, and 90 kDa (Figure 9). In support of this supposition, other studies have documented the vulnerability of α- and β-tubulin proteins (of ≈50 kDa) to either organophosphorylation or oxidation [55–57], but conﬁrmation of the identity of the major carbonylated proteins that accumulate in response to these neurotoxicants will require future study. Furthermore, future studies will need to consider the fate of these oxidatively-damaged proteins. If the levels of ROS overwhelm the cellular antioxidant system, cell death is triggered [26,28]. However, for cells that survive a neurotoxic insult, these carbonylated proteins may be detrimental to cellular functionality [58] and therefore represent a pesticide-induced cellular injury. Other studies have conﬁrmed increased carbonylation in response to CPF treatment in neuronal cells or tissue [59,60] but this is the ﬁrst report of a similar response from AZO and aldicarb. Furthermore, our data suggest that the target proteins for carbonylation may overlap for each of the neurotoxicants, indicative of a common mechanism of neurotoxicity (Figure 9). Carbonylated damage to proteins may contribute to the blunting of neuritic arborization we detected and is consistent with reports that exposure to certain (but not all) pesticides, including AZO, can inhibit neurite outgrowth in vitro [61]. However, in vitro Brain Sci. 4.3. Differentiated Neurons Are More Vulnerable to Non-Cholinergic Toxicity from CPO, AZO, and Aldicarb Funding: This research was funded by a UK Foreign, Commonwealth and Development Ofﬁce (FCDO) Commonwealth Scholarship Commission (UK) PhD award to A.W.M. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data is available on request from the first or last authors of the manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. 5. Chlorpyrifos technical fact sheet, National Pesticide Information Centre, USA. Available online: http://npic.orst.edu/factsheets/ archive/chlorptech.html (accessed on 28 March 2023). g 4. Eaton, D.L.; Daroff, R.B.; Autrup, H.; Bridges, J.; Bufﬂer, P.; Costa, L.G.; Coyle, J.; McKhann, G.; Mobley, W.C.; Nadel, L.; et al. Review of the Toxicology of Chlorpyrifos with an Emphasis on Human Exposure and Neurodevelopment. Crit. Rev. Toxicol. 2008, 38, 1–125. [CrossRef] 4.3. Differentiated Neurons Are More Vulnerable to Non-Cholinergic Toxicity from CPO, AZO, and Aldicarb 2023, 13, 728 17 of 20 17 of 20 studies, such as ours, are limited by the application of pesticides or their metabolites directly to cells and further in vivo studies are required to substantiate these research ﬁndings. studies, such as ours, are limited by the application of pesticides or their metabolites directly to cells and further in vivo studies are required to substantiate these research ﬁndings. 4.4. Cholinergic and Non-Cholinergic Toxicity: Considerations for Neurodevelopmental Effects, Treatment of Exposures, and Pesticide Risk Assessment The susceptibility of neurons to oxidative damage during neurogenesis and neuronal arborization has implications for the impact of pesticide exposures on the developing brain. Studies have indicated that prenatal and childhood exposures to chlorpyrifos can induce neurodevelopmental and neurobehavioural deﬁcits in children and adults [13–16,62] and this could in part reﬂect oxidative damage to key proteins involved in the production of neuritic projections and connections between neurons. Noteworthy is that acute intoxication from OP pesticides is usually through self-harm and is estimated to account for approximately 100,000 deaths per year [63]. The current treatment of OP pesticide exposure utilizes a muscarinic antagonist (typically atropine) and, depending upon the type of OP pesticide poisoning (if known), an oxime (such as prali- doxime) as an AChE reactivator [64]. However, given the importance of non-cholinergic toxicity mediated via redox stress, it would be prudent to also consider treatment with antioxidant co-therapy. Hence, a pesticide risk assessment should consider acute and potential long-lasting toxicity and the clinical sequelae that reﬂects both cholinergic and non-cholinergic mechanisms. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/brainsci13050728/s1, supplementary data, Table S1: comparison of the cytotoxicity induced by CPO, AZO, and aldicarb to undifferentiated versus differentiated SH-SY5Y cells. Author Contributions: Conceptualization, A.W.M. and W.G.C.; methodology, A.W.M., B.C.W. and A.K.; validation, A.W.M., B.C.W., A.K. and W.G.C.; formal analysis, A.W.M., B.C.W., A.K. and W.G.C.; investigation, A.W.M., B.C.W. and A.K.; resources, W.G.C.; data curation, A.W.M., B.C.W., A.K. and W.G.C.; writing—original draft preparation, A.W.M. and W.G.C.; writing—review and editing, A.W.M., B.C.W., A.K. and W.G.C.; supervision, W.G.C.; project administration, W.G.C.; funding acquisition, A.W.M. and W.G.C. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by a UK Foreign, Commonwealth and Development Ofﬁce (FCDO) Commonwealth Scholarship Commission (UK) PhD award to A.W.M. 3. Nicolopoulou-Stamati, P.; Maipas, S.; Kotampasi, C.; Stamatis, P.; Hens, L. Chemical Pesticides and H Need for a New Concept in Agriculture. Front. Public Health 2016, 4, 148. [CrossRef] p 3. Nicolopoulou-Stamati, P.; Maipas, S.; Kotampasi, C.; Stamatis, P.; Hens, L. Chemical Pesticides and Human Health: The Urgent Need for a New Concept in Agriculture. Front. Public Health 2016, 4, 148. [CrossRef] [ ] Casida, J.E.; Durkin, K.A. Anticholinesterase insecticide retrospective. Chem. Interact. 2013, 203, 221–225. [ References 1. Richardson, J.R.; Fitsanakis, V.; Westerink, R.H.S.; Kanthasamy, A.G. Neurotoxicity of pesticides. Acta Neuropathol. 2019, 138, 343–362. [CrossRef] Brain Sci. 2023, 13, 728 18 of 20 18 of 20 6. Tarhoni, M.H.; Lister, T.; Ray, D.E.; Carter, W.G. Albumin binding as a potential biomarker of exposure to moderately low levels of organophosphorus pesticides. Biomarkers 2008, 13, 343–363. [CrossRef] g p p p 7. Mangas, I.; Estevez, J.; Vilanova, E.; França, T.C.C. New insights on molecular interactions of organophosphorus pesticides with esterases. Toxicology 2017, 376, 30–43. [CrossRef] gy 8. Carter, W.G.; Tarhoni, M.; Rathbone, A.J.; Ray, D.E. Differential protein adduction by seven organophosphorus pesticides in both brain and thymus. Hum. Exp. Toxicol. 2007, 26, 347–354. [CrossRef] 9. Carter, W.G.; Tarhoni, M.H.; Ray, D.E. Analytical approaches to investigate protein–pesticide adducts. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2010, 878, 1312–1319. [CrossRef] f 10. Terry, A. Functional consequences of repeated organophosphate exposure: Potential non-choline Ther. 2012, 134, 355–365. [CrossRef] 10. Terry, A. Functional consequences of repeated organophosphate exposure: Potential non-cholinergic mechanisms. Pharmacol. Ther. 2012, 134, 355–365. [CrossRef] 10. Terry, A. Functional consequences of repeated organophosphate exposure: Potential non cholinergic mechanisms. Pharmacol. Ther. 2012, 134, 355–365. [CrossRef] 11 Costa L G Organophosphorus Compounds at 80: Some Old and New Issues Toxicol Sci 2018 162 24 35 [CrossRef] 11. Costa, L.G. Organophosphorus Compounds at 80: Some Old and New Issues. Toxicol. Sci. 2018, 16 nophosphorus Compounds at 80: Some Old and New Issues. Toxicol. Sci. 2018, 162, 24–35. [CrossRef] 12. Elmorsy, E.; Al-Ghafari, A.; Al Doghaither, H.; Salama, M.; Carter, W.G. An Investigation of the Neurotoxic Effects of Malathion, Chlorpyrifos, and Paraquat to Different Brain Regions. Brain Sci. 2022, 12, 975. [CrossRef] py q g 13. Rauh, V.A.; Perera, F.P.; Horton, M.K.; Whyatt, R.M.; Bansal, R.; Hao, X.; Liu, J.; Barr, D.B.; Slotkin, T.A.; Peterson, B.S. Brain anomalies in children exposed prenatally to a common organophosphate pesticide. Proc. Natl Acad. Sci. USA 2012, 109, 7871–7876. [CrossRef] 14. Ross, S.M.; McManus, I.C.; Harrison, V.; Mason, O. Neurobehavioral problems following low-level exposure to organophosphate pesticides: A systematic and meta-analytic review. Crit. Rev. Toxicol. 2013, 43, 21–44. [CrossRef] 15. Rauh, V.A.; Garcia, W.E.; Whyatt, R.M.; Horton, M.K.; Barr, D.B.; Louis, E.D. Prenatal exposure to the organophosphate pesticide chlorpyrifos and childhood tremor. Neurotoxicology 2015, 51, 80–86. [CrossRef] 16. Burke, R.D.; Todd, S.W.; Lumsden, E.; Mullins, R.J.; Mamczarz, J.; Fawcett, W.P.; Gullapalli, R.P.; Randall, W.R.; Pereira, E.F.R.; Albuquerque, E.X. References Developmental neurotoxicity of the organophosphorus insecticide chlorpyrifos: From clinical ﬁndings to preclinical models and potential mechanisms. J. Neurochem. 2017, 142, 162–177. [CrossRef] 17. Intorre, L.; Soldani, G.; Cognetti-Varriale, A.; Monni, G.; Meucci, V.; Pretti, C. Safety of azamethiphos in eel, seabass and trout. Pharmacol. Res. 2004, 49, 171–176. [CrossRef] 18. Tarhoni, M.H.; Vigneswara, V.; Smith, M.; Anderson, S.; Wigmore, P.; Lees, J.E.; Ray, D.E.; Carter, W.G. Detection, Quantiﬁcation, and Microlocalisation of Targets of Pesticides Using Microchannel Plate Autoradiographic Imagers. Molecules 2011, 16, 8535–8551. [CrossRef] 19. Barisic, J.; Cannon, S.; Quinn, B. Cumulative impact of anti-sea lice treatment (azamethiphos) on health status of Rainbow trout (Oncorhynchus mykiss, Walbaum 1792) in aquaculture. Sci. Rep. 2019, 9, 16217. [CrossRef] 20. Gupta, R.C. Carbamate Pesticides. In Encyclopedia of Toxicology, 3rd ed.; Wexler, P., Ed.; Oxford Academic Press: Oxford, UK, 2014; pp. 661–664. 21. Smith, J.F.; Catchot, A.L.; Musser, F.R.; Gore, J. Effects of aldicarb and neonicotinoid seed treatments on on cotton. J. Econ. Èntomol. 2013, 106, 807–815. [CrossRef] 22. Bauer, P.J.; Roof, M.E. Nitrogen, Aldicarb, and Cover Crop Effects on Cotton Yield and Fiber Properties. Agron. J. 2004, 96, 369–376. [CrossRef] 23. Risher, J.F.; Mink, F.L.; Stara, J.F. The toxicologic effects of the carbamate insecticide aldicarb in mammals: A review. Environ. Health Perspect. 1987, 72, 267–281. [CrossRef] 24. Fukuto, T.R. Mechanism of action of organophosphorus and carbamate insecticides. Environ. Health Perspect. 1990, 87, 245–254. [CrossRef] 25. Vale, A.; Lotti, M. Organophosphorus and carbamate insecticide poisoning. In Handbook of Clinical Neurology; Elsevier: Amsterdam, The Netherlands, 2015; Volume 131, pp. 149–168. 26. Tsai, Y.-H.; Lein, P.J. Mechanisms of organophosphate neurotoxicity. Curr. Opin. Toxicol. 2021, 26, 49–60 Mechanisms of organophosphate neurotoxicity. Curr. Opin. Toxicol. 2021, 26, 49–60. [CrossRef] 27. Richardson, R.J.; Fink, J.K.; Glynn, P.; Hufnagel, R.B.; Makhaeva, G.F.; Wijeyesakere, S.J. Neuropathy target esterase (NTE/PNPLA6) and organophosphorus compound-induced delayed neurotoxicity (OPIDN). Adv. Neurotoxicol. 2020, 4, 1–78. [CrossRef] [ ] 28. Sule, R.O.; Condon, L.; Gomes, A.V. A Common Feature of Pesticides: Oxidative Stress—The Role of Oxidative Stress in Pesticide-Induced Toxicity. Oxidative Med. Cell. Longev. 2022, 2022, 5563759. [CrossRef] [PubMed] y g 29. Chin-Chan, M.; Navarro-Yepes, J.; Quintanilla-Vega, B. Environmental pollutants as risk factors for ne l h d k d ll [ f] [ b d] y g 29. Chin-Chan, M.; Navarro-Yepes, J.; Quintanilla-Vega, B. Environmental pollutants as risk factor Alzheimer and Parkinson diseases. Front. Cell. Neurosci. 2015, 9, 124. [CrossRef] [PubMed] 29. References Chin-Chan, M.; Navarro-Yepes, J.; Quintanilla-Vega, B. Environmental pollutants as risk factors for neurodegenerative disorders: Alzheimer and Parkinson diseases. Front. Cell. Neurosci. 2015, 9, 124. [CrossRef] [PubMed] 29. Chin Chan, M.; Navarro Yepes, J.; Quintanilla Vega, B. Environmental pollutants as risk factors f Alzheimer and Parkinson diseases. Front. Cell. Neurosci. 2015, 9, 124. [CrossRef] [PubMed] 30. Jokanovi´c, M. Neurotoxic effects of organophosphorus pesticides and possible association with man: A review. Toxicology 2018, 410, 125–131. [CrossRef] 30. Jokanovi´c, M. Neurotoxic effects of organophosphorus pesticides and possible association with neurodegenerative diseases in man: A review. Toxicology 2018, 410, 125–131. [CrossRef] 31. Tang, B.L. Neuropathological Mechanisms Associated with Pesticides in Alzheimer’s Disease. Toxics 2020, 8, 21. [CrossRef] [PubMed] 32. Ellman, G.L.; Courtney, K.D.; Andres, V., Jr.; Featherstone, R.M. A new and rapid colorimetric cholinesterase activity. Biochem. Pharmacol. 1961, 7, 88–95. [CrossRef] 33. Flaskos, J.; Harris, W.; Sachana, M.; Muñoz, D.; Tack, J.; Hargreaves, A. The effects of diazinon and cypermethrin on the differentiation of neuronal and glial cell lines. Toxicol. Appl. Pharmacol. 2007, 219, 172–180. [CrossRef] 19 of 20 19 of 20 Brain Sci. 2023, 13, 728 34. Encinas, M.; Iglesias, M.; Liu, Y.; Wang, H.; Muhaisen, A.; Ceña, V.; Gallego, C.; Comella, J.X. Sequential Treatment of SH-SY5Y Cells with Retinoic Acid and Brain-Derived Neurotrophic Factor Gives Rise to Fully Differentiated, Neurotrophic Factor- Dependent, Human Neuron-Like Cells. J. Neurochem. 2000, 75, 991–1003. [CrossRef] 35. Shipley, M.M.; Mangold, C.A.; Szpara, M.L. Differentiation of the SH-SY5Y human neuroblastom 108, 53193. 36. Denizot, F.; Lang, R. Rapid colorimetric assay for cell growth and survival. Modiﬁcations to the tetrazolium dye procedure giving improved sensitivity and reliability. J. Immunol. Methods 1986, 89, 271–277. [CrossRef] [PubMed] 37. Erdozain, A.M.; Morentin, B.; Bedford, L.; King, E.; Tooth, D.; Brewer, C.; Wayne, D.; Johnson, L.; Gerdes, H.K.; Wigmore, P.; et al. Alcohol-Related Brain Damage in Humans. PLoS ONE 2014, 9, e93586. [CrossRef] g 38. Lowry, O.H.; Rosebrough, N.J.; Farr, A.L.; Randall, R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951, 193, 265–275. [CrossRef] [PubMed] 39. Augustyniak, E.; Adam, A.; Wojdyla, K.; Rogowska-Wrzesinska, A.; Willetts, R.; Korkmaz, A.; Atalay, M.; Weber, D.; Grune, T.; Borsa, C.; et al. Validation of protein carbonyl measurement: A multi-centre study. Redox Biol. 2015, 4, 149–157. [CrossRef] p y y 40. Colombo, G.; Clerici, M.; Garavaglia, M.E.; Giustarini, D.; Rossi, R.; Milzani, A.; Dalle-Donne, I. A step-by-step protocol for assaying protein carbonylation in biological samples. References J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2015, 1019, 178–190. [CrossRef] 41. Vigneswara, V.; Cass, S.; Wayne, D.; Bolt, E.L.; Ray, D.E.; Carter, W.G. Molecular Ageing of Alpha- and Beta-Synucleins: Protein Damage and Repair Mechanisms. PLoS ONE 2013, 8, e61442. [CrossRef] [PubMed] g p 42. Påhlman, S.; Hoehner, J.; Nånberg, E.; Hedborg, F.; Fagerström, S.; Gestblom, C.; Johansson, I.; Larsson, U.; Lavenius, E.; Örtoft, E.; et al. Differentiation and survival inﬂuences of growth factors in human neuroblastoma. Eur. J. Cancer 1995, 31, 453–458. [CrossRef] [PubMed] [ ] [ ] 43. Xie, H.R.; Hu, L.S.; Li, G.Y. SH-SY5Y human neuroblastoma cell line: In vitro cell model of dopaminergic neurons in Parkinson’s disease. Chin. Med. J. 2010, 123, 1086–1092. 44. Kovalevich, J.; Langford, D. Considerations for the use of SH-SY5Y neuroblastoma cells in neurobiology. Methods Mol. Biol. 2013, 1078, 9–21. [CrossRef] 45. Aldicarb, Pubchem. Available online: https://pubchem.ncbi.nlm.nih.gov/compound/9570071 (access em. Available online: https://pubchem.ncbi.nlm.nih.gov/compound/9570071 (accessed on 17 April 2023). Pubchem Available online: https://pubchem ncbi nlm nih gov/compound/71482 (accessed on 17 April 20 45. Aldicarb, Pubchem. Available online: https://pubchem.ncbi.nlm.nih.gov/compound/9570071 (accessed on 17 April 2023). 46. Azamethiphos, Pubchem. Available online: https://pubchem.ncbi.nlm.nih.gov/compound/71482 (accessed on 17 April 2023). 47. Chlorpyrifos, Pubchem. Available online: https://pubchem.ncbi.nlm.nih.gov/compound/2730 (accessed on 17 April 2023). 45. Aldicarb, Pubchem. Available online: https://pubchem.ncbi.nlm.nih.gov/compound/9570071 (accessed on 17 April 2023). 46. Azamethiphos, Pubchem. Available online: https://pubchem.ncbi.nlm.nih.gov/compound/71482 (accessed on 17 April 2023). hl f b h l bl l h // b h b l h / d/ ( d l ) 46. Azamethiphos, Pubchem. Available online: https://pubchem.ncbi.nlm.nih.gov/compound/71482 (ac 46. Azamethiphos, Pubchem. Available online: https://pubchem.ncbi.nlm.nih.gov/compound/71482 (accessed on 17 April 2023). 4 Chl f P b h A l bl l h // b h b l h / d/ ( d 1 A l ) 47. Chlorpyrifos, Pubchem. Available online: https://pubchem.ncbi.nlm.nih.gov/compound/2730 (acces 47. Chlorpyrifos, Pubchem. Available online: https://pubchem.ncbi.nlm.nih.gov/compound/2730 (accessed on 17 April 2023). 48. The WHO Recommended Classiﬁcation of Pesticides. 2019. Available online: https://www.who.int/publications/i/item/9789 240005662 (accessed on 28 March 2023). 49. Krejci, E.; Valenzuela, I.M.-P.Y.; Ameziane, R.; Akaaboune, M. Acetylcholinesterase Dynamics at the Neuromuscular Junction of Live Animals. J. Biol. Chem. 2006, 281, 10347–10354. [CrossRef] 50. Wilson, I.B.; Harrison, M.A. Turnover number of acetyl-cholinesterase. J. Biol. Chem. 1961, 236, 2292–22 50. Wilson, I.B.; Harrison, M.A. Turnover number of acetyl-cholinesterase. J. Biol. Chem. 1961, 236, 2292–2295. [CrossRef] [PubMed] 51. Namba, T. Cholinesterase inhibition by organophosphorus compounds and its clinical effects. Bull. World Health Organ. 1971, 44, 289–307. 51. References Namba, T. Cholinesterase inhibition by organophosphorus compounds and its clinical effects. Bull. World Health Organ. 1971, 44, 289–307. 52. Lockridge, O.; Schopfer, L.M. Review: Organophosphorus toxicants, in addition to inhibiting acetylcholinesterase activity, make covalent adducts on multiple proteins and promote protein crosslinking into high molecular weight aggregates. Chem. Interactions 2023, 376, 110460. [CrossRef] [PubMed] 53. Cheung, Y.-T.; Lau, W.K.-W.; Yu, M.-S.; Lai, C.S.-W.; Yeung, S.-C.; So, K.-F.; Chang, R.C.-C. Effects of all-trans-retinoic acid on human SH-SY5Y neuroblastoma as in vitro model in neurotoxicity research. NeuroToxicology 2009, 30, 127–135. [CrossRef] 53. Cheung, Y.-T.; Lau, W.K.-W.; Yu, M.-S.; Lai, C.S.-W.; Yeung, S.-C.; So, K.-F.; Chang, R.C.-C. Effects of all-trans-retinoic acid on human SH-SY5Y neuroblastoma as in vitro model in neurotoxicity research. NeuroToxicology 2009, 30, 127–135. [CrossRef] 54 Labisso W L ; Raulin A -C ; Nwidu L L ; Kocon A ; Wayne D ; Erdozain A M ; Morentin B ; Schwendener D ; Allen G ; 54. Labisso, W.L.; Raulin, A.-C.; Nwidu, L.L.; Kocon, A.; Wayne, D.; Erdozain, A.M.; Morentin, B.; Schwendener, D.; Allen, G.; Enticott, J.; et al. The Loss of α- and β-Tubulin Proteins Are a Pathological Hallmark of Chronic Alcohol Consumption and Natural Brain Ageing. Brain Sci. 2018, 8, 175. [CrossRef] g g 55. Grigoryan, H.; Schopfer, L.M.; Peeples, E.S.; Duysen, E.G.; Grigoryan, M.; Thompson, C.M.; Lockridge, O. Mass spectrometry identiﬁes multiple organophosphorylated sites on tubulin. Toxicol. Appl. Pharmacol. 2009, 240, 149–158. [CrossRef] 56. Zhang, Q.; Zhang, M.; Huang, X.; Liu, X.; Li, W. Inhibition of cytoskeletal protein carbonylation may protect against oxidative damage in traumatic brain injury. Exp. Ther. Med. 2016, 12, 4107–4112. [CrossRef] 57. Hernandez-Toledano, D.; Vega, L. The cytoskeleton as a non-cholinergic target of organophosphate compounds. Chem. Biol. Interact. 2021, 346, 109578. [CrossRef] [PubMed] 58. Dalle-Donne, I.; Aldini, G.; Carini, M.; Colombo, R.; Rossi, R.; Milzani, A.D.G. Protein carbonylation, disease progression. J. Cell. Mol. Med. 2006, 10, 389–406. [CrossRef] [PubMed] ini, G.; Carini, M.; Colombo, R.; Rossi, R.; Milzani, A.D.G. Protein carbonylation, cellular dysfunction, and J. Cell. Mol. Med. 2006, 10, 389–406. [CrossRef] [PubMed] 59. Brasil, F.B.; de Almeida, F.J.S.; Luckachaki, M.D.; Dall’oglio, E.L.; de Oliveira, M.R. A Pretreatment with Isoorientin Attenu- ates Redox Disruption, Mitochondrial Impairment, and Inﬂammation Caused by Chlorpyrifos in a Dopaminergic Cell Line: Involvement of the Nrf2/HO-1 Axis. Neurotox. Res. 2022, 40, 1043–1056. [CrossRef] [PubMed] 60. Xu, M.-Y.; Wang, P.; Sun, Y.-J.; Yang, L.; Wu, Y.-J. g 61. Christen, V.; Rusconi, M.; Crettaz, P.; Fent, K. Developmental neurotoxicity of different pesticides i Appl. Pharmacol. 2017, 325, 25–36. [CrossRef] [PubMed] 62. Guo, J.; Zhang, J.; Wu, C.; Lv, S.; Lu, D.; Qi, X.; Jiang, S.; Feng, C.; Yu, H.; Liang, W.; et al. Associations of prenatal and childhood chlorpyrifos exposure with Neurodevelopment of 3-year-old children. Environ. Pollut. 2019, 251, 538–546. [CrossRef] py p p y , , [ ] 63. Mew, E.J.; Padmanathan, P.; Konradsen, F.; Eddleston, M.; Chang, S.-S.; Phillips, M.R.; Gunnell, D. The global burden of fatal self-poisoning with pesticides 2006-15: Systematic review. J. Affect. Disord. 2017, 219, 93–104. [CrossRef] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. p g p y J ff , , [ ] 64. Eddleston, M.; Buckley, N.A.; Eyer, P.; Dawson, A.H. Management of acute organophosphorus pesticide poisoning. Lancet 2008, 371, 597–607. [CrossRef] [PubMed] py p p y [ ] 63. Mew, E.J.; Padmanathan, P.; Konradsen, F.; Eddleston, M.; Chang, S.-S.; Phillips, M.R.; Gunnell, D. The global burden of fatal self-poisoning with pesticides 2006-15: Systematic review. J. Affect. Disord. 2017, 219, 93–104. [CrossRef] 64. Eddleston, M.; Buckley, N.A.; Eyer, P.; Dawson, A.H. Management of acute organophosphorus pesticide poisoning. Lancet 2008, , J ; , ; , ; , ; g, ; p , ; , g self-poisoning with pesticides 2006-15: Systematic review. J. Affect. Disord. 2017, 219, 93–104. [CrossRef] 64. Eddleston, M.; Buckley, N.A.; Eyer, P.; Dawson, A.H. Management of acute organophosphorus pesticide poisoning. Lancet 2008, 371, 597–607. [CrossRef] [PubMed] self-poisoning with pesticides 2006-15: Systematic review. J. Affect. Disord. 2017, 219, 93–104. [Cros 64. Eddleston, M.; Buckley, N.A.; Eyer, P.; Dawson, A.H. Management of acute organophosphorus pes 371, 597–607. [CrossRef] [PubMed] References Joint toxicity of chlorpyrifos and cadmium mitochondrial damage in neuronal cells. Food Chem. Toxicol. 2017, 103, 246–252. [CrossRef] [PubMe 61. Christen, V.; Rusconi, M.; Crettaz, P.; Fent, K. Developmental neurotoxicity of different pesticides in PC-12 cells in vitro. Toxicol. Appl. Pharmacol. 2017, 325, 25–36. [CrossRef] [PubMed] 20 of 20 Brain Sci. 2023, 13, 728 Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
https://openalex.org/W4361880311	https://figshare.com/articles/journal_contribution/Supplementary_Figure_S1_from_Telomerase_Template_Antagonist_GRN163L_Disrupts_Telomere_Maintenance_Tumor_Growth_and_Metastasis_of_Breast_Cancer/22441221/1/files/39892092.pdf	English	null	Supplementary Figure S1 from Telomerase Template Antagonist GRN163L Disrupts Telomere Maintenance, Tumor Growth, and Metastasis of Breast Cancer	null	2,023	cc-by	244	Supplementary Fig. 1 Supplementary Fig. 1 Supplementary Fig. 1 A 0 5 10 15 20 25 0 20 40 60 80 Days CPDL Control +GRN163L 21NT 0 10 20 30 40 50 60 0 20 40 60 Days CPDL Untreated Mismatch +GRN163L MCF-7 0 5 10 15 20 25 30 35 0 20 40 60 Days CPDL Control +GRN163L HCC1937 0 10 20 30 40 50 60 0 20 40 60 80 Days C P D L Control +GRN163L MDA-MB-231 0 10 20 30 40 50 60 0 20 40 60 Days C PDL Control +GRN163L HME50-T 0 10 20 30 40 50 60 0 50 100 150 200 Days CPDL Control +GRN163L SKBR3 MCF-7 cells B 21NT cells C A 0 10 20 30 40 50 60 0 20 40 60 Days CPDL Untreated Mismatch +GRN163L MCF-7 A 0 5 10 15 20 25 0 20 40 60 80 Days CPDL Control +GRN163L 21NT 0 10 20 30 40 50 60 0 20 40 60 80 Days C P D L Control +GRN163L MDA-MB-231 0 5 10 15 20 25 30 35 0 20 40 60 Days CPDL Control +GRN163L HCC1937 0 10 20 30 40 50 60 0 20 40 60 Days C PDL Control +GRN163L HME50-T 0 10 20 30 40 50 60 0 50 100 150 200 Days CPDL Control +GRN163L SKBR3 control +GRN163L MCF-7 cells B control +GRN163L 21NT cells C control 21NT C C B control control +GRN163L
https://openalex.org/W4214903654	http://www.econ.uniurb.it/RePEc/urb/wpaper/WP_14_01.pdf	English	null	Industrial Policy in Europe	Routledge eBooks	2,003	public-domain	7,689	ISSN 1974-4110 (on line edition) ISSN 1594-7645 (print edition) ISSN 1974-4110 (on line edition) ISSN 1594-7645 (print edition) ISSN 1974-4110 (on line edition) ISSN 1594-7645 (print edition) WP-EMS Working Papers Series in Economics, Mathematics and Statistics • Mario Pianta (University of Urbino and Centro Linceo Interdisciplinare, Accademia dei Lincei, Italy) WP-EMS # 2014/01 “AN INDUSTRIAL POLICY FOR EUROPE” • Mario Pianta (University of Urbino and Centro Linceo Interdisciplinare, Accademia dei Lincei, Italy) WP-EMS # 2014/01 WP-EMS # 2014/01 Affiliation: Postal address: Università di Urbino “Carlo Bo” Dipartimento di Economia, Società, Politica Via Saffi 42, 61029 Urbino (PU) Abstract After Europe’s long stagnation, a debate is emerging on how industrial capacity could be reconstructed. The paper reviews current EU policies and provides the rationale for a new industrial policy at the European level. Such public action could help address current macroeconomic, industrial, innovation, cohesion and environmental problems and would be crucial for the recovery of countries of the “periphery” that have been hit hardest by the crisis. A range of proposals for organising, implementing and funding a new industrial policy – focusing on selected economic activities - are presented, combining action at the European, national and local levels. Keywords Industrial policy, Public policy, Europe Mario Pianta Affiliation: Università di Urbino “Carlo Bo” and Centro Linceo Interdisciplinare, Accademia die Lincei Affiliation: Università di Urbino “Carlo Bo” and Centro Linceo Interdisciplinare, Accademia die Lincei (Table 1 here) The “centre” – Germany and few neighbours - has preserved its industrial base and increased its exports to the “periphery” and emerging countries. Current accounts of “periphery” countries have avoided major unbalances only because of the severity of the depression, resulting in large import reductions. As soon as a recovery arrives, the loss of domestic production capacity is likely to result in mounting trade imbalances for many EU countries; they could be addressed either by continuing austerity policies - depressing again incomes and imports - or by renewed capital inflows, further expanding private and public debt. In both cases, Europe’s “periphery” is unlikely to avoid a spiral of losses of income, jobs, production and exports. Such a reshaping of Europe’s economy is driven by the restructuring of the international systems of production controlled by large firms and is affected by national and EU policies. Operating in the pursuit of short term profits, market power and financial rents – and with no attention to increasing environmental constraints - firms’ responses to the crisis have included the following: drastic downsizing and plant closing; reduction of R&D, innovation and investment; emergence of hierarchical production systems with extensive outsourcing and offshoring both in Europe’s “periphery” and in emerging countries with cost advantages and a large potential for growth in domestic markets; consolidation and acquisitions, leading to more oligopolistic market structures. These negative consequences have been concentrated in the countries of the “periphery” where the recession has hit hardest.1 In a context where European macroeconomic policies resist pressures to end austerity, stimulate new demand and redistribute income, a generalised return to growth is unlikely. Private investment continues to be negatively affected by expectations of low demand by firms; world export growth has not returned to pre-crisis levels and remains important for Germany and few other European countries only. This means that without a substantial increase in public demand an end of the current stagnation is unlikely. With a prolonged stagnation, Europe is likely to develop a more polarised industrial structure; “weak” countries, regions, industries and firms are becoming weaker; the “centre” may be negatively affected by lower demand; all countries will end up with a reduced ability to develop new technologies and economic activities. 1. Stagnation and polarisation in Europe The crisis of 2008 has brought Europe to a stagnation. The continent has been divided between a slow-growing “centre” with financial and political power, and a “periphery” in depression, with no political influence, high public debt, high unemployment. This polarisation is evident in the data on real industrial production shown in Table 1. With 2008 values equal to 100, in 2013 only Germany, Austria and the Netherlands had an index that had suffered limited slumps during the recession and had returned to pre-crisis levels. Progress was made by Poland alone, reaching 118. Ireland has returned to a 2013 value of 99 after dramatic losses in the midst of the crisis. Most countries in Central and Northern Europe failed to recover; France, the UK, Sweden, and Denmark have 2013 values equal to 89, Finland is at 83 (in Finland and the Netherlands GDP has been falling in 2012 and 2013). Southern Europe has experienced a dramatic loss of industrial production; 2013 values are 88 for Portugal, 79 for Italy, 76 for Spain, 73 for Greece. As a result of the prolonged European crisis, a permanent loss of production capacity is taking place in most industries and most countries, with a major destruction of economic activities in the Southers “periphery”. JEL Classification codes E6, L5, O4 Mario Pianta is Professor of Economic Policy at the University of Urbino and is a member of the Centro Linceo Interdisciplinare of the Accademia Nazionale dei Lincei, Italy’s Academy of Sciences. He has been visiting scholar at the European University Institute, the London School of Economics, Université de Paris 1 Panthéon-Sorbonne. He works on economic growth, innovation and inequality. http://works.bepress.com/mario_pianta 1 1 2 1 Analyses of the evolution of European industries in the recession include WIIW 2013; Simonazzi, Ginzburg and Nocella, 2013; Reinstaller et al. 2013; Aiginger, 2014. 2. Five reasons for a new industrial policy There is no need, however, to accept such an outcome as inevitable. Europe is now facing multiple challenges – ending the depression; upgrading its economic structure with new job creating activities; extending public action and public goods provision after decades of privatisations; reducing the polarisation between “centre” and “periphery” emerging from the crisis; moving towards an ecological transformation of the economy and society. An important, well known and effective tool that could contribute to address all these challenges is a new Europe-wide industrial policy. p y In Europe, industrial policy has driven the highly successful expansion of production from the 1950s to the 1970s. Then industrial policy fell out of fashion as governments largely left decisions on the evolution of the economy to markets - that is, to large multinational firms - with waves of liberalisations and privatisation of public enterprises. The argument of such neoliberal policies was that markets are able to operate efficiently both in the short term - allocating given resources - and in the long term - when the challenge is developing new activities, resources and markets. Policies lost their selectivity and were limited to automatic “horizontal” mechanisms, such as across-the- board tax incentives for R&D or for the acquisition of new machinery, or incentives to producers and consumers of goods. The result has been a general loss of policy influence on the direction of industrial change and development in Europe. g p p A widespread rethinking on the importance of industrial policy – and of manufacturing industry itself - is now under way. In new industrialised countries extensive public policies have been effective in combining public and private efforts to develop knowledge, acquire technologies, invest in new activities and expand foreign markets. Chang (1994) has provided a restatement of the need for industrial policy; as argued by Rodrik (2008), the question is not whether industrial policy makes sense, but the way in which it can be carried out. Its relevance for emerging countries is discussed by Cimoli, Dosi and Stiglitz (2009) and by Stiglitz and Lin Yifu (2013); the case of Korea and Asia is investigated in particular by Lee (2013a, 2013b). The European context is examined by Coriat (2004), Pianta (2010), WIIW (2013) and Aiginger (2014). Mazzucato (2013) emphasises the need for a broad role of ‘transformative’ public action in innovation and industrial change. (Table 1 here) Without growth, change is more difficult; Europe as a whole could be stuck in its traditional economic trajectory – with sluggish markets, a heavy environmental burden, cosmetic attention to climate change, and growing inequality - while other 2 advanced and emerging countries may move faster towards new knowledge, new products and processes, new sources of employment, supported by faster demand dynamics. The policy targets of Europe 2020 and the broader opportunity to develop in Europe a new trajectory of growth based on environmentally friendly activities and greater social justice would become more difficult to pursue. advanced and emerging countries may move faster towards new knowledge, new products and processes, new sources of employment, supported by faster demand dynamics. The policy targets of Europe 2020 and the broader opportunity to develop in Europe a new trajectory of growth based on environmentally friendly activities and greater social justice would become more difficult to pursue. 2. Five reasons for a new industrial policy Even mainstream perspectives have paid attention to the mechanisms for controlling and targeting industrial policies (Aghion et al. 2011, 2012). Building on such a debate, I would argue that, in the context of the current stagnation, there are five major reasons for developing a new industrial policy in Europe. The first one is rooted in macroeconomics. Exiting the current stagnation requires a substantial increase in demand, that could come from a Europe-wide investment plan driven by public policies, as argued by a growing range of voices (see below). The second one is associated with the changes in Europe’s economic structure resulting from the crisis; major losses are taking place in troubled industries, a downsizing is needed of the inflated financial sector and no new large economic activities that could offer new useful products and services and provide new employment are emerging. A EU-wide industrial policy could drive the rise of new environmentally sustainable, knowledge intensive, high skill and high wage economic activities. Specific activities that could be targeted include: a) the protection of the environment, sustainable transporation, energy efficiency and renewable energy sources; b) the production and dissemination of knowledge, applications of ICTs and web-based activities; c) health, welfare and caring activities. 3 Third, a new EU-wide industrial policy is needed in order to reverse the massive privatisations of past decades; an economy based on private, market based activities, with decisions left to the short term interests of firms – where finance is playing a dominant role - has failed to sustain investment, employment and environment-friendly growth. The new activities outlined above require a substantial action by the public sector – at the EU, national and local level - in setting priorities, investing, creating employment. Public action could provide direction and support to private activities – including the development of competences and entrepreneurship, access to capital, the organisation of new markets, etc. - and could directly produce public goods, such as knowledge, environmental quality, wellbeing, social integration and territorial cohesion. The need for greater cohesion and reduced imbalances within the EU and individual countries is the fourth reason for a new EU-wide industrial policy. Current changes in Europe’s industrial structure open up a growing divide between a relatively strong “centre” and a “periphery” where a large share of industrial capacity is being lost. 2. Five reasons for a new industrial policy This leads to deepening imbalances within the EU - and within individual countries - in terms of knowledge base, investment, trade, employment and incomes. A EU-wide industrial policy could have a specific aim of reducing such imbalances, concentrating action in the countries of the “periphery” and on the less favoured regions of the “centre”. Fifth, a new EU-wide industrial policy could become a major tool for addressing the urgent need for an ecological transformation of Europe. Turning Europe into a sustainable economy and society - reducing the use of non renewable resources, developing renewable energy sources and energy efficiency, protecting ecological systems and landscapes, lowering CO2 and other greenhouse gas emissions, reducing waste and generalising recycling - goes well beyond the emergence of specific environmentally friendly new activities; it is a transformation that concerns the whole economy and society. A combination is needed of direct public action with provision of environmental services, and appropriate regulations for private activities, including environmental taxation, incentives, public procurement and organisation of new markets. A new EU-wide industrial policy could provide the framework for integrating the different policy tools needed for making Europe sustainable. With a pioneering role along the road to ecological transformation, Europe could also substantially increase its role at the global level. Industrial policy can be an important and flexible tool for addressing all these priorities. In order to implement it effectively, there is a need for new institutional arrangements and funding sources, new mechanisms of accountable governance, efficient and effective operation, systematic links between the EU, national and local levels, as well as forms of democratic control with participatory practices. But let us consider first the current policies carried out by the European Union in this field. 3. Europe’s missing industrial policy Industrial policy has long had a marginal role in Europe’s policies. European Union policies on the evolution of economic activities are now framed in the Europe 2020 strategy, approved in June 2010 by the European Council. It provides the new framework for economic policy in Europe, replacing the Lisbon Strategy that was supposed to inspire Europe’s policies in the previous decade. In the Lisbon Strategy the EU set the goal “to become the most competitive and dynamic knowledge-based economy in the world capable of sustainable economic growth with more and better jobs and greater social cohesion”. A comprehensive economic strategy was expected to be developed “preparing the transition to a knowledge-based economy and society by better policies for the information society and research and development (R&D), as well as by stepping up the process of structural reform for competitiveness and innovation and by completing the internal market; modernising the European social model, investing in people and combating social exclusion; sustaining the healthy economic outlook and favourable growth prospects by applying an appropriate macro-economic policy mix”. As pointed out by Lundvall and Lorenz (2011), after the 4 mid-term evaluation of 2004-05 – and with right-wing governments replacing centre-left majorities in most European countries - the EU strategy was scaled down and focused on neoliberal policies for employment and economic growth. The Europe 2020 strategy follows this same trajectory identifying three priorities: ‘smart growth’: an economy based on knowledge and innovation; ‘sustainable growth’: a resource efficient, greener and more competitive economy; and ‘inclusive growth’ a high-employment economy with social and territorial cohesion. By 2020 the EU is expected to reach five “headlines targets” through a wide range of actions at the national and EU level, but the specific policy tools for achieving such goals appear limited. Eight “flagship” initiatives are associated to priority themes for re-launching Europe (European Commission, 2010a). p ( p ) The specific targets identified by Europe 2020 follow the footsteps of the Lisbon Agenda. The target of devoting 3% of EU GDP to R&D expenditure is maintained. In 2008, R&D in EU-27 amounted to 2.1%, with a highly uneven distribution across countries and no sign of convergence. Since then, the recession has led to falling expenditures and greater disparities. 3. Europe’s missing industrial policy Innovation capacity should be supported by the formation of human capital: the share of early school leavers should be under 10% in 2020 (it was 14,4% in 2009 in EU-27) and at least 40% of the younger generation should have a tertiary degree (32,2% in 2009 in EU-27). Again, progress towards such goals has been highly uneven and the recession has rolled back advances in “periphery” countries. g y p p y The strategy includes a set of indicators from the 20/20/20 climate/energy targets established in 2009 by the European Council. The first one is the 20% reduction of emissions by 2020 on the levels of 1990 (enlarged to 30% “if the conditions are right”); in 2009, the EU level has declined by 17%, largely due to the economic crisis that has deeply reduced output as well as emissions. The second target is the reduction of 20% in the use of renewable sources (in 2008, it was 10.3%); the third one is a rise of 20% in energy efficiency, with a move towards clean and efficient production systems the potential to create millions of jobs. The two “flagship” initiatives devoted by Europe 2020 to innovation and industrial policy include the “Innovation Union” (European Commission, 2010b) and “An integrated industrial policy for the globalization era” (European Commission, 2010c). The aim is to provide the best conditions for business to innovate and grow, as well as to support the transformation of the manufacturing system towards a low-carbon economy. As in the Lisbon agenda, industrial policy is based on a “horizontal” approach, where the main policy tools are the provision of infrastructures, the reduction of transaction costs across the EU, a more appropriate regulatory framework favouring competition and access to finance. A significant role is ascribed to the ability of small and medium enterprises to promote growth and create employment. Key issues include the need to fight protectionism, increase the flows of goods, capital and people within and outside the EU, to exploit a more open single market for services, to benefit from globalization. This strategy confirms the rejection by EU policy – first emerged in the 1980s - of targeted industrial policies and state action for developing particular sectors, choosing a market driven approach. Selective industrial policies continue to be considered ineffective by the EU, due to the difficulty of fine-tuning actions and evaluating results (Lerner, 2009). 3. Europe’s missing industrial policy When the crisis started in 2008 and austerity policies were imposed on Euro-area countries, the emphasis on fiscal consolidation and macroeconomic coordination has sidelined any serious discussion on industrial policy. Europe 2020 is now in line the neoliberal view that economic growth can be supported by the operation of markets and that fiscal consolidation and debt reduction create appropriate conditions for long term growth. Europe 2020 only suggests more resources for “growth-enhancing items” such as education, R&D and innovation, at the expense of social expenditure, that is considered to be unsustainable (European Commission, 2010a, 2010c). Such view has become explicit in the policy directives imposed in 2011 on weaker countries of the “periphery” of Europe - Greece, Portugal and Spain in particular – as conditions for granting them financial help facing their debt crisis. Cuts in government expenditures, public sector jobs and When the crisis started in 2008 and austerity policies were imposed on Euro-area countries, the emphasis on fiscal consolidation and macroeconomic coordination has sidelined any serious discussion on industrial policy. Europe 2020 is now in line the neoliberal view that economic growth can be supported by the operation of markets and that fiscal consolidation and debt reduction create appropriate conditions for long term growth. Europe 2020 only suggests more resources for “growth-enhancing items” such as education, R&D and innovation, at the expense of social expenditure, that is considered to be unsustainable (European Commission, 2010a, 2010c). Such view has become explicit in the policy directives imposed in 2011 on weaker countries of the “periphery” of Europe - Greece, Portugal and Spain in particular – as conditions for granting them financial help facing their debt crisis. Cuts in government expenditures, public sector jobs and 5 wages, liberalisation of labour markets and reduced workers’ protection have been key elements of the austerity plans imposed on these countries, with the result of worsening the recession, industrial decline and unemployment. The major losses in industrial production since the start of the crisis - documented in Table 1 -, however, have led the European Commission to introduce in January 2014 a new policy initiative called “Industrial Compact”, establishing the “target” of returning industrial activities to 20% of GDP by 2020, against the present 16% (European Commission, 2014a). German – and, to a lesser extent, Italian – industry and governments lobbied for such an action, which remains entirely within the Europe 2020 approach described above. 3. Europe’s missing industrial policy The only novelties include the call to support investment in fast growing, high value added industries such as energy efficiency, green industries and digital technologies, and the consideration of industrial research among the aims of already existing EU initiatives, such as the Horizon 2020 R&D programme, the Competitiveness of Enterprises and Small and Medium-sized Enterprises (COSME), and the Structural Funds (including national co-financing). Greater attention is also emerging towards the need to act at the EU level on climate change and energy, but again little additional resources are available and no change in the approach to industrial policy is in view (European Commission, 2014b). g pp p y ( p ) A major policy development in Europe, however, has emerged in 2013 with the negotiations for the Transatlantic Trade and Investment Partnership (TTIP) with the United States. The Treaty would move Europe further ahead along the road of trade liberalisation – the very process that has led to a more unbalanced and hierarchical industrial stucture. More importantly, it would offer a very strong protection for private foreign investment and scale back the scope for public policy and regulation in major fields, including environmental rules, GMOs, utilities and other public services. In fact, TTIP is bringing back the agenda of the Multilateral Agreement on Investment that was discussed at the OECD in the late 1990s and was stopped by the opposition of France and by mounting global mobilisations.2 TTIP has come under increasing criticism, and its future is uncertain. If it were approved, the scope for a European industrial policy would be definitely reduced, and the space for public action in the economy reduced to a minimum. The overall policy by the European Union has continued to disregard the seriousness of industrial decline and to rely on a policy frame where the priority is given to market liberalisation. Even after the dramatic effects of the crisis, ‘horizontal’ actions remain the main forms of ‘allowed’ public intervention, and no significant EU-wide resources have been made available to members states. Moreover, even the very mild tools of present EU industrial policies have lacked an adequate governance mechanism; industry lobbies exert a major influence and there is a lack of democratic processes and broad participation in decision making - a weakness that, unfortunately, is found in all fields of the present model of European integration. 6 2 On TTIP and the expected economic benefits see CEPR (2013); a critical review is in EuroMemo Group (2014, ch.7). On global activism on trade and investment see Utting, Ellersiek and Pianta (2012) and Pianta (2014). 4. How can we change what is produced? A different policy perspective is needed, addressing at the European level the joint needs to end the depression and rebuild sustainable economic activities in a less polarised continent. Decisions on the future of the industrial structure in Europe have to be brought back into the public domain. A new generation of Europe-wide industrial policy has to overcome the limitations and failures of past experiences - such as collusive practices between political and economic power, heavy bureaucracy, and lack of accountability and entrepreneurship. They should be creative and selective, with mechanisms of decision making based on the priorities for using public resources that are more democratic, inclusive of different social interests, and open to civil society and trade union voices. They have to introduce new institutions and economic agents, and new rules and business practices that may ensure an effective and efficient implementation of such policies. 6 The general principles of industrial policy are simple enough. It should favour the evolution of knowledge, technologies and economic activities towards directions that improve economic performances, social conditions and environmental sustainability. It should favour activities and industries characterised by learning processes – by individuals and in organisations -, rapid technological change, scale and scope economies, and a strong growth of demand and productivity. An obvious list would include activities centred on the environment and energy; knowledge and information and communication technologies (ICTs); health and welfare. Environment and energy: The current industrial model has to be deeply transformed in the direction of environmental sustainability. The technological paradigm of the future could be based on "green" products, processes and social organisations, that use much less energy, resources and land, have a much lighter effect on climate and eco-systems, move to renewable energy sources, organise transport systems beyond the dominance of cars with integrated mobility systems, rely on the repair and maintenance of existing goods and infrastructures, and protect nature and the Earth. Such a perspective raises enormous opportunities for research, innovation and new economic and social activities; a new set of coherent policies should address these complex, long-term challenges. Knowledge and ICTs: Current change is dominated by the diffusion throughout the economy of the paradigm based on ICTs. Its potential for wider applications, higher productivity and lower prices, and new goods and social benefits should be supported. 4. How can we change what is produced? However, ICTs and web-based activities are reshaping the boundaries between the economic and social spheres, as the success of open source software, copyleft, Wikipedia and peer-to-peer clearly show. Policies should encourage the practice of innovation as a social, cooperative and open process, easing the rules on the access and sharing of knowledge, rather than enforcing and restricting the intellectual property rules designed for a previous technological era. Health and welfare. Europe is an aging continent with the best health systems in the world, rooted in their nature of a public service outside the market. Advances in care systems, instrumentation, biotechnologies, genetics and drug research have to be supported and regulated considering their ethical and social consequences (as in the cases of GMOs, cloning, access to drugs in developing countries, etc.). Social innovation may spread in welfare services with a greater role of citizens, users and non-profit organisations, renewed public provision and new forms of self-organisation of communities. All these fields are characterised by labour intensive production processes and by a requirement of medium and high skills, with the potential to provide "good" jobs. But how could Europe change its economic activities in such directions? Industrial policy has long relied on different mechanisms. On the supply side, public funds have supported selected R&D, innovation and investment efforts. Public investment banks and public enterprises – as well as non profit foundations – have supported business start-ups in key fields with credits and venture capital and managed the restructuring of major production activities. Public, community and cooperative enterprises have a role in fields - such as knowledge-based activities, environmental and local services - where public goods and public procurement are prevalent. On the demand side, far-sighted public procurement, the organisation and regulation of markets with high growth potential, and support and incentives for early users of new technologies have helped “pull” innovation and investment through “mission oriented” policies (see Mazzucato, 2013, for a comprehensive review of recent initiatives). Similar policy tools have in some cases shifted production and consumption towards more sustainable patterns; in Europe the diffusion of wind and 7 solar energy is the result of the use of such instruments. In fewer cases policies have “empowered the users”, letting them define specific applications of existing technologies that may lead to new goods and services with large markets. 4. How can we change what is produced? Finally, policies have aimed at building closer relationships among all actors of national and European systems of innovation - firms, financial institutions, universities and policy makers - helping to coordinate decisions of public and private actors. The funding for such policies have generally come from national public expenditures, the granting of public capital to state banks and enterprises, and from financial markets through bonds with various degrees of public guarantee. Austerity policies, EU constraints and pressure for fiscal consolidation on national public budgets mean that different types of funding have now to be developed, with a focus on European-level initiatives. solar energy is the result of the use of such instruments. In fewer cases policies have “empowered the users”, letting them define specific applications of existing technologies that may lead to new goods and services with large markets. Finally, policies have aimed at building closer relationships among all actors of national and European systems of innovation - firms, financial institutions, universities and policy makers - helping to coordinate decisions of public and private actors. 5. A proposal for a new Europe-wide industrial policy The need for rebuilding and restructuring economic activities in Europe has recently led to a series of policy proposals. The German trade union confederation DGB has proposed “A Marshall Plan for Europe” (DGB, 2013), envisaging a public investment plan of the magnitude of 2% of Europe’s GDP per year over 10 years. Along the same lines the European Trade Union Confederation has developed the proposal of “A new path for Europe” (ETUC, 2013). Previous proposals were developed in Pianta (2010), Lucchese and Pianta (2013), EuroMemo Group (2013). p p Building on such a debate – and on previous experiences in Europe - we can argue that an ambitious but realistic proposal for a new industrial policy in Europe could be developed on the basis of the following institutions, governance mechanisms and funding arrangements. The institutional arrangements The new industrial policy has to be firmly set within the European Union and – if required – within the institutions of the Euro-zone. This is needed in order to coordinate industrial policy with macroeconomic, monetary, fiscal, trade, competition and other EU-wide policies, providing full legitimation to public action at the European level for influencing what is being produced (and how). Major changes are required in current EU regulations, in particular the ones that prevent public action from “distorting” the operation of markets. The expansion of economic activities that markets are unable to develop should become an explicit objective of EU policy. The EU level is crucial also for funding such policy (see below). As this policy is likely to meet opposition by some EU countries, a “variable geometry” EU policy could be envisaged, excluding the countries that do not wish to participate. A close integration has to be developed between the European dimension - providing policy coherence, overall priorities and funding -, the national dimension – where public agencies have to operate and an implementation strategy has to be defined - and the local dimension – where specific public and private actors have to be involved in the complex tasks associated to the development of new economic activities. Existing institutions could be renewed and integrated in such a new industrial policy, including – at the EU level – Structural Funds and the European Investment Bank (EIB). However, their mode of operation should be adapted to the different requirements of the role here proposed. While in the short term adapting existing institutions is the most effective way to proceed, in the longer term there is a need for a dedicated institution – either a European Public Investment Bank, or a European Industrial Agency - coherent with the mandate of reshaping economic activities in Europe. A system could be envisaged where EU governments and the European Parliament agree on the guidelines and funding of industrial policy, calling the EU Commission to implement appropriate policy tools and spending mechanisms. In each country a specific institution – either an existing or a new one, either a National Public Investment Bank, or a National Industrial Agency – could 8 assume the role of coordinating the implementation of industrial policies at the national level, interacting with the existing national innovation system, policy actors, the financial sector, etc. The funding of industrial policy Funds for a Europe-wide industrial policy should come from Europe-wide resources. It is essential that troubled national public budgets are not burdened with the need to provide additional resources and that national public debt is not increased. The order of magnitude of the funding for an industrial policy programme that could address the challenges identified in section 2 above is the one suggested by the DGB plan and by the ETUC proposal – 2% of EU GDP over a period of 10 years, that is about €260 billion per year. As terms of reference, we can note that the European Central Bank provided in the period December 2011-March 2012 alone €1,000 billion of special funds to private banks at 1% interest rate, with no success in turning them into real investment; EU Structural Funds in the period 2007-2013 reached €347 billion; annual lending by the European Investment Bank is €65 to 70 billion per year. An investment effort of about 2% of EU GDP appears to be feasible – considering the size and power of European institutions - and would be big enough to compensate – at the macroeconomic level - for the lack of private investment and low exports, effectively ending Europe’s stagnation. Different funding arrangements could be envisaged. As suggested by the DGB proposal “A Marshall Plan for Europe” (DGB, 2012) - funds could be raised on financial markets by a new European Public Agency; funds could come from the Europe-wide receipts of a once-for-all wealth tax and from the newly introduced Financial Transactions Tax. Such tax income could help cover interest payments for the necessary projects that are not profitable in market terms. This arrangement would not burden domestic public finances and could visibly make the connection between policies for downsizing finance, taxing the rich, reducing inequality, and the industrial policy that could lead to new economic activities and jobs. An alternative may come from a deeper European fiscal reform, introducing a EU-wide tax on corporations, thus effectively eliminating fiscal competition between EU countries. Perhaps 15% of proceedings could go to fund industrial policy, public investment, knowledge generation and diffusion at the EU level; the rest could be transferred to the countries’ Treasuries. For the group of Euro-zone countries, financing through EMU mechanisms could be considered. The institutional arrangements More specific Agencies, Consortia or Enterprises, with a flexible status but a strong public orientation, could be created (or adapted, if already in place) for action at the local and regional level and for initiatives in particular fields. The institutions at the national and local level would take responsibility for spending decisions, identifying the private firms to be supported – either with low interest loans of with a share of ownership -, the projects to be developed, the new public activites that are required. And they would be subject to the strict monitoring described below. The governance system The different options outlined above are associated to different governance arrangements of EU- wide industrial policy. As an example, we can assume that a European Public Investment Bank or Agency – let us call it European Public Investment (EPI) – is created and similar organisations – National Public Investment (NPI) – act in each country. The European institution should be accountable to the European Parliament, who appoints its board where representatives from business, research organisations, trade unions, environmental civil society organisations should be included. No “revolving door” between industrial policy institutions and private firms and banks would be allowed. The European institution should engage in consultation with EU political, economic and social actors for developing its proposed industrial policy, that should be approved by the European Parliament. Funds would then become available, and could be assigned to national institutions and specific targets and activities. Funds could be used for a variety of activities, possibly in combination with private investment that could be attracted to the creation of new economic activities and markets. In particular, in each country the National Public Investment organisation could use the EU funds - for the economic activities outlined in section 4 - in the following ways: - fund R&D in universities, public and private institutions; - fund innovation and its diffusion in private and public organisations; - procurement programmes for innovative products relevant for public services; - minority ownership of new start-up firms in high risk, high innovation fields; the shares could then be sold if the start-ups are successful and attract private finance; y p p g g then be sold if the start-ups are successful and attract private finance; - fund and organise networks of innovators, producers and users in new activities, in order to consolidate economic relationships and create markets; p - continue to provide ‘horizontal’ support to firms with the existing policy instruments. The lessons from successful experiences outside Europe, such as ARPA-E in the US, the Brasilian Development Bank BNDES – discussed at length by Mazzucato (2013) – could lead to a more specific and effective forms of public action. Transparency in decisions would be required, monitoring and evaluation procedures – similar to those required by EU Structural Funds - would be arranged. g The same governance system could be introduced in the implementation of activities at the country level. The funding of industrial policy Eurobonds could be created to fund industrial policy; a new European Public Investment Bank could borrow funds directly from the ECB; the ECB could directly provide funds for industrial policy to the spending agencies concerned. Moreover, funding arrangements could be different according the relevance of the “public” dimension: a) the priority of public funds should go to public investment in non-market activities – such as public goods provision, infrastructures, knowledge, education and health; a) the priority of public funds should go to public investment in non-market activities – such as public goods provision, infrastructures, knowledge, education and health; b) public funds and long term private investment should be combined in funding new “strategic” market activities, such as the provision of public capital for new activities in emerging sectors; c) public support could stimulate financial markets to invest in private firms and nonprofit organisations developing “good” market activities that could more easily repay the investment. In all cases, the rationale for financing industrial policy cannot be reduced to the financial logic of b) public funds and long term private investment should be combined in funding new “strategic” market activities, such as the provision of public capital for new activities in emerging sectors; ) bli t ld ti l t fi i l k t t i t i i t fi d fit b) public funds and long term private investment should be combined in funding new “strategic” market activities, such as the provision of public capital for new activities in emerging sectors; c) public support could stimulate financial markets to invest in private firms and nonprofit organisations developing “good” market activities that could more easily repay the investment. In all cases, the rationale for financing industrial policy cannot be reduced to the financial logic of the “return on investment”. The benefits in terms of environmental quality, social welfare, greater c) public support could stimulate financial markets to invest in private firms and nonprofit organisations developing “good” market activities that could more easily repay the investment. In all cases, the rationale for financing industrial policy cannot be reduced to the financial logic of the “return on investment”. The benefits in terms of environmental quality, social welfare, greater 9 territorial cohesion, more diffused growth at the European level have to be considered, and the costs have to be shared accordingly. The governance system The National Public Investment organisation could identify partners - both private, nonprofit and public – operating at the local level and in specific policy fields, who could become key players in the implementation of specific investment programmes. The fields that could be eligible for such industrial policy programmes can be identified within the broad areas outlined in section 4 above. In order to reduce the scope for ‘pork barrel politics’, the countries and regions where such investments could be carried out have to be defined in advance, with the explicit aim to reduce the polarisation that is weakening the industrial base of Europe’s “periphery”. For instance, 75% of funds could go to activities located in “periphery” countries (Eastern and Southern Europe, plus Ireland); at least 50% of them should be devoted to the poorer regions of such countries; 25% could go to the poorer regions of the countries of the “centre”. These criteria for operation, transparency in decision making, accountability to the EU Parliament and citizens may contribute to overcome the collusion between industrial policy and economic and political power that has characterised past European and national experiences. Extensive public consultations and a democratic debate about what and how we produce could support these policy initiatives, building consensus and credibility for a EU-wide industrial policy. 10 Opening up a debate on industrial policy in Europe is an urgent task. A wide range of ideas and proposals have to be shared and discussed. The political obstacles for such a new industrial policy are indeed huge, and major changes would be required in order to implement it. But the results of such efforts could be very important – ending stagnation, creating new high wage jobs where they are most needed, greater EU cohesion and public action, progress towards an ecological transformation of Europe, greater democracy in economic decision making. References Aghion, P., Boulanger, J., Cohen, E. Rethinking industrial policy, Bruegel Policy Brief, Issue 2011/04, 2011. Aghion, P., Dewatripont, M., Du, L., Harrison, A., and Legros, P. Industrial Policy and Competition, NBER Working Paper No. 18048, 2012. Aiginger, Karl. Industrial Policy for a Sustainable Growth Path, WIFO Working Papers, No. 469, 2014. Botta, Alberto. The Road to Structural Convergence in the Eurozone. Industrial Policies in a future Eurozone Federal State. Paper for the EuroMemorandum conference, London September 2013. CEPR, “Reducing transatlantic barriers to trade and investment: an economic assessment”, Brussels: CEPR, 2013. Chang, Ha-Joon. The political economy of industrial policy. Basingstoke: Macmillan, 1994. Cimoli, Mario, Dosi, Giovanni and Stiglitz, Joseph (eds). Industrial policy and development, Oxford: Oxford University Press, 2009. Coriat, Benjamin. Politique de la concurrence et politique industrielle dans l’Union européenne. Un rééquilibrage est-il possible? Cahiers français. Paris: La Documentation française, 2004 DGB. A Marshall Plan for Europe: Proposal by the DGB for an economic stimulus, investment and development programme for Europe, 2012. http://www.dgb.de/themen/++co++d92f2d46-5590- 11e2-8327-00188b4dc422/# EuroMemo Group. EuroMemorandum 2014. The deepening divisions in Europe and the need for a radical alternative to EU policies, 2013. http://www.euromemo.eu/euromemorandum/euromemorandum_2014/index.html European Commission. Europe 2020. A strategy for smart, sustainable and inclusive growth, COM (2010) 2020 final, Brussels, 2010a. European Commission. Innovation Union, COM(2010) 546, Brussels, 2010b. European Commission. An integrated industrial policy for the globalization era, COM (2010) 614, Brussels, 2010c. European Commission. For a European Industrial Renaissance. COM (2014) 14/2, 2014a. European Commission. For a European Industrial Renaissance. COM (2014) 14/2, 2014a. 11 European Commission. A policy framework for climate and energy in the period from 2020 to 2030. COM (2014) 15, 2014b. European Trade Union Confederation. A new path for Europe: ETUC plan for investment, sustainable growth and quality jobs, 7 November 2013. http://www.etuc.org/sites/www.etuc.org/files/EN-A-new-path-for-europe_3.pdf Greenwald, Bruce and Stiglitz, Joseph. Industrial policies, the creation of a learning society and economic development. In Stiglitz, Joseph and Lin Yifu, Justin (eds), 2013. Independent Annual Growth Survey (IAGS). Second Report 2014. Paris: OFCE, ECLM, IMK, 2013. Lee, Keun. Capability failure and industrial policy to move beyond the middle-income trap: from trade-based to technology-based specialization. In Stiglitz and Lin Yifu (eds), 2013a. Lee, Keun. Schumpeterian Analysis of Economic Catch-up. Knowledge, Path-Creation, and the Middle-Income Trap. Cambridge: Cambridge University Press, 2013b. Lerner, J. Utting, Peter, Ellersiek, Anne and Pianta, Mario (eds). Global justice activism and policy reform in Europe. Understanding how change happens, London: Routledge, 2012. References Boulevard of Broken Dreams, Why Public Efforts to Boost Entrepreneurship and Venture Capital Have Failed and What to Do About It, Princeton: Princeton University Press, 2009. Lundvall, Bengt-Åke and Lorenz, Edward. From the Lisbon Strategy to Europe 2020; in: Morel, N., Palier, B., Palme, J. (eds.): Social Investment, London: Policy Press, 2011. Mazzucato, Mariana.The entrepreneurial state, London: Anthem Press, 2013. Pianta, Mario. Industrial and innovation policies in Europe; in: Watt, A./Botsch, A. (eds.): After the crisis: towards a sustainable growth model, Brussels, ETUI, 92-95, 2010. Pianta, Mario. “Slowing Trade: Global Activism Against Trade Liberalization”. Global Policy, vol.5, 2, pp.214-221, 2014. Pianta, Mario and Lucchese, Matteo. Industrial and innovation policies in the European Union; in: Garibaldo, F., Baglioni, M., Telljohann V., Casey C. (eds.): Workers, Citizens, Governance: Socio- Cultural Innovation at Work, Berlin, Peter Lang, 2012. Reinstaller, A., Hölzl, W., Kutsam, J., and Schmid, C. The development of productive structures of EU Member States and their international competitiveness, WIFO research study, 2013. Rodrik, Dani. Normalizing industrial policy, The International Bank for Reconstruction and Development/The World Bank, Commission on Growth and Development, Working Paper, no. 3, 2008. Simonazzi, Annamaria, Ginzburg, Andrea and Nocella, Gianluigi. Economic relations between Germany and southern Europe, Cambridge Journal of Economics, 37 (3):653-675, 2013. Stiglitz, Joseph and Lin Yifu, Justin (eds). The industrial policy revolution 1. The role of government beyond ideology. Basingstoke: Palgrave Macmillan, 2013. 12 Utting, Peter, Ellersiek, Anne and Pianta, Mario (eds). Global justice activism and policy reform in Europe. Understanding how change happens, London: Routledge, 2012. Vivarelli, M., Pianta, M. (eds.) The Employment Impact of Innovation, London: Routledge, 2000. Wade, Robert H. ‘Return of industrial policy?’, International Review of Applied Economics, 26(2), pp. 223-239. 2012. WIIW (2013) European Competitiveness Report. A ‘Manufacturing imperative’ in the EU - Europe’s position in global manufacturing and the role of industrial policy. Vienna: WIIW. WIIW (2013) European Competitiveness Report. A ‘Manufacturing imperative’ in the EU - Europe’s position in global manufacturing and the role of industrial policy. Vienna: WIIW. 13 Table 1 Real Industrial Production in Europe in 2013 Pre-crisis data for 2008 = 100 Germany 98 Austria 101 Poland 118 Netherlands 99 Ireland 99 France 89 United Kingdom 89 Sweden 89 Denmark 89 Finland 83 Italy 79 Portugal 88 Spain 76 Greece 73 Real output in mining, manufacturing, public utilities. Construction is excluded. Source: Eurostat, Unece. References Real Industrial Production in Europe in 2013 Pre-crisis data for 2008 = 100 Germany 98 Austria 101 Poland 118 Netherlands 99 Ireland 99 France 89 United Kingdom 89 Sweden 89 Denmark 89 Finland 83 Italy 79 Portugal 88 Spain 76 Greece 73 Real output in mining, manufacturing, public utilities. Construction is excluded. Source: Eurostat, Unece. 14
https://openalex.org/W3005786891	https://www.e3s-conferences.org/10.1051/e3sconf/201912405019/pdf	English	null	Development of an intelligent system for integrated management of hydroelectric cascade modes	E3S web of conferences	2,019	cc-by	3,958	A G Rusina1, Dzh Kh Khudzhasaidov2, O V Naumov3 and A N Gorlov4 A G Rusina1, Dzh Kh Khudzhasaidov2, O V Naumov3 and A N Gorlov4 1Novosibirsk State Technical University, Novosibirsk, Russia 2Tajik Technical University, Tajikistan 3Kazan State Power Engineering University, Kazan, Russia 4The Southwest State University, Kursk, Russia Abstract. The paper deals with an isolated electric power system (EPS) based on hydroelectric power plants. The analysis of the existing methods and approaches for investigation of modes of EPS which comprise hydroelectric power plants is presented. A mathematical model of a hydroelectric station cascade has been developed, which allows taking into account the hydraulic connection during calculation of electrical modes. A software tool for optimization the operating modes of hydroelectric power plants as part of the power system was developed. It uses redistribution the load between hydraulic units both inside the station and between hydroelectric complexes of cascade hydroelectric stations. The task of modeling power consumption and load graphs of EPS with specific properties, based on the application of artificial intelligence methods, is considered. electricity modes for a system of cascade of hydropower plants, to improve the efficiency of its operation. * Corresponding author: 311670@list.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 (http://creativecommons.org/licenses/by/4.0/). 1 Introduction Electric power systems (EPS), which contain hydroelectric power plants (HPPs) in their generating structures, are currently competitive power complexes. The characteristic features possessed by hydroelectric power plants play the decisive role. The question related to the task of HPPs optimization should be considered as system-wide and inextricably linked with the characteristic features of the entire fuel and energy complex (FEC). 3 Model of the modes’ optimization The power system of the Pamirs is a system in which 100% of electricity is generated by hydroelectric power stations, which makes it necessary to correct the mechanism of hydroelectric modes optimization in specific conditions. In such an energy system, fuel efficiency is a factor characterizing the efficient use of hydropower resource, i.e. the primary task is to optimally distribute the load between the system stations, while the natural variability and uncertainty of hydraulic processes are an objective reality. Thus, the amount of runoff and hydrograph cannot be affected, since this is a natural non-repeating process. If the domestic flow rate or the mode of usage the reservoir water resources changes, then the capability of hydroelectric power plants also changes. Thus, rational actions related to the optimal regime can lead to a significant increase in the production of electricity at hydropower plants. The composition of the main hydroelectric complex of the considered Pamir energy system is given in table 1. EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 icenses/by/4.0/). E3S Web of Conferences 124, 05019 (2019) SES-2019 E3S Web of Conferences 124, 05019 (2019) SES-2019 https://doi.org/10.1051/e3sconf/201912405019 2 Object of study Load stations Nj, MW Load of Units, MW 1 2 3 4 Qi, m3/s 1-7 1-7 – – – 2.2-10.1 7.5-13.5 4.5-6.5 3-7 – – 11-19.4 14-18.5 5-6.5 5-6.5 4-5.5 – 20.09-26.685 19-27.5 5-7 5-7 4-6.5 5-7 27.44-40.57 28 7 7 7 7 41.41 The joint operation of hydroelectric stations depends on the degree of flow regulation, installed capacity, cascade location of stations with hydraulic connections, Table 1. Characteristics of HPPs of the main hydroelectric complex of the Pamir EPS. № Name of HPP Installed power, MW Number of hydraulic units Calculated head, m Water consumption through the unit, m3/s 1 Pamir-1 28 4 79.6 10.1 2 Khorog 9 5 59 3.55 3 Namadhut 2.5 2 36 3.5 Table 1. Characteristics of HPPs of the main hydroelectric complex of the Pamir EPS. № Name of HPP Installed power, MW Number of hydraulic units Calculated head, m Water consumption through the unit, m3/s 1 Pamir-1 28 4 79.6 10.1 2 Khorog 9 5 59 3.55 3 Namadhut 2.5 2 36 3.5 Table 1. Characteristics of HPPs of the main hydroelectric complex of the Pamir EPS. Table 2. The optimal mode of Pamir-1, with H=79.6 m. Load stations Nj, MW Load of Units, MW 1 2 3 4 Qi, m3/s 1-7 1-7 – – – 2.2-10.1 7.5-13.5 4.5-6.5 3-7 – – 11-19.4 14-18.5 5-6.5 5-6.5 4-5.5 – 20.09-26.685 19-27.5 5-7 5-7 4-6.5 5-7 27.44-40.57 28 7 7 7 7 41.41 Table 2. The optimal mode of Pamir-1, with H=79.6 m. 9.81 i i i i N Q H =  , (1) In the program, this is analyzed by value d. With d = 0, performance is converted to consumption. For other values of d, this conversion is not performed. In the program, this is analyzed by value d. With d = 0, performance is converted to consumption. For other values of d, this conversion is not performed. 2 Object of study Depending on the selected step t, the number of calculation points l is calculated in the interval from power Pmin to Pmax of one unit: p Depending on the selected step t, the number of calculation points l is calculated in the interval from power Pmin to Pmax of one unit: The joint operation of hydroelectric stations depends on the degree of flow regulation, installed capacity, cascade location of stations with hydraulic connections, single location of hydroelectric power stations on various watercourses, equipment characteristics, etc. The task should be solved according to the criterion of maximum advantage of using all hydroelectric stations in the system, which requires consideration of their mutual influence. To distribute the load between the stations located in a cascade (Pamir-1 HPP and Khorog HPP), the dynamic programming method was also used, and the calculation results are presented in table 3. 1 min max + − = t P P l , (2) (2) (2) Depending on the obtained value l, the cost matrix of all aggregates B[i, j] is calculated for all fixed power values, where i is the unit number; j is the number of power values. For each power value, we select the unit, the flow of which is less: )} ( min{ ) ( i i s i P Q P f = , (3) (3) Table 3. Load distribution between stations. Table 3. Load distribution between stations. Load power, MW Stations load, MW Water consumption Pamir-1 Khorog Qi, m3/s 1-15 15 0 2.1-21 16 15 1 23 17-20 17-20 0 24.3-28 21 20 1 30.1 22-28 22-28 0 30.8-40.4 29-37 28 1-9 42.5-53.8 where Pmin ≤Pi ≤ Pmax, Ps is the station load. The value Qi(Ni) is determined by the points of the characteristics of any unit consumption of which is minimum for the corresponding power value. The calculation is performed starting from the minimum power station, which can be provided with two units to the maximum: max min 2 2 N P N s   . 2 Object of study At present, Pamir Energy operates 11 hydroelectric power plants, of which Pamir-1 and Khorog HPPs are the larger ones, as well as 9 small hydropower plants with a total installed capacity of 43.5 MW. It is worth noting that small hydropower plants are characterized by insufficient water supply during the winter months. In this regard, hydropower plants, working to a given schedule and using the river’s natural flow without redistribution in the daily context, cannot cover the load curve during periods of peak loads. Uncertainty of processes, development of the system and the importance of taking into account the time factor in the absence of tools allowing decision-making while planning and managing the EPS modes results in the necessity for creation an imitation model. This model should allow one to carry out a series of simulation calculations taking into account the time factor and make decisions on the analysis and planning of normal Characteristics of the units are presented in the form of consumption Qi=Qi(Ni) or performance ηi= ηi(Ni) at Hi=const. Calculations are made according to the expenditure characteristics, if one knows the performance data, it is recalculated according to the formula: E3S Web of Conferences 124, 05019 (2019) E3S Web of Conferences 124, 05019 (2019) SES-2019 https://doi.org/10.1051/e3sconf/201912405019 9.81 i i i i N Q H =  , (1) Table 2. The optimal mode of Pamir-1, with H=79.6 m. Load stations Nj, MW Load of Units, MW 1 2 3 4 Qi, m3/s Table 1. Characteristics of HPPs of the main hydroelectric complex of the Pamir EPS. № Name of HPP Installed power, MW Number of hydraulic units Calculated head, m Water consumption through the unit, m3/s 1 Pamir-1 28 4 79.6 10.1 2 Khorog 9 5 59 3.55 3 Namadhut 2.5 2 36 3.5 9.81 i i i i N Q H =  , (1) In the program, this is analyzed by value d. With d = 0, performance is converted to consumption. For other values of d, this conversion is not performed. Depending on the selected step t, the number of calculation points l is calculated in the interval from power Pmin to Pmax of one unit: 1 min max + − = t P P l , (2) Table 2. The optimal mode of Pamir-1, with H=79.6 m. 4 Modeling of electric consumption and load schedules of EPS 18.95 0.0043 8.8sin(0.5237 1.009) 2.59sin(1.047 0.785) P t t t = + + + + + + . (9) (9) The operation of electric power system as a whole depends on the accuracy of the energy balances. Load graphs in a power system are sequences of calculated values that reflect changes in power over a specific time interval. Over the years, the most common way to describe the process of power consumption is the time series. The change in daily, weekly and annual power consumption in this case is often modeled by dividing the time series into trend, periodic, and random components: The coefficients of the time series can be selected both expertly and using various methods. Within the framework of solving problems related to power consumption modeling, the application of three methods for finding optimal coefficients for the time series configuration was evaluated: the gradient method, particle digging method and bee digging method. To automate the selection process, a software implementation of the “Prediction” block has been developed, which allows determining time series coefficients based on the listed methods. ) ( ) ( ) ( ) ( t U t S t Q t X + + = , (6) (6) When analyzing power consumption using a time series by asking initial approximations, it is possible to reduce the modeling error using the gradient optimization method. This method will begin the process with the solution found by the DFT, and then improve the model, reducing its average error. The resulting model is characterized by the equation: where Q(t) is the trend-steady systematic changes; S(t) is the periodic component, i.e. fluctuations relative to the trend; U(t) is the irregular component, i.e. random noise. The initial information is data on electricity consumption (monthly average power values) for 8 years, in the periods from 2007-2011 and from 2014- 2016, i.e. there is some “gap” in the source data, which is an additional subject for assessment of the “sustainability” of the considered modeling methods. Analysis, modeling and forecasting of power consumption in energy issues are most often carried out on the basis of time series, written in general form by the following expressions: 18.95 0.0048 8.8sin(0.5239 1.009) 2.59sin(1.047 0.785) P t t t = + + + + + + . (10) (10) The gradient method is sensitive to initial approximations that may affect the resulting solution, and, therefore, the modeling error. 2 Object of study (4) (4) For each load value Ps there is at least f2, and the power of the second unit P2 varies from Pmin to Pmax: As it can be seen from table 3, when distributing the load between stations located in a cascade, for the effective use of hydro-resource, it is necessary to load the Pamir-1 HPP, since with such a distribution of load, it is possible to achieve the lowest water consumption. )} ( ) ( min{ ) ( 2 1 2 2 N P f N Q P f s s − + = . (5) (5) The determination of the optimal station mode is carried out using the reverse procedure. So, for the known power station it is determined by the optimal composition of the units and their power, which is clearly shown in table 2. To solve this problem, a software implementation of the “Optimization” block was created, which is based on the dynamic programming method. This unit is used in the work not only in terms of system and in-station optimization issues, but also during the simulation calculations of the normal modes of the power system. The block “Optimization” allows performing the distribution of load between the units of hydroelectric power plants in the automated mode, as well as to 2 2 E3S Web of Conferences 124, 05019 (2019) SES-2019 E3S Web of Conferences 124, 05019 (2019) https://doi.org/10.1051/e3sconf/201912405019 choose the composition of the units included in the operation. choose the composition of the units included in the operation. The phases of selected harmonics can be obtained from the phase spectrum, and the general model of power consumption is characterized by the equation: 4 Modeling of electric consumption and load schedules of EPS The model obtained by the particle swarm method is described by the following equation: 3 E3S Web of Conferences 124, 05019 (2019) https://doi.org/10.1051/e3sconf/201912405019 E3S Web of Conferences 124, 05019 (2019) SES-2019 18.9 0.0047 2.49sin(5.2382 2.135) 8.59sin(5.67 2.172) P t t t = + + + + + + , (12) (winter maximum and summer minimum) for all substations of the Pamir power grid. (12) As a result of the comparison, the dependences of changes in hourly capacities and their time shift are obtained: The model obtained by the particle swarm method is represented by the equation: )1 ( 5 ) ( .s . w .s − − = j P P P P i i i ji (14) )1 ( 5 ) ( .s . w .s − − = j Т Т Т Т i i i ji . (15) 19.25 0.0022 7.48sin(6.8 0.61) 0.11sin(4.57 0.25) P t t t = − + + − − + , (13) (14) (13) Comparison of the implemented methods is shown in table 4. (15) From table 4 it can be seen that the most accurate was the model obtained using the particle swarm method, while the swarm of bEPS showed the biggest error. It should be noted that in the absence of information for two years, all models showed a good ability to recover data. where j is month, i is the day time. where j is month, i is the day time. where j is month, i is the day time. The derived law of changing the configuration of the load graph allows one to determine the load power in every hour of any day (month) in each node of the load of the power system, which makes it possible to carry out simulation calculations not of the instantaneous values of normal EPS modes, but taking into account the time factor. The derived law of changing the configuration of the load graph allows one to determine the load power in every hour of any day (month) in each node of the load y y y ( ) of the power system, which makes it possible to carry out simulation calculations not of the instantaneous values of normal EPS modes, but taking into account the time factor. Table 4. Time series coefficients and average model error. Table 4. Time series coefficients and average model error. 4 Modeling of electric consumption and load schedules of EPS Coefficients DFT DFT+ Gradient Swarm of particles Swarm of bEPS a0, MW 18.95 18.95 18.9 19.25 a1, MW 0.0043 0.0048 0.0047 -0.0022 a2, MW 8.8 8.8 2.49 7.48 ɷ2, Hz 0.5237 0.5239 5.2382 6.8 φ2, ° 1.009 1.009 2.135 0.61 a3, MW 2.59 2.59 8.59 -0.11 ɷ3, Hz 1.047 1.047 5.76 4.57 φ3, ° 0.785 0.785 2.172 0.25 δ,% 6.95 6.94 6.8 11.38 A model of the Pamir power system was created in the Eurostag PVC, daily power generation and load schedule. Simulation of load graphs at each node allows simulating any modes of the electric power system depending on water resources. 4 Modeling of electric consumption and load schedules of EPS To reduce the error, the use of two methods of artificial intelligence was considered: a swarm of bEPS and a swarm of particles. The methods of swarm intelligences describe the collective behavior of various objects, each of which performs a number of simple functions, while interacting with other objects. The algorithm of swarm intelligence can be represented as a mathematical system: 0 1 2 2 2 3 3 3 ( ) sin( ) sin( ) P t a a t a t a t     = + + + + + + , (7) (7) (7) where a0 is the constant component; an is the amplitude; ω is the frequency; φ is the phase shift. The average model error is defined as the average value of the model deviation from the actual data for the entire sample: } O I, P, A, M, S, { = I S , (11) (11) where S is many agents; M is an object intended for the exchange of experience between agents (most often it is a certain matrix or vector to which all agents of the swarm have access and which is used in A); A are the rules of creation, modification, behavior of agents; P are parameters that are used in formulas from A; % 100 act mod act P P P − =  , (8) (8) where Pact is the actual power value, MW; Pmod is the value of power consumption, obtained using the model, MW. } , { fb 1 I I I = , A time series reflecting a change in power consumption always contains a trend showing steady systematic changes and a periodic component – seasonal fluctuations relative to the trend. To obtain its approximate model, one can apply the discrete Fourier transform (DFT). I1 is the system input to which the objective function and restrictions are applied; Ifb is input for feedback; } , { fb 1 O O O = , After applying the DFT, an array of complex numbers was obtained, each of which contains information about the corresponding harmonic of the original time series. O1 is output (the best-found solution to the problem); Ofb is output for feedback. Table 5. Average model errors for different EPS, in percent. Table 5. Average model errors for different EPS, in percent. Table 5. Average model errors for different EPS, in percen Power grid DFT DFT+G radient Swarm of particles Swarm of bEPS EPS Pamir 6.5 6.94 6.8 11.4 UES Siberia 4.5 4 2.38 10.8 As a result of the calculations, an unacceptable voltage drop was detected in some narrow power systems, which corresponds to the real situation in the Pamir power system Thus, for the considered objects, the adequacy of the methods is equivalent, i.e. the particle swarm method gives the smallest error, and the swarm of the bee’s method gives the greatest error. The model created using DFT shows a satisfactory error, which is significantly reduced by the gradient optimization performed sequentially with the DFT. 5 Imitations modeling of electric consumption and load schedules of EPS To simulate the joint control of the HPP cascade in the power system based on the transit flow for them, the functional capabilities of the Eurostag RVC were used to calculate the normal conditions. The maximum power limits developed in the speed control macro-model for the Pamir-1 and Khorog HPPs were used. To verify the universality of the considered methods for selecting coefficients, an additional object was considered – the unified energy system of Siberia (UES of Siberia), for which similar calculations were carried out. Comparative results of calculations for two objects are shown in table 5. To check the correct functioning of the developed model, verification was carried out by testing macromodels at various water consumption values, calculated idle discharges, and stations through turbines, station power, and each hydraulic unit power at specified water consumption, also taking into account the time of wave arrival. 6 Conclusion The performed analysis of the existing methods and approaches to the study of operating modes of power systems with cascading hydroelectric power plants showed that the existing tools do not fully provide the simulation calculations and decision-making on EPS modes planning taking into account their features. A mathematical model of a hydroelectric power plant cascade has been developed, which takes into account the hydraulic connections during calculations of electric The next task of the study of a simulation model development was to determine the load graphs in each node of the power system for analyzing the modes of its operation, taking into account the changes in parameters over time. In this case, the initial data were measurements of daily load graphs on regime days 4 4 E3S Web of Conferences 124, 05019 (2019) E3S Web of Conferences 124, 05019 (2019) SES-2019 https://doi.org/10.1051/e3sconf/201912405019 equipment parameters on the value of the equivalent resistance the intrashop power supply systems, Elektrika, 7, 2-6 (2013) equipment parameters on the value of the equivalent resistance the intrashop power supply systems, Elektrika, 7, 2-6 (2013) modes, which increases the efficiency of the hydro resources usage. The model of power consumption of the load graph was created using the methods of artificial intelligence, which allows simulation modeling of changes in power at each node at any time with discrete hour, day, and month. The simulation results confirm the adequacy of the model obtained, which allows it to be used in solving related problems of optimal water flow through the hydraulic system, taking into account hydrological limitations and external influences. References 1. Yu.M. Sidorkin, A.G. Rusina, M.Sh. Misrikhanov, T.A. Filippova, Hydropower: studies for universities, 640 (2012) 2. O.V. Petinov, E.F. Shcherbakov, Testing of electrical apparatus, 215 (1985) 3. V.A. Dulin, Methods for studying the reliability of low-voltage devices, 152 (1970) 4. T.T. Namitokov, Testing of low voltage apparatus, 248 (1985) 5. M.G. Koblenz, Study of electrical wear resistance power contacts of contactors when operating in intermittent mode, 59-71 (1962) 6. E.A. Konyukhova, Reliability of power supply to industrial enterprises, 92 (2001) 7. V.V. Shevchenko, E.I. Gracheva, Determination of electric power losses in shop networks with voltage up to 1000V, Industrial power engineering, 10, 33- 35 (2001) 8. Yu.S. Zhelezko, A.V. Artemyev, O.V. Savchenko, Calculation, analysis and rationing of electricity losses in electric networks (2003) 9. O.V. Fedorov, Assessment of influence parameters of electric equipment at a size of losses the electric power in intra factory networks of low voltage, 2nd Int. Conf. on Industrial Engineering, Applications and Manufacturing, CFP16F42-ART (2016) DOI: 10.1109/ICIEAM.2016.7911463 10. G.V. Butkevich, N.A. Belkin, N.A. Vedeshenkov, Electric erosion of high-current contacts and electrodes, 256 (1978) 11. A.N. Shpiganovich, A.A. Shpiganovich, E.P. Zatsepin, Features of the functioning multi-level power supply systems, News from higher educational institutions of the Black Earth Region, 3(53), 12-24 (2018) 12. A.N. Shpiganovich, A.A. Shpiganovich, To assess the reliability parameters of the equipment for power supply systems, News of the higher educational institutions of the Black Earth Region, 4(46), 48-56 (2016) 13. E.I. Gracheva, N.A. Kopytova, Analysis structure of systems shop power supply of the enterprises the machine-building industry, News of higher educational institutions. Power problems, 5/6, 73-78 (2011) 14. R. Holm, Electric contacts, 456 (1978) 15. E.I. Gracheva, A.R. Safin, A.V. Shagidullin, Modeling of the influence changes in electrical 5 5
https://openalex.org/W4289836337	https://journals.bahiana.edu.br/index.php/educacao/article/download/3831/4698	English	null	Cognitive competence: comparing learning between traditional classroom and active classroom	Revista Internacional de Educação e Saúde	2,022	cc-by	6,804	Samantha Bicalho de Oliveira Cavalier4 Glauce Dias da Costa5 Tiago Ricardo Moreira6 Para a coleta de dados, os autores utilizaram um instrumento de alta confiabi- lidade baseado na escala Likert de 4 pontos, contendo questões referentes às competências a serem desenvolvidas pelos alunos. O teste de normalidade de Kolmogorov-Smirnov foi realizado e o teste de Wilcoxon foi utilizado para identificar as diferenças entre os dois métodos. O nível de significância foi de 5%. RESULTADOS: A média atribuída a cada um dos dois métodos investigados foi significativamente maior (p <0,001) no Mapa Conceitual com Sala de Aula Invertida (3,38) do que na Sala de Aula Tradicional (2,75). CONCLUSÕES: Enquanto o Mapa Conceitual favorece o processo de aprendizagem significativa com compreensão, integração e assimilação dos conteúdos de forma autônoma e corresponsável pelos alunos, a Sala de Aula Tradicional baseia-se na memoriza- ção passiva dos conteúdos ministrada pelos professores. Por isso, sugere-se que o Mapa Conceitual com Sala de Aula Invertida, na percepção dos alunos, foi mais eficaz quando comparado à Sala de Aula Tradicional, por proporcionar a assimilação, interpretação e integração dos conteúdos. ABSTRACT \| INTRODUCTION: The new student profile has required new formats of teaching and learning, with interconnected knowledge based on a pedagogical practice founded on reflection OBJECTIVE: This study aimed to analyze how students evaluate what they learned using the Concept Map associated with the Inverted Classroom in the teaching, learning and assessment process compared to the Traditional Classroom. METHODS AND MATERIALS: A cross-sectional study was carried out with 90 students at a public university in Brazil from the years 2016 to 2018. For data collection, the authors used a high-reliability tool based on the Likert scale of 4 points containing questions regarding the competencies to be developed by the students. The Kolmogorov- Smirnov normality test was performed, and the Wilcoxon test was used to identify the differences between the two methods. The level of significance was 5%. RESULTS: The mean attributed to each of the two investigated methods was significantly higher (p <0.001) in the Concept Map with Flipped Classroom (3.38) than in the Traditional Classroom (2.75). CONCLUSIONS: While the Concept Map favors the process of meaningful learning with comprehension, integration, and assimilation of content in an autonomous and co-responsible form by the students, the Traditional Classroom is based on the passive memorization of the content given by the teachers. Samantha Bicalho de Oliveira Cavalier4 Glauce Dias da Costa5 Tiago Ricardo Moreira6 1,3-6Universidade Federal de Viçosa (Viçosa). Minas Gerais, Brazil. rosangelaminardi@gmail.com, rogerioprado777@gmail.com, samantha.cavalier27@gmail.com, glaucedcosta@gmail.com, tiagoricardomoreira@gmail.com 2Autora para correspondência. Universidade Federal de Viçosa (Viçosa). Minas Gerais, Brazil. emilynutufv@gmail.com Submitted 05/13/2021, Accepted 06/10/2022, Published 08/04/2022 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 ISSN: 2594-7907 ABSTRACT \| INTRODUCTION: The new student profile has required new formats of teaching and learning, with interconnected knowledge based on a pedagogical practice founded on reflection OBJECTIVE: This study aimed to analyze how students evaluate what they learned using the Concept Map associated with the Inverted Classroom in the teaching, learning and assessment process compared to the Traditional Classroom. METHODS AND MATERIALS: A cross-sectional study was carried out with 90 students at a public university in Brazil from the years 2016 to 2018. For data collection, the authors used a high-reliability tool based on the Likert scale of 4 points containing questions regarding the competencies to be developed by the students. The Kolmogorov- Smirnov normality test was performed, and the Wilcoxon test was used to identify the differences between the two methods. The level of significance was 5%. RESULTS: The mean attributed to each of the two investigated methods was significantly higher (p <0.001) in the Concept Map with Flipped Classroom (3.38) than in the Traditional Classroom (2.75). CONCLUSIONS: While the Concept Map favors the process of meaningful learning with comprehension, integration, and assimilation of content in an autonomous and co-responsible form by the students, the Traditional Classroom is based on the passive memorization of the content given by the teachers. Because of this, it is suggested that the Concept Map with a Flipped Classroom, according to students’ perception, was more effective when compared to the Traditional Classroom by providing contents assimilation, interpretation, and integration. KEYWORDS: Teaching assessment. University education. Teaching methods. Health education. Public health education. RESUMO \| INTRODUÇÃO: O novo perfil do aluno tem exigido no- vos formatos de ensino e aprendizagem, com saberes interligados a partir de uma prática pedagógica alicerçada na reflexão. Objeti- vo: O objetivo deste estudo foi analisar como os alunos avaliam o que aprenderam utilizando o Mapa Conceitual associado à Sala de Aula Invertida no processo de ensino, aprendizagem e avaliação em comparação com a Sala de Aula Tradicional. MATERIAIS E MÉ- TODOS: Estudo transversal realizado com 90 alunos de uma uni- versidade pública do Brasil nos anos de 2016 a 2018. How to cite this article: Cotta RMM, Ferreira ES, Prado REG, Cavalier SBO, Costa GD, Moreira TR. Cognitive competence: comparing learning between traditional classroom and active classroom. J. Educ. Health. 2022;6:e3743. http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 Original Article Original Article Original Article Cognitive competence: comparing learning between traditional classroom and active classroom Cognitive competence: comparing learning between traditional classroom and active classroom Competência cognitiva: comparando a aprendizagem entre sala de aula tradicional e sala de aula ativa Rosângela Minardi Mitre Cotta1 Emily de Souza Ferreira2 Rogério Elias Gomes do Prado3 1,3-6Universidade Federal de Viçosa (Viçosa). Minas Gerais, Brazil. rosangelaminardi@gmail.com, rogerioprado777@gmail.com, samantha.cavalier27@gmail.com, glaucedcosta@gmail.com, tiagoricardomoreira@gmail.com 2Autora para correspondência. Universidade Federal de Viçosa (Viçosa). Minas Gerais, Brazil. emilynutufv@gmail.com Samantha Bicalho de Oliveira Cavalier4 Glauce Dias da Costa5 Tiago Ricardo Moreira6 Rosângela Minardi Mitre Cotta1 Emily de Souza Ferreira2 Rogério Elias Gomes do Prado3 1,3-6Universidade Federal de Viçosa (Viçosa). Minas Gerais, Brazil. rosangelaminardi@gmail.com, rogerioprado777@gmail.com, samantha.cavalier27@gmail.com, glaucedcosta@gmail.com, tiagoricardomoreira@gmail.com 2Autora para correspondência. Universidade Federal de Viçosa (Viçosa). Minas Gerais, Brazil. emilynutufv@gmail.com Samantha Bicalho de Oliveira Cavalier4 Glauce Dias da Costa5 Tiago Ricardo Moreira6 Rosângela Minardi Mitre Cotta1 Emily de Souza Ferreira2 Rogério Elias Gomes do Prado3 1,3-6Universidade Federal de Viçosa (Viçosa). Minas Gerais, Brazil. rosangelaminardi@gmail.com, rogerioprado777@gmail.com, samantha.cavalier27@gmail.com, glaucedcosta@gmail.com, tiagoricardomoreira@gmail.com 2Autora para correspondência. Universidade Federal de Viçosa (Viçosa). Minas Gerais, Brazil. emilynutufv@gmail.com Samantha Bicalho de Oliveira Cavalier4 Glauce Dias da Costa5 Tiago Ricardo Moreira6 Samantha Bicalho de Oliveira Cavalier4 Glauce Dias da Costa5 Tiago Ricardo Moreira6 Introduction connecting students and teachers in the construction of knowledge and the development of competencies (knowledge, skills, and attitudes). Among these methods, we highlight and work in this study with the Concept Map using the Flipped Classroom, which aims to organize, represent and integrate knowledge in a collaborative way.11 The transformations arising from the new society of knowledge and the incorporation of Information and Communication Technology put in check not only the traditional way of teaching but also the learning process itself. The new student profile has required new formats of teaching and learning, with interconnected knowledge based on a pedagogical practice founded on reflection. In the twenty- first century, changes are required that demand different university agents (teachers, students, and managers) to act with audacity and courage in order to transform.1-3 In the context of competency learning, the emphasis of both methods (the Concept Map and Traditional Classroom) is on cognitive development (knowledge and content). Introduction However, while the Concept Map favors the process of meaningful learning with comprehension, integration, and assimilation of content in an autonomous and co-responsible form by the students, the Traditional Classroom is based on the passive memorization of the content given by the teachers.1,12 We need to implement formative processes with a humanistic focus centered on the human person, to overcome the technical-positivist (traditional) paradigm, towards the critical-reflexive (interpretative) paradigm, based on active participation.1,4-6 These transformations require a significant turnaround from the pedagogical, epistemological and psychosocial points of view, preparing the student for the dynamicity of life, which interferes with living conditions and the production of knowledge.5 In this study, the process of constructing the Concept Map was with a Flipped Classroom, a pedagogical approach by which the acquisition of knowledge is facilitated before its application in the classroom, where the students first study the content (at a distance) and then, in the classroom, discuss with their peers (other students) in a collaborative way, clarifying doubts and doing exercises, under the teacher’s guidance.13 The Concept Map, with a Flipped Classroom, provides active and meaningful learning, as well as the student's autonomy and student’s commitment to the teaching, learning, and evaluation process.3,14 In health, international guidelines recommend the formation of generalist, critical, reflexive, creative, humanized, and socially responsible professionals to work in universal, integral, and equitable systems, based on the demands of local communities, without losing sight of the global panorama.6-9 For the purposes of this study, effectiveness is understood as the effect of an activity (in the case of this study, a method) and its final results, benefits, and consequences for a particular group or study population (students), when compared to the established objectives and sustainability of the process and the initiative.15 In the case of our study, we call the effect of the use of teaching, learning, and assessment methods in the teaching of students in Nursing, Medicine, and Nutrition courses effective. In view of this, the active teaching, learning, and evaluation methods have been shown as strategies to stimulate the exercise of reflexive, critical, creative, and innovative professional development. This puts into question the need to carry out studies that evaluate the effectiveness of these methods, when compared to traditional ones, from the perspective of the main stakeholders of the learning process, that is, the students, since few studies in the literature consider the students' perception. Samantha Bicalho de Oliveira Cavalier4 Glauce Dias da Costa5 Tiago Ricardo Moreira6 Because of this, it is suggested that the Concept Map with a Flipped Classroom, according to students’ perception, was more effective when compared to the Traditional Classroom by providing contents assimilation, interpretation, and integration. PALAVRAS-CHAVE: Avaliação do Ensino. Ensino Superior. Méto- dos de Ensino. Ensino em Saúde. Educação em Saúde Pública. KEYWORDS: Teaching assessment. University education. Teaching methods. Health education. Public health education. Submitted 05/13/2021, Accepted 06/10/2022, Published 08/04/2022 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 ISSN: 2594-7907 Introduction Setting For data collection, we used a questionnaire called: Student Perception Assessment Tool – SPAT (see Annex 1), built to assess the perception of students of health courses on the teaching process, learning, and evaluation through the Concept Map with a Flipped Classroom (active classroom) and the traditional classroom (expository classes and exams). The overall reliability of the questionnaire was calculated using Cronbach's alpha coefficient and was 0,917. More specifically, the reliability of the concept map with a flipped classroom was 0,880, and the traditional classroom was 0,931, which is very high. According to the scale proposed by Prado (2015)16, all these values from 0,81 to 1,00 are considered very high. This study was developed for three years (2016, 2017, and 2018) with all 90 students of the 4th and 5th semesters of the Nursing, Medicine, and Nutrition courses that attended the discipline of Health Policies at the Federal University of Viçosa, a public university in Brazil. 40% of the contents were developed through the traditional classroom (expository classes and exams) and 60% through the active classroom (concept map and flipped classroom and other methods not deepened in this study); thus, all students experienced both methods and the same contents. The teaching and learning process referring to the traditional classroom is carried out individually, and the concept map and flipped classroom are in small groups (which are divided at the beginning of the school semester and remain the same until the end). The evaluation process also differs. In the active classroom, the evaluation is formative, occurring at six moments during the school semester, with assertive and timely feedback for correction of directions and achievement of the required competencies, followed by self-assessment by students and evaluation by teachers. The evaluation of the traditional classroom occurs in a punctual and summative way, using two exams, one in the middle of the semester and the other at the end, in addition to other exercises throughout the semester and lectures. The SPAT was built based on the Likert scale in which students identify their level of agreement from 4 propositions followed in ascending order: I totally disagree, disagree, agree, and totally agree. Methods Methods Setting The statements were constructed from the competencies necessary for the training of students in the health area and the learning objectives to be achieved with the methods analyzed.17-20 Sixteen questions were included, with 8 referring to the concept map with a flipped classroom and 8 on the traditional classroom. At the end of each semester, after completing all the activities of the discipline, the students were asked, anonymously, to answer the questionnaire, thus avoiding possible biases. In our study, we highlight the interprofessional nature of small groups. As we have three different courses, the orientation during the formation of the groups was that it should be as interprofessional as possible, that is, it should have at least one student from each course. This expands the learning and vision of colleagues who experience different areas, strengthening interprofessional teamwork, a common practice after the graduation of students at the university. Introduction This study aimed to analyze how students evaluate what they learned using the Concept Map associated with the Inverted Classroom in the teaching, learning, and assessment process compared to the Traditional Classroom. In the Traditional Classroom, it is understood that lectures are given by the teachers, whose methods of evaluation are quizzes or exams (summative evaluation).10 The Active Classrooms, in turn, play a central role in the learning process in a dialogical way, 2 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 Teaching, learning, and evaluation methods The evaluation process of the concept map follows the precepts of the formative evaluation: it is built at six specific moments in the classroom, throughout the school semester, and with a consecutive process of self-evaluation by students and evaluation and feedback by teachers. At the end of the semester, each team builds a single concept map containing the entire content of the discipline, so students gradually incorporate new concepts, and some key concepts become secondary to the extent that students assimilate them and new concepts. Table 1 shows the moments of the construction of the concept map with a flipped classroom. Performance assessment In addition to analyzing the students' perceptions through the answers of the questionnaires applied (SPAT), we also conducted a performance analysis. This performance was evaluated at each stage of the development of the concept map with a flipped classroom (six moments of construction and evaluation with assertive feedback during the school semester) using the Concept map Assessment Tool (see Annex 2). At the end of the school semester, the students present a single concept map of all discipline content, demonstrating the synthesis capacity and the integration of content. The Research Ethics Committee of the Federal University of Viçosa approved the study under protocol 2.230.939. Students who agreed to participate were informed about the study before signing the informed consent form. All participants signed the form. Minors were not included in this study. 3 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 realization of connections between the new knowledge studied and those already existing, the review of ideas, and the organization of the content autonomously. During the school semester, the same contents are worked both in the traditional classroom and in the active classroom, concomitantly, that is, the student is introduced and studies at the same time through both forms of teaching. The difference is the way they are approached from learning to evaluation (summative or formative), which allows us to compare which real learning was more effective. We measured the performance based on the entire teaching, learning, and evaluation process of students, throughout the school semester. In the discipline of policies and health, the concept map is collectively constructed (collaborative learning) by teams consisting of 5 to 7 students and is based on the chapters of the book used as a reference of the discipline, titled "Health Policy: Designs, Models and Paradigms".23 The construction of the concept maps occurs with the use of the CMap Tools Software, which is a free online available tool that allows the construction of concept schemes and represents them graphically. Performance assessment In the discipline of policies and health, the concept map is collectively constructed (collaborative learning) by teams consisting of 5 to 7 students and is based on the chapters of the book used as a reference of the discipline, titled "Health Policy: Designs, Models and Paradigms".23 The construction of the concept maps occurs with the use of the CMap Tools Software, which is a free online available tool that allows the construction of concept schemes and represents them graphically. Concept map and formative evaluation The concept map was developed by Novak (2008)21 as a graphical tool to organize and represent knowledge (cognitive dimension of competence), aiming to adapt greater understanding and assimilation of a particular theme or content. It is based on Ausubel's theory of Meaningful Learning (1980)22, which highlights that effective learning demands that knowledge be understood, significantly relevant, and integrated. For Cotta et al. (2015)5, the concept map enables the Table 1. Description of the Moments of Flipped Classroom Development and Concept Map Construction Table 1. Description of the Moments of Flipped Classroom Development and Concept Map Construction 4 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 Flipped Classroom The flipped classroom is a pedagogical approach by which students study the contents previously to the classroom. The classroom is designed for students to discuss with their peers (peer learning) and perform exercises under the tutoring and guidance of teachers.14,24 Anderson et al. (2001)25 presents a new structure of the revised Bloom's Taxonomy where the learning objectives are hierarchically classified, from which one can understand the difference between the traditional classroom and the flipped classroom. More specifically, in the traditional classroom, the easiest categories of Bloom's Taxonomy (remember and understand) are worked in the classroom, in the presence of the teacher. In turn, in the flipped classroom, the opposite occurs, students study the easiest categories at home, and in the classroom, in the presence of the teacher, students practice more complex cognitive processes (apply, analyze, evaluate and create) (Figure 1).26 earning objectives, according to the revised Bloom’s Taxonomy, worked in the traditional classroom and in the flipped classroom Figure 1. Learning objectives, according to the revised Bloom’s Taxonomy, worked in the traditional classroom and in the flipped classroom Source: Adapted from Bergmann 2018. Figure 1. Learning objectives, according to the revised Bloom’s Taxonomy, worked in the traditional classroom and in the flipped Source: Adapted from Bergmann 2018. Traditional classroom and summative evaluation In the traditional classroom, it usually occurs through lectures delivered by the teacher. In this model, students passively receive the content, memorize it, and then prove that they have learned through cognitive and punctual assessments. The reference is the Technicist-positivist model. This type of evaluation is usually called summative evaluation and aims to compare, categorize and classify students, here the final result expressed by means of grades is concerned.1,4 5 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 5 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 Results The skills worked on active methods can be seen in Graph 1, which addresses the questions provided in the SPAT questionnaire that all students answered at the end of the semester. These questions are the competencies that students develop when using the Concept Map and the Flipped Classroom for teaching, learning, and assessment. For each question presented in the graph (on the left), there is the percentage of agreement of all students. According to the frequency analysis, all competencies worked on in the Concept Map and the Flipped Classroom obtained a high approval rate (above 90%), with answers given 3 and 4 points (agree and totally agree, respectively). The students emphasize that the Concept Map contributes significantly to the exercise of the following competencies: "Facilitated the assimilation of the course content" (95,96%); "Facilitated the integration of the course content" (96,97%); "Encouraged teamwork" (88,89%); "Contributed to significant learning" (93,94%). The Graph 2 addresses the use of the traditional classroom; the indices were lower when compared with the d h fl d l l f h d f l d h h Graph 1. Students' perception using the Concept Map with the Flipped Classroom for learning Graph 1. Students' perception using the Concept Map with the Flipped Classroom for learning Graph 1. Students' perception using the Concept Map with the Flipped Classroom for learning According to the frequency analysis, all competencies worked on in the Concept Map and the Flipped Classroom obtained a high approval rate (above 90%), with answers given 3 and 4 points (agree and totally agree, respectively). The students emphasize that the Concept Map contributes significantly to the exercise of the following competencies: "Facilitated the assimilation of the course content" (95,96%); "Facilitated the integration of the course content" (96,97%); "Encouraged teamwork" (88,89%); "Contributed to significant learning" (93,94%). According to the frequency analysis, all competencies worked on in the Concept Map and the Flipped Classroom obtained a high approval rate (above 90%), with answers given 3 and 4 points (agree and totally agree, respectively). The students emphasize that the Concept Map contributes significantly to the exercise of the following competencies: "Facilitated the assimilation of the course content" (95,96%); "Facilitated the integration of the course content" (96,97%); "Encouraged teamwork" (88,89%); "Contributed to significant learning" (93,94%). The Graph 2 addresses the use of the traditional classroom; the indices were lower when compared with the concept map and the flipped classroom. Data analysis strategy From the questionnaire of Evaluation Tool for Active and Traditional Teaching, Learning and Assessment Methods, we assigned values (scores) from 1 to 4 for the answers to the questions about each method (1 - totally disagree, 2 - disagree, 3 - agree and 4 - totally agree). In the description of the quantitative variables, the mean and median were used as measures of central tendency and interquartile range (25 and 75%) as a measure of the dispersion of each method. The Kolmogorov-Smirnov normality test was performed, and the Wilcoxon test was used to identify the differences between the two methods. The level of significance was 5%. Statistical analysis was performed using SPSS software for Windows (Version 23.0; SPSS, Chicago). Results For example, 48,0% of the students felt unmotivated when using this method. 6 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 Our results indicate that students working with the Concept Map with a Flipped Classroom had a significantly higher degree of satisfaction when compared to the Traditional Classroom. Considering the sum of the minimum value (8 points) and maximum (32 points) of the adopted Likert scale, both the sum of the eight items that make up the tool and the average of them, presented higher medians when comparing the use of the Concept Map and Flipped Classroom with the Traditional Classroom (Table 2). Graph 2. Students' perception using the Traditional Classroom Graph 2. Students' perception using the Traditional Classroom Graph 2. Students' perception using the Traditional Classroom Graph 2. Students' perception using the Traditional Classroom Our results indicate that students working with the Concept Map with a Flipped Classroom had a significantly higher degree of satisfaction when compared to the Traditional Classroom. Considering the sum of the minimum value (8 points) and maximum (32 points) of the adopted Likert scale, both the sum of the eight items that make up the tool and the average of them, presented higher medians when comparing the use of the Concept Map and Flipped Classroom with the Traditional Classroom (Table 2). Our results indicate that students working with the Concept Map with a Flipped Classroom had a significantly higher degree of satisfaction when compared to the Traditional Classroom. Considering the sum of the minimum value (8 points) and maximum (32 points) of the adopted Likert scale, both the sum of the eight items that make up the tool and the average of them, presented higher medians when comparing the use of the Concept Map and Flipped Classroom with the Traditional Classroom (Table 2). Table 2. Concept Map and Flipped Classroom compare to the Traditional Method Table 2. Concept Map and Flipped Classroom compare to the Traditional Method Regarding Table 3, we made a comparison of learning, according to the student's perception, in the methods used: active (Conceptual Map and Flipped Classroom) and traditional (traditional classroom). Discussion The results of our study point to a better effectiveness in the development and exercise of cognitive competence, through the concept map and flipped classroom, than with the traditional classroom. The combination of the flipped classroom with the process of constructing the concept map was well evaluated by the students, mainly because they built the concept maps throughout the school semester and in the classroom in the presence of the teacher, who was available to guide them and discuss with them about important points. Our results also demonstrated the development of the habit of regularly studying, when they made the summaries of the book chapters and listed the key concepts, first individually followed, in a second moment, by the discussion with their respective teams. The results of this study show too that 90% of the students assessed the significant contribution of the Concept Map with the Flipped Classroom to the development of the competencies worked on in the Health Policy course. Comparing the two methods with the evaluation tool’s six competencies in common, the Concept Map provided better assimilation and integration of the content, and the students reported that they felt more motivated with the teaching, learning, and evaluation processes. On the other hand, 48% of the students did not feel motivated in the Traditional Classroom. The active classrooms are inserted in the context of the exercise of cognitive and/or metacognitive competencies. Thus, the exercise of reasoning, analysis, and synthesis, aims at the stimulation of thinking, understanding, and interpreting, leading to the improvement of strategies for the resolution of real-life problems.27 The integration of new knowledge into an existing student structure (meaningful learning) can help in the absorption, integration, and application of important content, as well as allowing them to learn and assimilate new concepts and relate them to each other.11 In our study, the Concept Map as an active classroom provided additional resources for the formative teaching and learning process through assertive and timely feedback from teachers, as well as constructing concrete strategies for the correction of direction and scope of the competencies outlined in partnership with the students, with a stimulus to self-evaluation of the teaching and learning process.28 In a study developed by Ho et al. (2014)11, besides students not resisting the use of the Concept Map, they also evaluated this method better than the Traditional Classroom. In another study by Hawkins et al. Results It shows that for each of the questions in common with the two methods, the students preferred active methods to the traditional one, with this difference being significant in all questions (p<0.001). 7 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 7 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 Table 3. Learning comparison between the Concept Map and Flipped Classroom with the Traditional Method Table 3. Learning comparison between the Concept Map and Flipped Classroom with the Traditional Method Conclusion In this way, the Concept Map and Flipped Classroom, to student's perceptions, are more adequate because they transform the teaching and learning praxis mechanistic and fragmented into innovative, critical, and reflexive, transforming educational experiences, facilitating the learning and comprehension of key concepts of the themes studied, besides providing connection between various content as well as teamwork stimulus with autonomy.5 In this way, the Concept Map and Flipped Classroom, to student's perceptions, are more adequate because they transform the teaching and learning praxis mechanistic and fragmented into innovative, critical, and reflexive, transforming educational experiences, facilitating the learning and comprehension of key concepts of the themes studied, besides providing connection between various content as well as teamwork stimulus with autonomy.5 Ethics approval and consent to participate The study was approved by the Ethics and Research Committee with Human Beings of the Federal University of Viçosa, and all the participants signed the Term of Free and Informed Consent. Discussion (2015)28, which implemented competency-based medical education, three scoring systems were used to assess the quality, the importance of propositions, and the complexity characteristics of the Concept Map. The authors reached the conclusion that this method can be reliably administered in education. 8 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 Both the studies by Ho et al. (2014)11 and Hawkins et al. (2015)28 concur with the results of the present research, showing that the use of the Concept Map was assessed by students as important for the acquisition of competencies related to Health Policies, especially cognitive ones. the methods are applied in a single school subject, which is why all students try all methods: active and traditional. On the other hand, our study was able to provide enough statistical power to adequately demonstrate significant differences between the two methods and the high reliability of the Cronbach alpha coefficient (α), from 0,880 for the Concept Map and Flipped Classroom and 0,931 for the Traditional Classroom, decreases possible biases of the study. A study carried out by Salah and Kassab (2016)12, whose objective was to evaluate the generalization and the sources of variance in evaluation scores of the Concept Map, obtained high reliability. This result upholds the results of our study, in which all the competencies addressed by the use of the Concept Map and Flipped Classroom obtained a score equal to or above 90%. Conclusion We evaluated and compared two methods whose emphasis is on the teaching and learning of cognitive competence, that is, in learning content and theoretical knowledge, one being an active and innovative method (Concept Map and Flipped Classroom) and the other technical-positivist (Traditional Classroom). The results of this study indicate that the use of Concept Map with a Flipped Classroom is superior to that of the Traditional Classroom by providing content assimilation and integration, teamwork stimulation, meaningful learning, and the development of autonomy. In a complementary way, the study developed by Schwartzstein (2017)29 also demonstrated that the Traditional Classroom, by placing the student in a situation of mechanical memorization and accumulation of information, may even be efficient from the teaching point of view (for the teacher), but it is not likely to be an effective way of learning (for students). Thus, the use of the Concept Map with the Flipped Classroom as an innovative and active classroom meets the needs and learning profile of the students in the 21st century by the following: stimulating the search for information autonomously and outside the classroom; strengthening the student-teacher and student-student bond and partnership; and providing continuous and timely feedback to make corrections. This active classroom, in addition to driving students to focus on more complex content, make the teaching and learning process more dynamic, and the teachers and students co-responsible for a project built and reconstructed constantly in partnership.3,12 We infer, therefore, that the Concept Map with a Flipped Classroom, when used in the context of formative teaching, learning, and evaluation, provides co-responsibility of the students and places them in a situation of the protagonist, having its strong point in the formative evaluation carried out longitudinally during the semester, with assertive and timely feedback, assisting in didactic planning and course correction. Authors' contributions 7. Delors J. La educación encierra un tesoro. Madrid: Santillana; 1996. 7. Delors J. La educación encierra un tesoro. Madrid: Santillana; 1996. 7. Delors J. La educación encierra un tesoro. Madrid: Santillana; 1996. 7. Delors J. La educación encierra un tesoro. Madrid: Santillana; 1996. Cotta RMM coordinated the study, participated in the data collection, the article's topic design, and review throughout its construction. Ferreira ES participated in the collection and analysis of the data and revision of the article during its production. Prado REG and Cavalier SBO participated in the collection and analysis of data and scientific writing. Moreira TR participated in the statistical analysis of the data and final review of the article. Costa GD participated in the review of the article during its production. All authors read and approved the final manuscript. 8. Frye AW, Hemmer PA. Program evaluation models and related theories: AMEE guide no. 67. Med Teach. 2012;34(67):288–99. https://doi.org/10.3109/0142159x.2012.668637 9. Conselho Nacional de Saúde (Brasil). Resolução MS/CNS nº 569, de 08 de dezembro de 2017 [Internet]. Brasília: Conselho Nacional de Saúde; 2017. Avaible from: https://conselho.saude.gov.br/ resolucoes/2017/Reso569.pdf 10. Blanco A. Desarrollo y evaluación de competencias en educación superior. Madrid: Narcea S.A. de Ediciones; 2009, p. 17-34. Acknowledgements We thank the Coordination for the Improvement of Higher Education Personnel - CAPES, Brazil for have supported related research projects and the research group of the Innovation Program in University Teaching. As a limitation of our study, we can highlight the small sample size of this study and the impossibility of using a control group and an intervention group since 9 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 References 13. Flipped Learning Network. Definition of Flipped Learning [Internet]; 2014. [uptaded 2014 mar. 12; cited 2019 jun. 01]. Available from: https://flippedlearning.org/definition-of-flipped- learning/ 1. Cotta RMM, Costa GD. Portfólio reflexivo: método de ensino, aprendizagem e avaliação. Minas Gerais: Editora UFV; 2016. 1. Cotta RMM, Costa GD. Portfólio reflexivo: método de ensino, aprendizagem e avaliação. Minas Gerais: Editora UFV; 2016. 2. Delphino FBB, Oliveira E, Felisbino AM, Sgorbissa ML, Souza DR. A utilização de metodologias ativas em cursos superiores para uma aprendizagem significativa. In: Jerez O, Silva C, organizators. Innovando en la educación superior: experiencias clave em Latinoamérica y el Caribe 2016-2017 (Volumen 3: Integración de TIC’s). Santiago: Facultad de Economía y Negocios, Universidad de Chile; 2017. pp. 67-77. https://doi.org/10.34720/7khq-d689 14. Chen F, Lui AM, Martinelli SM. A systematic review of the effectiveness of flipped classrooms in medical education. Med. Educ. 2017;51(6):585-97. https://doi.org/10.1111/medu.13272 15. Organización Mundial de la Salud. Evaluación de la promoción de la salud: principios y perspectivas. Metodologías para la promoción de la salud [Internet]. Washington, D.C.: OPS; 2007. 524 p. Available from: https://iris.paho.org/handle/10665.2/3070 15. Organización Mundial de la Salud. Evaluación de la promoción de la salud: principios y perspectivas. Metodologías para la promoción de la salud [Internet]. Washington, D.C.: OPS; 2007. 524 p. Available from: https://iris.paho.org/handle/10665.2/3070 3. Costa GD, Driessen E, Silva LS, Campos AAO, Costa TMT, Donateli CP, et al. Collective portfolio: assessment of teaching and learning in health undergraduate courses. Ciênc Saúde Colet. 2018;23(11):3779-787. https://doi.org/10.1590/1413- 812320182311.27072015 16. Prado LB, Avila NR, Llobet MP, Canut MTL, Rodriguez, SF, Lajara MAG. Escala de Autopercepción del Pensamiento Crítico en Alumnos de Enfermería [Internet]. III Congreso Internacional sobre Aprendizaje, Innovación y Competitividad (CINAIC); 2015 oct. 14-16; Madrid, Spain. [cited 2021 oct. 01]. Available from: http://138.4.83.137/dmami/documentos/liti/Actas_CINAIC_2015. pdf 4. Roget AD, Serés MVG. La práctica reflexiva: bases, modelos e instrumentos. Madrid: Narcea S.A. de Ediciones; 2014. 5. Cotta RMM, Costa GD, Mendonça ET. Critical and reflective portfolios: a pedagogical approach centered on cognitive and metacognitive skills. Interf. 2015;19(54):573-88. https://doi. org/10.1590/1807-57622014.0399 17. Zabalza MA. Competencias docentes del profesorado universitário: calidad y desarrollo profesional. 2a. ed. Madrid: Narcea S.A. de Ediciones; 2009. 6. Cotta RMM, Silva LS, Cotta RM, Cotta FM, Bastos MAP, Campos AAO, et al. The Conceptual Map as a tool for teaching and meaningful learning about the Unified Health System. J Manag Prim Health Care. 2015;6(2):264-81. https://doi.org/10.14295/ jmphc.v6i2.306 18. Lizarraga MLSA. Competencias cognitivas en Educación Superior. 2a. ed. Madrid: Narcea S.A. Competing interests No financial, legal or political competing interests with third parties (government, commercial, private foundation, etc.) were disclosed for any aspect of the submitted work (including but not limited to grants, data monitoring board, study design, manuscript preparation, statistical analysis, etc.). 11. Ho V, Kumar RK, Velan G. Online testable concept maps: benefits for learning about the pathogenesis of disease. Med Educ. 2014;48(7):687-97. https://doi.org/10.1111/medu.12422 12. Kassab SE, Fida M, Radwan A, Hassan AB, Abu-Hijleh M, O'Connor BP. Generalisability theory analyses of concept mapping assessment scores in a problem-based medical curriculum. Med Educ. 2016;50(7):730-37. https://doi.org/10.1111/medu.13054 References de Ediciones; 2010. 10 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 24. Cotta RMM, Ferreira ES. Mapas conceituais e aula invertida: benefícios para o processo de ensino e aprendizagem sobre as políticas de saúde. Rev Invest Educ Univ [Internet]. 2019;2(1):22- 32. Available from: http://revistas.educacioneditora.net/index. php/RIEU/article/view/26 19. Cotta RMM, Costa GD. Assessment instruments and self- evaluation of reflective portfolios: a theoretical-conceptual construction. Interf. 2016;20(56):171-83. https://doi. org/10.1590/1807-57622014.1303 20. Cotta RMM, Silva LS, Costa GD, Cotta FM, Cotta RM. Portfólio coletivo reflexivo: ferramenta potencializadora do trabalho em equipe, raciocínio crítico e tomada de decisões. Rev S Greg [Internet]. 2017;16:12-21. Available from: https://revista. sangregorio.edu.ec/index.php/REVISTASANGREGORIO/article/ view/416 25. Anderson LW, Krathwohl DR. A taxonomy for learning, teaching and assessing: a revision of Bloom’s Taxonomy of Educational Objectives. Nova York: Addison Wesley Longman; 2001. 26. Bergmann, J. Aprendizagem invertida para resolver o problema do dever de casa. Porto Alegre: Penso; 2018. 21. Novak JD, Cañas AJ. The theory underlying Concept Maps and how to construct them, Technical Report IHMC CmapTools 2006- 01. Florida Institute for Human and Machine Cognition [Internet]. 2006;(6):1-36. Disponível em: https://cmap.ihmc.us/publications/ researchpapers/theorycmaps/TheoryUnderlyingConceptMaps. bck-11-01-06.htm 21. Novak JD, Cañas AJ. The theory underlying Concept Maps and how to construct them, Technical Report IHMC CmapTools 2006- 01. Florida Institute for Human and Machine Cognition [Internet]. 2006;(6):1-36. Disponível em: https://cmap.ihmc.us/publications/ researchpapers/theorycmaps/TheoryUnderlyingConceptMaps. bck-11-01-06.htm 27. Daley BJ, Torre DM. Concept maps in medical education: an analytical literature review. Med Educ. 2010;44(5):440-48. https:// doi.org/10.1111/j.1365-2923.2010.03628.x 27. Daley BJ, Torre DM. Concept maps in medical education: an analytical literature review. Med Educ. 2010;44(5):440-48. https:// doi.org/10.1111/j.1365-2923.2010.03628.x 28. Hawkins RE, Welcher CM, Holmboe ES, Kirk LM, Norcini JJ, Simons KB, et al. Implementation of competency-based medical education: are we addressing the concerns and challenges? Med Educ. 2015;49(11):1086-102. https://doi.org/10.1111/medu.12831 29. Schwartzstein RM, Roberts DH. Saying goodbye to lectures in medical school - paradigm shift or passing fad? N Engl J Med. 2017;377(7):605-07. https://doi.org/10.1056/nejmp1706474 28. Hawkins RE, Welcher CM, Holmboe ES, Kirk LM, Norcini JJ, Simons KB, et al. Implementation of competency-based medical education: are we addressing the concerns and challenges? Med Educ. 2015;49(11):1086-102. https://doi.org/10.1111/medu.12831 28. Hawkins RE, Welcher CM, Holmboe ES, Kirk LM, Norcini JJ, Simons KB, et al. Implementation of competency-based medical education: are we addressing the concerns and challenges? Med Educ. 2015;49(11):1086-102. https://doi.org/10.1111/medu.12831 22. Ausubel DP, Novak JD, Hanesian H. Educational psychology. 2a. ed. New York: Holt, Rinehart and Winston; 1980. Inter. J. Educ. Health, Salvador, 2022;6:e3831 References 23. Cotta RMM, Costa GD, Mendonça ET. Reflective portfolio: a proposal for teaching and learning geared on competencies. Ciênc Saúde Colet. 2013;18(6):1847-56. https://doi.org/10.1590/ S1413-81232013000600035 23. Cotta RMM, Costa GD, Mendonça ET. Reflective portfolio: a proposal for teaching and learning geared on competencies. Ciênc Saúde Colet. 2013;18(6):1847-56. https://doi.org/10.1590/ S1413-81232013000600035 29. Schwartzstein RM, Roberts DH. Saying goodbye to lectures in medical school - paradigm shift or passing fad? N Engl J Med. 2017;377(7):605-07. https://doi.org/10.1056/nejmp1706474 11 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 11 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 Annexes Annexes Annex 1. Student Perception Assessment Tool (to be continued) Annex 1. Student Perception Assessment Tool (to be continued) Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 12 Annex 1. Student Perception Assessment Tool (conclusion) Annex 1. Student Perception Assessment Tool (conclusion) Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 13 Annex 2. Conceptual Map Assessment Tool (to be continued) Annex 2. Conceptual Map Assessment Tool (to be continued) Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 14 Annex 2. Conceptual Map Assessment Tool (continuation) Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 15 Attachments 2. Conceptual Map Assessment Tool (conclusion) p p ( ) Inter. J. Educ. Health, Salvador, 2022;6:e3831 http://dx.doi.org/10.17267/2594-7907ijeh.2022.e3831 \| ISSN: 2594-7907 16
https://openalex.org/W4281258181	https://jurnal.saburai.id/index.php/teknik/article/download/1497/1183	Indonesian	null	Analisa Perbandingan Rencana Anggaran Biaya Menggunakan Indeks Harga Satuan Pekerjaan Standar SNI 2008 Dan Standar BOW Pada Proyek Pembangunan Talud Pantai 1 Bintuhan	Teknika sains	2,022	cc-by-sa	3,737	Comparative Analysis of the Budget Plan Using the 2008 SNI Standard Work Unit Price Index and BOW Standards on the Bintuhan 1 Coastal Coast Construction Project Yan Juansyah1*, Dewi Fadilasari2, Joni Imron3 1Program studi Teknik Sipil, Fakultas Teknik, Universitas Malahayati, Lampung Email: 1juansyah1@yahoo.com, 2d.fadilasari@gmail.com, 3joniimron29@gmail.com Abstrak Rencana Anggaran Biaya (RAB) merupakan perhitungan besarnya biaya yang dibutuhkan untuk bahan dan upah. Di Indonesia terdapat metode untuk merencanakan harga satuan biaya anggaran proyek yaitu BOW (Burgelijke Openbare Werken) dan SNI (Standar Nasional Indonesia). Tujuan penelitian ini untuk mengetahui perbandingan rencana anggaran biaya antara metode perhitungan SNI dan perhitungan BOW, sehingga di peroleh RAB yang lebih efisien.Jenis penelitian yang dilakukan bersifat studi literatur dan studi Kasus. Penulis melakukan studi kasus pada Proyek Pembangunan Talud Pantai 1 Bintuhan untuk meneliti biaya konstruksi dalam hal ini penulis menganalisa dan menghitung ulang rencana anggaran biaya pembangunan Talud dengan membandingkan analisa harga satuan pekerjaan SNI dan analisa harga satuan pekerjaan BOW. Dari hasil perhitungan pada pembahasan tugas akhir tentang analisa perbandingan rencana anggaran biaya pembangunan talud Dengan menggunakan Metode BOW dan SNI. Hasil akhir dari penelitian menunjukan bahwa perhitungan biaya pembangunan Talud Pantai 1 Bintuhan dengan menggunakan metode BOW sebesar Rp 6.351.650.000,00, sedangkan hasil estimasi biaya menggunakan metode SNI sebesar Rp 4.894.800.000,00. Dari hasil perhitungan, perbandingan estimasi anggaran biaya antara metode BOW dan SNI yakni metode BOW lebih mahal sebesar Rp 1.463.400.000,00 dari metode SNI. Kata kunci: Analisa BOW, Analisa SNI, Rencana Anggaran Biaya, Talud, Manajemen Konstruksi Jurnal Teknika Sains Volume 07, Nomor 01, 2022 Jurnal Teknika Sains Volume 07, Nomor 01, 2022 Keywords: BOW analysis, SNI analysis, Budget Plan, Talud, Construction Management PENDAHULUAN selanjutnya dikalikan harga material dan upah yang berlaku pada saat itu[5]. Metode SNI merupakan pembaharuan dari analisis BOW 1921, bahwasanya analisis SNI merupakan anlisa BOW yang diperbaharui, sistem penyusunan mengguanakan analisis SNI hampir sama dengan menggunakan analisis BOW[6]. Prinsip yang mendasar pada metode SNI adalah, daftar koefisien bahan dan upah tenaga sudah ditetapkan untuk menganalisis harga atau biaya yang diperlukan dalam membuat harga satuan pekerjaan bangunan[7]. Dari kedua koefisien tersebut akan didapatkan kalkulasi bahan-bahan yang diperlukan dan kalkulasi upah. Komposisi perbandingan dan susunan material serta tenaga kerja pada satu pekerjaan sudah ditetapkan yang selanjutnya dikalikan dengan harga material dan upah yang berlaku dipasaran[8]. Dalam merencanakan anggaran suatu proyek diperlukan analisa harga satuan pekerjaan sebagai pedoman dalam menghitung rencana anggaran biaya[9]. Perkiraan jumlah material dan kebutuhan tenaga kerja dalam proses pekerjaan konstruksi memegang peranan cukup penting untuk control kualitas dan kuantitas pekerjaan, maka dari itu perlu ditinjau ulang analisa harga satuan anggaran biaya terutama pada Proyek Pembangunan Talud Pantai 1 Bintuhan. Dimana dalam penelitian ini akan menghitung analisa harga satuan pekerjaan dengan perbandingan antara hasil analisa SNI 2008 dan BOW. Rencana Anggaran Biaya (RAB) suatu gedung atau proyek merupakan perhitungan besarnya biaya yang dibutuhkan untuk material dan upah, serta biaya lain yang terkait dengan pelaksanaan bangunan atau proyek tersebut[1]. Sedangkan anggaran adalah harga bangunan yang diperhitungkan dengan cermat, tepat, dan memenuhi persyaratan[2]. Anggaran untuk jenis bangunan yang sama dapat bervariasi tergantung pada harga bahan dan upah tenaga kerja yang berlaku di setiap daerah. Tujuan pembuatan RAB adalah untuk menentukan harga suatu bagian atau benda kerja sebagai pedoman untuk mengeluarkan biaya selama masa konstruksi[3]. Selain itu, juga dimaksudkan agar gedung yang akan didirikan dapat dilaksanakan secara efektif dan efisien. Efektif dan efisien disini dimaksudkan agar kita dapat membangun gedung dengan perhitungan biaya yang akurat dan ekonomis, namun bangunan yang dihasilkan tetap berkualitas sesuai dengan standar yang berlaku. Sedangkan fungsi RAB adalah sebagai pedoman pelaksanaan pekerjaan dan sebagai sarana pengendalian pelaksanaan pekerjaan[4]. Melalui RAB ini kita dapat menghitung dan mengetahui secara pasti berapa biaya yang dibutuhkan untuk membangun sebuah gedung sesuai dengan permintaan pemilik proyek. Abstract The Cost Budget Plan (RAB) is used as a material for calculating the Cost Budget Plan (RAB). In Indonesia, there are methods for planning the unit price of project budget costs, namely BOW (Burgelijke Openbare Werken) and SNI (Indonesian National Standard). The purpose of this study is to determine the comparison of the budget plan between the SNI calculation method and the BOW calculation, so that a more efficient RAB is obtained. The type of research carried out is a literature study and case study. The author conducts a case study on the Coastal 1 Bintuhan Coastal Development Project to examine construction costs in this case the author analyzes and recalculates the budget plan for the construction of the Talud by comparing the analysis of the SNI work unit price and the BOW work unit price analysis. From the calculation results in the discussion of the final project on comparative analysis of the budget plan for the construction of the embankment by using the BOW and SNI methods. The final result of the research shows that the calculation of the cost of constructing the Coastal Channel 1 Bintuhan using the BOW method is Rp. 6,351,650,000.00, while the result of the estimated cost using the SNI method is Rp. 4,894,800,000.00. From the calculation results, the comparison of the estimated cost budget between the BOW method and the SNI method, namely the BOW method is more expensive by Rp. 1,463,400,000.00 than the SNI method. Jurnal Teknika Sains Volume 07, Nomor 01, 2022 Jurnal Teknika Sains Volume 07, Nomor 01, 2022 PENDAHULUAN Dalam menghitung Analisis Harga Satuan Pekerjaan (HSP) dapat dihitung dengan beberapa metode yaitu Metode BOW, dalam analisa BOW telah ditetapkan angka jumlah tenaga kerja dan bahan untuk suatu pekerjaan yang terdapat dalam metode BOW mencakup daftar koefisien upah dan bahan yang telah ditetapkan, dari koefisien tersebut akan didapatkan kalkulasi upah yang mengerjakan komposisi perbandingan dan susunan material serta tenaga kerja pada suatu pekerjaan sudah ditetapkan Faktor yang mempengaruhi analisis harga satuan pekerjaan ini adalah angka koefisien kebutuhan bahan, tenaga kerja dan mungkin juga beserta alat yang di perlukan untuk menyelesaikan suatu pekerjaan per satu satuan tertentu[10]. Kemudian akan dilakukan perbandingan antara metode perhitungan AHSP berstandar SNI dengan perhitungan AHSP berstandar BOW. Analisa ini bertujuan untuk mengetahui 2 Jurnal Teknika Sains Volume 07, Nomor 01, 2022 Jurnal Teknika Sains Volume 07, Nomor 01, 2022 manakah harga satuan bahan dan upah tenaga kerja pada Proyek Pembangunan Talud Pantai 1 Bintuhan yang lebih efisien. rinci perhitungan volume dari tiap-tiap satuan pekerjaan. Setelah melihat hasil perhitungan tiap-tiap volume selanjutnya dapat dilihat pada lampiran tabel volume hasil tiap-tiap satuan pekerjaan pada bangunan studi kasus. Volume satuan pekerjaan sudah didapat dari hasil perhitungan luasan maupun jumlah pekerjaan yang akan dilakukan di masing- masing jenis pekerjaannya, yang mana nantinya volume satuan pekerjaan tersebut akan di kalikan dengan analisis satuan pekerjaan baik SNI ataupun BOW. METODE PENELITIAN Jenis penelitian yang dilakukan bersifat studi literature dan studi Kasus[11]. Penulis melakukan studi kasus pada Proyek Pembangunan Talud Pantai 1 Bintuhan untuk meneliti biaya konstruksi dalam hal ini penulis menganalisa dan menghitung ulang rencana anggaran biaya pembangunan Proyek Pembangunan Talud Pantai 1 Bintuhan dengan membandingkan analisa harga satuan pekerjaan SNI dan analisa harga satuan pekerjaan BOW. Untuk detail perhitungan pekerjaan pemakaian besi yang di lakukan pada bangunan studi kasus ini untuk lebih jelasnya dapat dilihat juga pada pada lampiran pembesian. Perhitungan besi tersebut didapat dengan melihat gambar kerja dan menghitung total panjang besi yang dipakai serta ukurannya dan dikonversi ke dalan berat dengan satuan kilogram (kg) dengan cara satuan dengan cara menggunakan panduan dari SNI tetang analisa besi dan almunium. Hasil Perhitungan Volume Satuan Pekerjaan Dalam penelitian ini, penulis menghitung volume pekerjaan untuk setiap pekerjaan dari awal pengembangan hingga akhir pengembangan. Penulis menghitung ulang volume pekerjaan dengan cara membagi tiap-tiap satuan pekerjaan yang dari awal pekerjaan hingga akhir agar memudahkan dalam menentukan analisis pekerjaan yang akan dipakai baik menggunakan metode sni ataupun metode bow. Tabel 1. Hasil Pehitungan Volume Satuan Pekerjaan Uraian Pekerjaan Satuan Volume PEKERJAAN PENDAHULUAN Papan Proyek Ls 1 Pengukuran dan Pemasangan Bowplank Ls 1 Lansiran Material dan Mobilisasi Ls 1 Kayu Penahan Ombak Ls 1 Pembongkaran Existing Ls 1 PEKERJAAN TANAH , PONDASI DAN PLESTERAN Pekerjaan Galian Tanah m3 2019,24 Urugan Tanah m3 178,92 Pas. Batu Kosong 1 : 2 m3 357,84 Pas. Batu Kali 1 : 4 m3 3802,05 Plesteran Siar 1 : 2 m2 3834,00 Plesteran Top Saluran 1 : 2 m2 639,00 Pipa Peresapan Ø 12” M 1993,68 PEKERJAAN BETON Sloef 20 x 25 cm a. Beton (K-200) m3 63,90 b. Besi kg 9059,97 c. Bekisting m2 639,00 Kolom 15 x 15 cm Tabel 1. Hasil Pehitungan Volume Satuan Pekerjaan Tabel 1. Hasil Pehitungan Volume Satuan Pekerjaan Uraian Pekerjaan Satuan Volume Uraian Pekerjaan Satuan Volume PEKERJAAN PENDAHULUAN Papan Proyek Ls 1 Pengukuran dan Pemasangan Bowplank Ls 1 Lansiran Material dan Mobilisasi Ls 1 Kayu Penahan Ombak Ls 1 Pembongkaran Existing Ls 1 Sebagai contoh perhitungan volume pekerjaan galian tanah dengan penampang atas 1,2 m, penampang bawah 1,2 m, tinggi 1 m dan penampang panjang 1278 m. Sehingga didapatkan luas penampang = ½ × (1,2 m + 1,2 m) × 1 m = 1,2 m2, untuk menapatkan volume keseluruhan pekerjaan galian tanah pondasi = 1,2 m2 x 1278 m = 1533,60 m3 volume pekerjaan galian tanah pondasi. adapun untuk melihat rincian dan detail perhitungan dari setiap satuan pekerjaan dapat di lihat pada lampiran volume pekerjaan yang menjelaskan secara g g PEKERJAAN TANAH , PONDASI DAN PLESTERAN PEKERJAAN TANAH , PONDASI DAN PLESTERAN Pekerjaan Galian Tanah m3 2019,24 Urugan Tanah m3 178,92 Pas. Batu Kosong 1 : 2 m3 357,84 Pas. Batu Kali 1 : 4 m3 3802,05 Plesteran Siar 1 : 2 m2 3834,00 Plesteran Top Saluran 1 : 2 m2 639,00 Pipa Peresapan Ø 12” M 1993,68 PEKERJAAN BETON Sloef 20 x 25 cm a. Beton (K-200) m3 63,90 b. Besi kg 9059,97 c. Analisa Harga Satuan Pekerjaan Tabel 2. Rencana Anggaran Biaya Menurut BOW Uraian Pekerjaan Sa tu an Volu me Biaya Kontruk si Jumlah Harga Pekerjaan Pendahuluan Papan Proyek Ls 1 250.000, 00 250.000,0 0 Pengukura n dan Pemasanga n Bowplank Ls 1 3.000.00 0,00 3.000.000, 00 Lansiran Material dan Mobilisasi Ls 1 8.500.00 0,00 8.500.000, 00 Kayu Penahan Ombak Ls 1 9.500.00 0,00 9.500.000, 00 Pembongk aran Existing Ls 1 30.000.0 00,00 30.000.00 0,00 Total 51.250.00 0,00 Pekerjaan Tanah , Pondasi Dan Plesteran Pekerjaan Galian Tanah m 3 2019 ,24 99.187,5 0 200.283.3 67,50 Urugan Tanah m 3 178, 92 121.181, 25 21.681.74 9,84 Pas. Batu Kosong 1 : 2 m 3 357, 84 457.975, 01 163.881.7 77,94 Pas. Batu Kali 1 : 4 m 3 3802 ,05 952.864, 37 3.622.837. 964,65 Plesteran Siar 1 : 2 m 2 3834 ,00 89.529,0 6 343.254.4 31,38 Plesteran Top Saluran 1 : 2 m 2 639, 00 89.866,6 8 57.424.80 9,16 Tabel 2. Rencana Anggaran Biaya Menurut BOW Analisa harga satuan pekerjaan adalah perhitungan analisis harga untuk suatu jenis pekerjaan yang terdiri dari biaya tenaga kerja, biaya bahan atau bahan habis pakai, dan biaya peralatan. Untuk menghitung estimasi biaya metode BOW dan SNI, dapat dihitung sesuai dengan analisa masing- masing. Secara umum, analisis harga satuan dapat dirumuskan sebagai berikut: Harga Satuan Pekerjaan = Indeks Koefisien x Harga Satuan Tenaga/Alat Analisis harga satuan ini menetapkan suatu perhitungan harga satuan upah dan bahan, peralatan dan tenaga kerja, yang secara teknis dirinci berdasarkan cara kerja dan asumsi-asumsi sesuai dengan yang diuraikan dalam spesifikasi teknis, gambar desain dan komponen harga satuan. Analisis ini menjadi dasar perhitungan perkiraan harga itu sendiri dan perkiraan biaya perancang, yang dituangkan sebagai satuan harga untuk pekerjaan, seperti: bahan (m, m2, m3, kg, ton, zak, dll.), peralatan (unit, jam, hari, dll.) dan remunerasi untuk pekerjaan (jam, hari, bulan, dll.). 0,00 Pekerjaan Tanah , Pondasi Dan Plesteran Pekerjaan Galian Tanah m 3 2019 ,24 99.187,5 0 200.283.3 67,50 Urugan Tanah m 3 178, 92 121.181, 25 21.681.74 9,84 Pas. Batu Kosong 1 : 2 m 3 357, 84 457.975, 01 163.881.7 77,94 Pas. Batu Kali 1 : 4 m 3 3802 ,05 952.864, 37 3.622.837. 964,65 Plesteran Siar 1 : 2 m 2 3834 ,00 89.529,0 6 343.254.4 31,38 Plesteran Top Saluran 1 : 2 m 2 639, 00 89.866,6 8 57.424.80 9,16 Hasil Perhitungan Volume Satuan Pekerjaan Bekisting m2 639,00 Kolom 15 x 15 cm 3 Jurnal Teknika Sains Volume 07, Nomor 01, 2022 Jurnal Teknika Sains Volume 07, Nomor 01, 2022 a. Beton (K-200) m3 27,32 b. Besi kg 6881,30 c. Bekisting m2 971,28 Balok 15 x 40 cm a. Beton (K-200) m3 76,68 b. Besi kg 8387,64 c. Bekisting m2 1022,40 PEKERJAAN AKHIR Pembersihan Sisa Pekerjaan Ls 1 Asbuild Drawing dan Backup Data Ls 1 Sumber : Analisa Kontraktor Untuk melihat hasil melihat hasil dan lebih jelasnya dapat dilihat hasil dari rancangan anggaran biaya bangunan menggunakan metode BOW pada Tabel 2, ditersebut juga di masukan jenis pekerjaan atau bahan bangunan yang memakai sistem kerja langsam atau bahan yang dipakai langsung untuk pembangunan proyek talud pada studi kasus. Hasil harga bangunan tersebut menggunakan metode BOW terdapat juga pada lampiran. Rencana Anggaran Biaya Metode BOW Setelah didapat analisa harga satuan pekerjaan menurut tiap-tiap jenis pekerjaannya, selanjutnya adalah membuat rencana anggaran biaya dengan cara mengalikan analisa harga satuan pekerjaan dengan volume dari tiap tiap jenis pekerjaan. 4 Jurnal Teknika Sains Volume 07, Nomor 01, 2022 Pipa Peresapan Ø 12” m 1993 ,68 4.950,00 9.868.716, 00 Total 4.395.821. 133,31 Pekerjaan Beton Sloef 20 x 25 cm a. Beton (K-200) m 3 63,9 0 1.505.29 0,51 96.188.06 3,42 b. Besi kg 9059 ,97 18.716,2 5 169.568.6 55,61 c. Bekisting m 2 639, 00 354.978, 41 226.831.2 01,59 Jumlah 492.587.9 20,62 Kolom 15 x 15 cm a. Beton (K-200) m 3 27,3 2 1.505.29 0,51 41.120.39 7,11 b. Besi kg 6881 ,30 18.716,2 5 128.792.1 31,24 c. Bekisting m 2 971, 28 447.511, 72 434.659.1 82,19 Jumlah 604.571.7 10,54 Balok 15 x 40 cm a. Beton (K-200) m 3 76,6 8 1.505.29 0,51 115.425.6 76,10 b. Besi kg 8387 ,64 18.716,2 5 156.985.1 54,75 c. Bekisting m 2 1022 ,40 479.446, 64 490.186.2 48,57 Jumlah 762.597.0 79,42 Total 1.859.756. 710,59 Pekerjaan Akhir Pembersih an Sisa Pekerjaan Ls 1 200.000, 00 200.000,0 0 Asbuild Drawing dan Backup Data Ls 1 2.500.00 0,00 2.500.000, 00 Total 2.700.000, 00 Real Cost 6.309.527. 843,90 Pada pekerjaan ini Didapat hasil perhitungan dengan metode BOW pekerjaan talud pantai 1 bintuhan di dapat hasil Estimasi anggaran biaya dengan metode BOW sebesar Rp 6.307.701.534,13. Rencana Anggaran Biaya Metode SNI Sama seperti rencana anggaran biaya bangunan menggunakan metode Bow Pada metode SNI juga sam tetapi analisa harga satuan pekerjaan yang dikalikan dengan volume jenis pekerjaannya adalah analisa yang memakai sistem SNI yang untuk lebih jelasnya melihat hasil perhitungan harganya dapat dilihat pada tabel 4.33, biaya akhir dari pembangunan talud pada studi kasus juga terdapat dalam lampiran tersebut dengan biaya menggunakan metode SNI yang seterusnya akan di bandingkan dengan biaya pembangunan dengan metode BOW. Tabel 3.Rekapitulasi Perbandingan Rencana Anggaran Biaya Uraian Pekerjaan Volu me BOW SNI Seli sih Pekerjaan Pendahuluan Papan Proyek 1 250.000, 00 250.000, 00 Pengukura n dan Pemasanga n Bowplank 1 3.000.00 0,00 3.000.00 0,00 Lansiran Material dan Mobilisasi 1 8.500.00 0,00 8.500.00 0,00 Kayu Penahan Ombak 1 9.500.00 0,00 9.500.00 0,00 Pembongk aran Existing 1 30.000.0 00,00 30.000.0 00,00 Jumlah 51.250.0 00,00 51.250.0 00,00 Pekerjaan Tanah, Pondasi Dan Plesteran Pekerjaan Galian Tanah 2019, 24 200.283. 367,50 200.283. 367,50 - Urugan Tanah 178,9 2 21.681.7 49,84 21.681.7 49,25 Pas. Batu Kosong 1 : 2 357,8 4 163.881. 777,94 123.314. 884,56 Pas. Batu Kali 1 : 4 3802, 05 3.622.83 7.964,65 2.584.20 2.216,79 Plesteran Siar 1 : 2 3834, 00 343.254. 431,38 296.830. 312,02 Tabel 3.Rekapitulasi Perbandingan Rencana Anggaran Biaya Uraian Pekerjaan Volu me BOW SNI Seli sih Pekerjaan Pendahuluan Papan Proyek 1 250.000, 00 250.000, 00 Pengukura n dan Pemasanga n Bowplank 1 3.000.00 0,00 3.000.00 0,00 Lansiran Material dan Mobilisasi 1 8.500.00 0,00 8.500.00 0,00 Kayu Penahan Ombak 1 9.500.00 0,00 9.500.00 0,00 Pembongk aran Existing 1 30.000.0 00,00 30.000.0 00,00 Jumlah 51.250.0 00,00 51.250.0 00,00 Tabel 3.Rekapitulasi Perbandingan Rencana Anggaran Biaya Pada pekerjaan ini Didapat hasil perhitungan dengan metode BOW pekerjaan talud pantai 1 bintuhan di dapat hasil Estimasi anggaran biaya dengan metode BOW sebesar Rp 6.307.701.534,13. 5 Jurnal Teknika Sains Volume 07, Nomor 01, 2022 Volume 07, Nomor 01, 2022 Plesteran Top 1 : 2 639,0 0 57.424.8 09,16 54.193.6 59,01 Pipa Peresapan Ø 12” 1993, 68 9.868.71 6,00 9.868.71 6,00 Jumlah 4.415.96 2.292,46 3.290. 290.625, 31 3.29 0. 290. 625, 31 Pekerjaan Beton Sloef 20 x 25 a.Beton (K-200) 63,90 m3 96.188.0 63,42 80.053.2 35,60 b.Besi 9059, 97 kg 169.568. 655,61 169.568. 655,61 c.Bekisting 639,0 0 m2 226.831. 201,59 139.428. 601,88 Jumlah 492.587. 920,62 389.050. 493,09 103. 537. 427, 53 Kolom 20 x 20 a.Beton (K-200) 27,32 m3 41.120.3 97,11 34.222.7 57,96 b.Besi 6881, 30 kg 128.792. 131,24 128.792. Rencana Anggaran Biaya Metode SNI 131,24 c.Bekisting 971,2 8 m2 434.659. 182,19 329.108. 818,73 Jumlah 604.571. 710,54 518.741. 408,57 85.8 30.3 01,9 7 Balok 15 x 40 a.Beton (K-200) 76,68 m3 115.425. 676,10 96.063.8 82,00 b.Besi 8387, 64 kg 156.985. 154,75 156.985. 154,75 c.Bekisting 1022, 40 m2 490.186. 248,57 393.073. 498,5794 Jumlah 762.597. 079,42 646.122. 535,33 116.47 4.544, 09 Total 1.859.75 6.710,59 1.527.29 6.736,12 337.82 4.804, 91 Pekerjaan Akhir Pembersih an Sisa Pekerjaan 1 200.000, 00 200.000, 00 Asbuild Drawing dan Backup Data 1 2.500.00 0,00 2.500.00 0,00 Plesteran Top 1 : 2 639,0 0 57.424.8 09,16 54.193.6 59,01 Pipa Peresapan Ø 12” 1993, 68 9.868.71 6,00 9.868.71 6,00 Jumlah 4.415.96 2.292,46 3.290. 290.625, 31 3.29 0. 290. 625, Jumlah 2.700.00 0,00 2.700.00 0,00 Real Cost (Rc) 6.309.52 7.843,90 4.860.12 8.815,18 1.449. 399.02 8,72 Dibulatkan 6.309.50 0.000,00 4.860.00 0.000,00 1.449. 500.00 0,00 Dari hasil perhitungan dengan metode SNI dan metode BOW pekerjaan talud pantai 1 bintuhan di dapat hasil estimasi anggaran biaya sebagai berikut : gg y g a) Estimasi anggaran biaya dengan metode SNI sebesar Rp 4.860.000.000,00 b) Estimasi anggaran biaya dengan metode BOW sebesar Rp 6.309.500.000,00 Dari data diatas terdapat selisih estimasi anggaran biaya antara metode SNI dengan BOW sebesar : Rp.4.860.000.000,00 -Rp.6.309.500.000,00 = Rp.1.449.500.000,00 Tabel 4. Rekapitulasi Rencana Anggaran Biaya Tabel 4. Rekapitulasi Rencana Anggaran Biaya Menurut BOW Uraian Pekerjaan SNI Pekerjaan Pendahuluan 51.250.000,00 Pekerjaan Tanah, Pondasi Dan Plesteran 4.415.962.292,46 Pekerjaan Beton 1.859.756.710,59 Pekerjaan Akhir 2.700.000,00 Real Cost (RC) 6.309.527.843,90 Dibulatkan 6.309.500.000,00 Tabel 4. Rekapitulasi Rencana Anggaran Biaya Menurut BOW Tabel 5. Rekapitulasi Rencana Anggaran Biaya Menurut SNI Tabel 5. Rekapitulasi Rencana Anggaran Biaya Menurut SNI Uraian Pekerjaan SNI Pekerjaan Pendahuluan 51.250.000,00 Pekerjaan Tanah, Pondasi Dan Plesteran 3.290. 290.625,31 Pekerjaan Beton 1.527.296.736,12 Pekerjaan Akhir 2.700.000,00 Real Cost (RC) 4.860.128.815,18 Dibulatkan 4.860.000.000,00 Grafik Hasil Estimasi Anggaran Biaya Grafik Hasil Estimasi Anggaran Biaya Metode SNI Dan Metode BOW Grafik Hasil Estimasi Anggaran Biaya Metode SNI Dan Metode BOW Hasil estimasi angaran biaya dengan metode SNI dan BOW dapat di buat dalam sebuah grafik. Adapun grafik tersebut dapat dilihat pada gambar 4.2 dalam pembuatan 6 Jurnal Teknika Sains Volume 07, Nomor 01, 2022 Volume 07, Nomor 01, 2022 grafik berdasarkan tabel 6 yang menunjukkan hasil estimasi anggaran biaya antara metode SNI dan BOW. Gambar 2. Grafik Rencana Anggaran Biaya SNI, BOW Dan Kontraktor Gambar 3. Grafik Rekapitulasi Rencana Anggaran Biaya SNI, BOW Dan Kontraktor Tabel 6. Hasil Estimasi Anggaran Biaya Metode Real Cost (RC) SNI 4.860.000.000,00 BOW 6.309.500.000,00 KONTRAKTOR 4.698.600.000,00 Sumber : Analisa Penulis Gambar 2. Grafik Rencana Anggaran Biaya SNI, BOW Dan Kontraktor Ga ba . G a e ca a gga a aya SN , BOW Dan Kontraktor Gambar 3. Grafik Rekapitulasi Rencana Anggaran Biaya SNI, BOW Dan Kontraktor Hasil akhir dari penelitian menunjukan bahwa perhitungan biaya pembangunan Talud Pantai 1 Bintuhan dengan menggunakan metode BOW sebesar Rp. 6.309.500.000,00, sedangkan hasil estimasi biaya menggunakan metode SNI 2008 sebesar Rp. 4.860.000.000,00 dan untuk hasil estimasi biaya KONTRAKTOR sebesar Rp. 4.698.600.000,00. Gambar 3. Grafik Rekapitulasi Rencana Anggaran Biaya SNI, BOW Dan Kontraktor KESIMPULAN Gambar 1. Hasil estimasi angaran biaya 0,00 5.000.000.000,00 10.000.000.000,00 BOW SNI KONTRAKTOR Hasil akhir dari penelitian menunjukan bahwa perhitungan biaya pem-bangunan Talud Pantai 1 Bintuhan dengan menggunakan metode BOW sebesar Rp. 6.309.500.000,00, sedangkan hasil estimasi biaya menggunakan metode SNI 2008 sebesar Rp. 4.860.000.000,00. Dari hasil perhitungan, perbandingan estimasi anggaran biaya antara metode BOW dan metode SNI yakni metode BOW lebih mahal sebesar Rp. 1.449.500.000,00 dari metode SNI. Dari hasil perhitungan rencana anggaran proyek pembangunan Talud Pantai 1 Bintuhan dengan kedua metode, selisih harga tersebut di dapat karena perbedaadaan pemakaian koefisien upah dan bahan material pada kedua analisis, sedangkan untuk pemakaian harga dan bahan material tetap sama menggunakan harga upah dan bahan yang dikeluarkan pemerintah Kab. Kaur. Gambar 1. Hasil estimasi angaran biaya Gambar 1. Hasil estimasi angaran biaya 7 7 Jurnal Teknika Sains Volume 07, Nomor 01, 2022 DAFTAR PUSTAKA Talud Pantai Desa Talaga Besar,” SCEJ (Shell Civ. Eng. Journal), vol. 3, no. 1, pp. 39–47, 2018. [1] S. N. Sari, “Evaluasi Anggaran Biaya menggunakan Batu Bata Merah dan Batu Bata Ringan Gedung Kantor Kelurahan Bareng Kecamatan Klaten Tengah Kabupaten Klaten,” J. Qua Tek., vol. 9, no. 1, pp. 1–10, 2019. [8] A. H. Rizal, D. B. A. Nisnoni, and I. M. Udiana, “Perbandingan Produktivitas Tenaga Kerja Tukang Batu Antara Metode Lapangan Terhadap Permen Pupr Tahun 2016,” J. Tek. Sipil, vol. 9, no. 2, pp. 323–334, 2020. [2] G. Oktarian and M. Fauzan, “Perancangan Gedung Kuliah Fakultas Ekonomi Universitas Islam Negeri Raden Fatah Kampus B Di Jakabaring Palembang.” Politeknik Negeri Sriwijaya, 2019. [9] Y. U. Yati, M. S. Amin, and S. W. Utami, “Pelatihan Penyusunan Rencana Anggaran Biaya Infrastruktur Desa Bagi Perangkat Desa Di Desa Karangbendo Kecamatan Rogojampi,” J-Dinamika J. Pengabdi. Masy., vol. 3, no. 1, 2018. [3] V. Aprilia, “Analisis Perbandingan Sistem Struktur Pelat Konvensional Dan Half Slab Ditinjau Dari Segi Biaya Dan Waktu.” Universitas Komputer Indonesia, 2021. [10] I. K. WIJAYANTO, “Analisis Produktivitas Tenaga Kerja Pada Pekerjaan Pemasangan Keramik Lantai,” 2021. [4] M. Y. Ina and L. Langga, “Proyek Anggaran Sebagai Alat Pengendalian Biaya Pada Kantor Satuan Kerja Pelaksanaan Jalan Nasional Wilayah Iv Provinsi Nusa Tenggara Timur,” ANALISIS, vol. 11, no. 2, pp. 239–250, 2021. [11] T. Hidayat and U. M. Purwokerto, “Pembahasan studi kasus sebagai bagian metodologi penelitian,” J. Study Kasus, pp. 1–13, 2019. [5] D. Asmaroni and S. Wahyuni, “Analisis Perbandingan Rencana Anggaran Biaya Dengan Menggunakan Metode Analisa Standart Kementerian Pupr Tahun 2016 Dan Sni Tahun 2018 Pada Proyek Pembangunan Kantor Djarum Dso (Districk Sales Office) Di Kota Pamekasan,” Rekayasa J. Tek. Sipil, vol. 6, no. 2, pp. 25–29, 2022. [6] Y. Juansyah, D. Oktarina, and M. Zulfiqar, “Analisis perbandingan Rencana Anggaran Biaya bangunan menggunakan metode SNI dan BOW (Studi kasus: Rencana Anggaran Biaya bangunan gedung Kwarda Pramuka Lampung),” J. Rekayasa, Teknol. dan Sains, vol. 1, no. 1, 2017. [7] L. Sianto, M. Takdir, M. Maswanto, and S. Karsidi, “Analisa Perbandingan Estimasi Anggaran Biaya antara Metode SNI Dan BOW pada Pekerjaan 8 8
https://openalex.org/W4320731205	https://biblio.ugent.be/publication/01GSDFBQ53Y0G93FTB3BWNA3SW/file/01GSW7D6RV4WD4QAYHD0AVG79Y	English	null	Detection of Breast Cancer-Specific Extracellular Vesicles with Fiber-Optic SPR Biosensor	International journal of molecular sciences	2,023	cc-by	13,783	Detection of Breast Cancer-Speciﬁc Extracellular Vesicles with Fiber-Optic SPR Biosensor Yagmur Yildizhan 1 , Kaat Driessens 1, Hong Shen Kevin Tsao 1, Robin Boiy 2, Debby Thomas 3 Nick Geukens 3, An Hendrix 2 , Jeroen Lammertyn 1,* and Dragana Spasic 1 Yagmur Yildizhan 1 , Kaat Driessens 1, Hong Shen Kevin Tsao 1, Robin Boiy 2, Debby Thomas 3 , Nick Geukens 3, An Hendrix 2 , Jeroen Lammertyn 1,* and Dragana Spasic 1 1 Department of Biosystems, Biosensors Group, Katholieke Universiteit Leuven, 3001 Leuven, Belgium 2 Laboratory of Experimental Cancer Research, Cancer Research Institute Ghent, Department of Human Structure and Repair, Ghent University, 9000 Ghent, Belgium 3 PharmAbs, The KU Leuven Antibody Center, University of Leuven, 3000 Leuven, Belgium * Correspondence: jeroen.lammertyn@kuleuven.be; Tel.: +32-16-32-14-59 1 Department of Biosystems, Biosensors Group, Katholieke Universiteit Leuven, 3001 Leuven, Belgium 2 Laboratory of Experimental Cancer Research, Cancer Research Institute Ghent, Department of Human Structure and Repair, Ghent University, 9000 Ghent, Belgium 3 PharmAbs, The KU Leuven Antibody Center, University of Leuven, 3000 Leuven, Belgium * Correspondence: jeroen.lammertyn@kuleuven.be; Tel.: +32-16-32-14-59 1 Department of Biosystems, Biosensors Group, Katholieke Universiteit Leuven, 3001 Leuven, Belgium 2 Laboratory of Experimental Cancer Research, Cancer Research Institute Ghent, Department of Human Structure and Repair, Ghent University, 9000 Ghent, Belgium 3 PharmAbs, The KU Leuven Antibody Center, University of Leuven, 3000 Leuven, Belgium * Correspondence: jeroen.lammertyn@kuleuven.be; Tel.: +32-16-32-14-59 * Correspondence: jeroen.lammertyn@kuleuven.be; Tel.: +32-16-32-14-59 Abstract: Extracellular vesicles (EVs) have attracted great attention as potential biomarkers for cancer diagnostics. Although several technologies have been developed for EV detection, many of them are still not applicable to clinical settings as they rely on complex EV isolation processes, while lacking sensitivity, speciﬁcity or standardization. To solve this problem, we have developed a sensitive breast cancer-speciﬁc EV detection bioassay directly in blood plasma using a ﬁber-optic surface plasmon resonance (FO-SPR) biosensor, previously calibrated with recombinant EVs. First, we established a sandwich bioassay to detect SK-BR-3 EVs by functionalizing the FO-SPR probes with anti-HER2 antibodies. A calibration curve was built using an anti-HER2/Banti-CD9 combination, resulting in an LOD of 2.1 × 107 particles/mL in buffer and 7 × 108 particles/mL in blood plasma. Next, we investigated the potential of the bioassay to detect MCF7 EVs in blood plasma using an anti- EpCAM/Banti-mix combination, obtaining an LOD of 1.1 × 10 8 particles/mL. Citation: Yildizhan, Y.; Driessens, K.; Tsao, H.S.K.; Boiy, R.; Thomas, D.; Geukens, N.; Hendrix, A.; Lammertyn, J.; Spasic, D. Detection of Breast Cancer-Speciﬁc Extracellular Vesicles with Fiber-Optic SPR Biosensor. Int. J. Mol. Sci. 2023, 24, 3764. https://doi.org/ 10.3390/ijms24043764 Keywords: extracellular vesicles; biosensors; ﬁber-optic surface plasmon resonance; breast cancer; HER2; EpCAM International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences Detection of Breast Cancer-Speciﬁc Extracellular Vesicles with Fiber-Optic SPR Biosensor Finally, the speciﬁcity of the bioassay was proven by the absence of signal when testing plasma samples from 10 healthy people unknown to be diagnosed with breast cancer. The remarkable sensitivity and speciﬁcity of the developed sandwich bioassay together with the advantages of the standardized FO-SPR biosensor highlight outstanding potential for the future of EV analysis. 1. Introduction Academic Editors: Giorgio Rispoli and Pierpaolo Greco Received: 14 January 2023 Revised: 2 February 2023 Accepted: 9 February 2023 Published: 13 February 2023 Extracellular vesicles (EVs) hold a crucial role as mediators of cell-to-cell communica- tion by carrying the diverse molecular cargo of their parental cells, including RNA, DNA, lipids, metabolites and proteins [1]. As such, they are involved in several physiological and pathological processes within the body, from cell maintenance to tissue regeneration, as well as tumor invasion, progression, metastasis, and even activation of immunogenic responses for cancer immunotherapy [2–5]. Their effect on cancer development and po- tential use as noninvasive cancer biomarkers has been continuously triggering interest among researchers, offering a great prospect for cancer diagnostics, prognostics and thera- peutics [2,6]. That is why the accurate and reliable characterization and detection of EVs have become crucial to meet the growing demands of clinical applications [7]. Academic Editors: Giorgio Rispoli and Pierpaolo Greco Received: 14 January 2023 Revised: 2 February 2023 Accepted: 9 February 2023 Published: 13 February 2023 Copyright: © 2023 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/). However, EV studies remain challenging because of their inherently complex biogen- esis and extensive heterogeneity in size, composition, and origin [8]. As a consequence, currently there are no speciﬁc universal sets of proteins that can be used for the accurate characterization of different EV subpopulations. When EV samples originating from dif- ferent sources need to be analyzed, difﬁculties arise in terms of accurate comparison of data. Therefore, the International Society for Extracellular Vesicles recommends careful https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2023, 24, 3764. https://doi.org/10.3390/ijms24043764 Int. J. Mol. Sci. 2023, 24, 3764 2 of 14 characterization of EV proteins to avoid: (1) overestimation of total protein concentration and (2) false assumptions about the uniform presence of proteins on the EVs (that might be caused by contamination with high-abundance matrix proteins like albumin [9,10] or as a result of EV lysis required for some analytical approaches). y q y pp Even though there are many well-established conventional methods and emerging technologies for EV characterization and detection, the absence of analytical instruments well calibrated with reference material is still a signiﬁcant problem in the ﬁeld [9,11]. Among the most favored conventional methods, Western blotting (WB) is the most pre- ferred EV analysis technique that can identify the size of the different proteins and allows semiquantitative assessment of proteins of choice. The second most preferred technique in this context is enzyme-linked immunosorbent assay (ELISA) as it offers signiﬁcant ﬂex- ibility with respect to the bioassay formats [9]. However, while WB or ELISA may give an accurate insight for a highly puriﬁed EV population, this becomes more challenging when working directly in a complex biological ﬂuid because of the presence of various molecules with sizes and physical properties overlapping with the EVs [12]. Consequently, the exploitation of these methods relies profoundly on the purity of the EV sample to obtain a reliable and reproducible analysis that can be transferred to clinical settings. Furthermore, both approaches are limited in their use in clinics due to lengthy preparation steps and analysis time, as well as requirement for a well-equipped facility. Besides these two, mass spectroscopy (MS) is a crucial analysis method that can achieve high-throughput, quantita- tive, and comparative proteomic but also lipidomic analyses of EVs [13,14]. Moreover, it can uncover the functional activities of EV cargo and their role in intercellular communica- tion [13]. Despite these beneﬁts, MS has several disadvantages, such as the requirement for highly puriﬁed EV samples to avoid contamination by other soluble biomolecules, which can cause aspeciﬁc signals. In addition, there is a prerequisite for peptide proﬁling, which entails complex processing of EVs by separating peptides via enzymatic digestion. As such, MS needs proper protein proﬁling, quantiﬁcation, and validation through other techniques that should be calibrated with a reference material [15]. Therefore, these techniques, al- though conventional, still suffer from the lack of standardization prior to analyzing complex biological samples in order to ensure reproducible quality independently of the internal complexity of the measured samples. p y p In addition to these conventional technologies, many emerging techniques have been developed throughout the years based on different detection principles from magnetic to electrochemical and plasmonic sensing considering the great potential of EVs to be utilized for liquid biopsy and therapy of cancer. Shao et al. developed a technology for EV analysis through magnetic detection in which an on-chip micro-nuclear magnetic resonance (µNMR) detection system is integrated with immunocapture for quantitative EV detection and protein proﬁling [16,17]. Their study demonstrated rapid and highly sensitive detection of glioblastoma-derived circulating microvesicles, a subtype of EVs, in clinical samples. Although the detection sensitivity of µNMR surpassed the sensitivity of WB, ELISA, and nanoparticle tracking analysis (NTA) by 104-, 103-, and 102-fold, respectively, this technology was developed using only EVs originating from cell culture or human body ﬂuids. The development of biosensing technologies based only on speciﬁc biological samples could create bias, since it will be established considering the intrinsic features of only a certain sample, which could harm the reproducibility and reliability of the biosensor when another biological sample of different origin is analyzed. Another example of sensitive EV detection (<105 EVs) are electrochemistry-based biosensors, which have even been integrated into a single iMEX (integrated magnetic–electrochemical exosome detection) platform together with magnetic enrichment, yielding fast and simpliﬁed EV analysis starting from only 10 µL of the sample [18,19]. Nevertheless, like µNMR, iMEX was also developed using only speciﬁc biological materials, such as ovarian cancer cell line- derived EVs and plasma from patients with ovarian and colorectal cancer. Consequently, the main limitation of these approaches is the lack of a standardized material during their development, which can seriously affect the reproducibility of the results. Int. J. Mol. Sci. 2023, 24, 3764 3 of 14 3 of 14 Among various EV detection principles, surface plasmon resonance (SPR) stands out with its capacity to enable real-time and label-free kinetics and afﬁnity measurements. Currently, there are many conventional SPR platforms, developed based on Kretschmann prism conﬁguration or alternative approaches, such as SPR coupled with imaging (SPRi) or microscopy (SPRM), to achieve even higher throughput in EV analysis [20]. For example, SPRi enables parallel measurements dependent on the number of sensing spots and can be combined with microﬂuidics to support the use of fewer reagents. An SPRi biosensor was used to detect EVs isolated from various non–small cell lung cancer and normal lung cell lines with an LOD of 107 EV/mL [21]. However, SPRi platforms may suffer from insufﬁcient image resolution. In this context, SPRM technologies have been introduced for higher-resolution imaging that can even enable single-level EV detection. Yang et al. presented an SPRM platform that can detect human lung cancer cell line-derived EVs providing information regarding the size, concentration and biochemical properties of EV membrane proteins [22]. Even though these techniques enable visualization of the EVs, they are mostly suited for research purposes rather than clinical use, since the small ﬁeld of view prevents parallel measurements. p p To overcome the common limitations in the ﬁeld (i.e., lack of standardization, require- ment for highly puriﬁed samples, and lengthy workﬂows not being suitable for clinical use) that hinder the progression of EV-based diagnostics, in this study we used our ﬁber-optic surface plasmon resonance (FO-SPR) biosensor (commercialized by FOx Biosystems). This biosensor was previously carefully calibrated with well-characterized recombinant EVs (rEVs), i.e., biological reference materials aiming to support EV isolation and analysis, assay development, and device calibration [23,24]. Moreover, in that study we preliminary showed its potential to detect EVs in complex matrices by measuring EVs spiked at single concentration. This potential is further elaborated in this work, where we develop sensitive and speciﬁc FO-SPR bioassays for the detection of two types of breast cancer-speciﬁc EVs, both in buffer and complex biological matrix, namely, human blood plasma (Scheme 1). To achieve this, we ﬁrst select two different (commonly used) breast cancer cell lines, being SK-BR-3 and MCF7, with the aim to have model systems for two breast cancer biomarkers, i.e., HER2 and EpCAM, respectively. To speciﬁcally detect EVs from both cell lines, we ﬁrst screen combinations of capture and detection antibodies, which also offers us a possibility to study colocalization of proteins on EVs. Next, we use the most optimal combinations to build for the ﬁrst time calibration curves for detecting EVs in 100-fold diluted blood plasma (in this case EVs originating from SK-BR-3 and MCF7 cell lines). Finally, the speciﬁcity of the established bioassays is tested with plasma samples from 10 healthy individuals as well as pooled plasma from more than 25 healthy people. Compared to other EV detection plat- forms, our FO-SPR sensor offers several advantages by (1) having low-cost sensor probes, (2) requiring low sample volume, (3) enabling both label-free and sandwich bioassays, and (4) being compatible with diverse complex matrices, including serum, plasma, whole blood and cell culture media [25,26]. Int. J. Mol. Sci. 2023, 24, 3764 ER REVIEW 4 of 14 of 14 Scheme 1. Schematic of the different steps from the FO-SPR EV detection sandwich bioassay. The EVs (originating from SK-BR-3 or MCF7 cell lines) are specifically captured by the capture antibod- ies immobilized on the FO-SPR surface (i.e., anti-HER2 or anti-EpCAM, respectively). Biotinylated detection antibodies (Banti-CD9, Banti-CD63 or Banti-CD81, introduced separately or as a mixture) are used to detect CD9, CD63 and CD81 tetraspanins on the EV surface. Finally, the AuNPs func- tionalized with anti-biotin antibodies, which recognize multiple biotin labels on the detection anti- bodies, are used for signal amplification. Antibodies on the FO-SPR surface as well as on the AuNPs have random orientation due to their immobilization through covalent bonds or physical adsorp- tion, respectively. The EVs are depicted in different sizes and colors to simulate the EV heterogene- Scheme 1. Schematic of the different steps from the FO-SPR EV detection sandwich bioassay. The EVs (originating from SK-BR-3 or MCF7 cell lines) are speciﬁcally captured by the capture antibodies immobilized on the FO-SPR surface (i.e., anti-HER2 or anti-EpCAM, respectively). Biotinylated detection antibodies (Banti-CD9, Banti-CD63 or Banti-CD81, introduced separately or as a mixture) are used to detect CD9, CD63 and CD81 tetraspanins on the EV surface. Finally, the AuNPs functionalized with anti-biotin antibodies, which recognize multiple biotin labels on the detection antibodies, are used for signal ampliﬁcation. Antibodies on the FO-SPR surface as well as on the AuNPs have random orientation due to their immobilization through covalent bonds or physical adsorption, respectively. The EVs are depicted in different sizes and colors to simulate the EV heterogeneity in a biological sample. This image was created with Biorender.com (not drawn to scale). of the different steps from the FO-SPR EV detection sandwich bioassay. The SK-BR-3 or MCF7 cell lines) are specifically captured by the capture antibod- FO-SPR surface (i.e., anti-HER2 or anti-EpCAM, respectively). Biotinylated anti-CD9, Banti-CD63 or Banti-CD81, introduced separately or as a mixture) 9, CD63 and CD81 tetraspanins on the EV surface. Finally, the AuNPs func- otin antibodies, which recognize multiple biotin labels on the detection anti- nal amplification. Antibodies on the FO-SPR surface as well as on the AuNPs on due to their immobilization through covalent bonds or physical adsorp- EVs are depicted in different sizes and colors to simulate the EV heterogene- Scheme 1. Schematic of the different steps from the FO-SPR EV detection sandwich bioassay. The EVs (originating from SK-BR-3 or MCF7 cell lines) are speciﬁcally captured by the capture antibodies immobilized on the FO-SPR surface (i.e., anti-HER2 or anti-EpCAM, respectively). Biotinylated detection antibodies (Banti-CD9, Banti-CD63 or Banti-CD81, introduced separately or as a mixture) are used to detect CD9, CD63 and CD81 tetraspanins on the EV surface. Finally, the AuNPs functionalized with anti-biotin antibodies, which recognize multiple biotin labels on the detection antibodies, are used for signal ampliﬁcation. Antibodies on the FO-SPR surface as well as on the AuNPs have random orientation due to their immobilization through covalent bonds or physical adsorption, respectively. The EVs are depicted in different sizes and colors to simulate the EV heterogeneity in a biological sample. This image was created with Biorender.com (not drawn to scale). p le. This image was create 2. Results and Discussion g ( 2.1. FO-SPR Surface Functionalization with HER2-Speciﬁc Antibodies g ( 2.1. FO-SPR Surface Functionalization with HER2-Speciﬁc Antibodies ussion Functionalization with HER2-Specific Antibodies detect EVs originating from the SK-BR-3 breast cancer cell line, FO- ctionalized with a HER2-specific antibody, given that HER2 (human actor receptor 2) has a critical role in the mediation of growth and cancer cells and is overexpressed in the SK-BR-3 cell line [27,28]. The y immobilization depends on several factors, including antibody con- zation time, buffer ionic strength, and pH value. Here, 20 μg/mL was mal antibody concentration based on saturated FO-SPR surface previ- t f th t t d tib di h i COOH f h i t To speciﬁcally detect EVs originating from the SK-BR-3 breast cancer cell line, FO-SPR surface was functionalized with a HER2-speciﬁc antibody, given that HER2 (human epider- mal growth factor receptor 2) has a critical role in the mediation of growth and progression of breast cancer cells and is overexpressed in the SK-BR-3 cell line [27,28]. The efﬁciency of antibody immobilization depends on several factors, including antibody concentration, immobilization time, buffer ionic strength, and pH value. Here, 20 µg/mL was selected as the optimal antibody concentration based on saturated FO-SPR surface previously obtained for most of the tested antibodies when using COOH surface chemistry [23,29,30]. Buffers with different pH values were examined, since the isoelectric point (pI) of the selected antibody was unknown. In order to maximize the immobilization efﬁciency, i.e., to obtain the maximum FO-SPR shift, we screened buffers with lower ionic strength, being 10 mM sodium acetate (NaAc) buffer (pH 5.2, 5.4 and 5.6) and 50 mM MES buffer (pH 6.0). Based on this screening experiment, 10 mM NaAc buffer with pH 5.4 resulted in the highest FO- Int. J. Mol. Sci. 2023, 24, 3764 5 of 14 SPR shift compared to the other three buffers (Figure 1) and was selected for immobilizing anti-HER2 antibody in further experiments. SPR shift compared to the other three buffers (Figure 1) and was selected for immobilizing anti-HER2 antibody in further experiments. Figure 1. Bar graphs representing the FO-SPR shifts for the immobilization of using 4 immobilization buffers (10 mM NaAc buffer pH 5.2, 5.4, 5.6, and 50 mM Figure 1. Bar graphs representing the FO-SPR shifts for the immobilization of anti-HER2 antibody using 4 immobilization buffers (10 mM NaAc buffer pH 5.2, 5.4, 5.6, and 50 mM MES buffer pH 6.0) obtained from two independent measurements. Figure 1. g ( 2.1. FO-SPR Surface Functionalization with HER2-Speciﬁc Antibodies Bar graphs representing the FO-SPR shifts for the immobilization of using 4 immobilization buffers (10 mM NaAc buffer pH 5.2, 5.4, 5.6, and 50 mM Figure 1. Bar graphs representing the FO-SPR shifts for the immobilization of anti-HER2 antibody using 4 immobilization buffers (10 mM NaAc buffer pH 5.2, 5.4, 5.6, and 50 mM MES buffer pH 6.0) obtained from two independent measurements. obtained from two independent measurements. 2.2. Development of an FO-SPR Sandwich Bioassay for Detecting SK-BR-3 EVs in Buffer 2.2. Development of an FO-SPR Sandwich Bioassay for Detecting SK-BR-3 E An FO-SPR sandwich bioassay was first built in buffer in order to sensitive detection of SK-BR-3 EVs. Starting from the FO-SPR probes fu anti-HER2 antibody (as described in Section 3.4), the sandwich bioass using our previously established 2-step signal amplification approach tails the implementation of biotinylated detection antibody in the first AuNPs functionalized with anti-biotin antibody. In this context, we ut detection antibodies against one of the tetraspanins often used in EV r nylated anti-CD9 (Banti-CD9), biotinylated anti-CD63 (Banti-CD63) or CD81 (Banti-CD81), either separately or an equal mixture of all three (i.e EVs have multiple copies of these proteins in their membranes [31]. A tions were probed in buffer with a fixed concentration of SK-BR-3 EV cles/mL) and detection antibodies at 10 μg/mL concentration. Figure 2 SPR signal shift after subtracting the negative control, the latter being tected in the sample without spiked EVs (i.e., 0 particles/mL). Based An FO-SPR sandwich bioassay was ﬁrst built in buffer in order to enable speciﬁc and sensitive detection of SK-BR-3 EVs. Starting from the FO-SPR probes functionalized with anti-HER2 antibody (as described in Section 3.4), the sandwich bioassay was developed using our previously established 2-step signal ampliﬁcation approach [23]. The latter entails the implementation of biotinylated detection antibody in the ﬁrst step, followed by AuNPs functionalized with anti-biotin antibody. In this context, we utilized biotinylated detection antibodies against one of the tetraspanins often used in EV research, i.e., biotinylated anti- CD9 (Banti-CD9), biotinylated anti-CD63 (Banti-CD63) or biotinylated anti-CD81 (Banti- CD81), either separately or an equal mixture of all three (i.e., Banti-mix), since EVs have multiple copies of these proteins in their membranes [31]. All these combinations were probed in buffer with a ﬁxed concentration of SK-BR-3 EVs (1.55 × 108 particles/mL) and detection antibodies at 10 µg/mL concentration. g ( 2.1. FO-SPR Surface Functionalization with HER2-Speciﬁc Antibodies The dotted lines indicate the 95% prediction band w observation Error bars represent one standard deviation (n = 3) independent experiments). (B) FO-SPR-based detection of a series of SK-BR-3 EV concentrations spiked in the detection buffer (50 mM MES pH 6, 0.01% BSA, 0.01% Tween 20) when using the anti-HER2/Banti-CD9 antibody combination (obtained after subtracting the negative control, i.e., SPR signal for 0 particles/mL). Simple linear regression ﬁtting was performed by GraphPad Prism soft- ware (Version 8.0.1, GraphPad Software Inc., MA, USA). The dotted lines indicate the 95% prediction bands for a new observation. Error bars represent one standard deviation (n = 3). The combination of anti-HER2/Banti-CD9 was exploited for building a calib ve by spiking in buffer different SK-BR-3 EV concentrations ranging from 9.7 × 5 × 108 particles/mL and also including a negative control without any EVs—0 s/mL. The obtained FO-SPR signal shifts after subtracting the negative control re plotted as a function of SK-BR-3 EV concentration (Figure 2B). The calibration owed that the FO-SPR biosensor can detect SK-BR-3 EVs over the entire tested c tion range with a limit of detection (LOD) of 2.1 × 107 particles/mL. The results ind t the obtained LOD was 1.5-fold lower than the previously reported LOD value o 13 × 107 particles/mL) detected in buffer using FO-SPR technology [24]. This im nt can be related to several factors, such as (1) strong affinity of selected anti ibody against isolated SK-BR-3 EVs (2) the number of HER2 oncogenic proteins The combination of anti-HER2/Banti-CD9 was exploited for building a calibration curve by spiking in buffer different SK-BR-3 EV concentrations ranging from 9.7 × 106 to 1.55 × 108 particles/mL and also including a negative control without any EVs—0 particles/mL. The obtained FO-SPR signal shifts after subtracting the negative control (n = 3) were plotted as a function of SK-BR-3 EV concentration (Figure 2B). The calibration curves showed that the FO-SPR biosensor can detect SK-BR-3 EVs over the entire tested concentration range with a limit of detection (LOD) of 2.1 × 107 particles/mL. The results indicated that the obtained LOD was 1.5-fold lower than the previously re- ported LOD value of rEVs (3.13 × 107 particles/mL) detected in buffer using FO-SPR technology [24]. g ( 2.1. FO-SPR Surface Functionalization with HER2-Speciﬁc Antibodies The plasma samples were spiked with an order of magnitude higher EV concentration (1.55 × 109 particles/mL) compared to the experiments in buffer to ensure EVs will be detected in such a complex sample. As per Figure 3A, 7 nm signal shifts were obtained after subtracting the negative control when using Banti-CD9, which matched previous results obtained in buffer (Figure 2A). Although the combination with Banti-mix gave a higher signal shift in one of the repetitions (10.10 nm), the poor reproducibility of this condition (mainly due to the usage of Banti-CD81 antibody) together with larger signal shifts obtained from the negative control (Supplementary Materials Figure S2) made it a less favorable condition, similar to the results obtained in buffer. Figure 2. (A) FO-SPR sandwich bioassay established with different antibody combinations tecting SK-BR-3 EVs in buffer. Bar graphs represent the FO-SPR shifts (obtained after subtr the negative control, i.e., SPR signal for 0 particles/mL) by combining anti-HER2 as capture an with different detection antibodies (Banti-CD9, Banti-CD63, Banti-CD81 or Banti-mix) for de SK-BR-3 EVs at 1.55 × 108 particles/mL concentration (measurements shown from two indepe experiments). (B) FO-SPR-based detection of a series of SK-BR-3 EV concentrations spiked detection buffer (50 mM MES pH 6, 0.01% BSA, 0.01% Tween 20) when using the anti-HER2 CD9 antibody combination (obtained after subtracting the negative control, i.e., SPR signa particles/mL). Simple linear regression fitting was performed by GraphPad Prism software (V 8.0.1, GraphPad Software Inc., MA, USA). The dotted lines indicate the 95% prediction band new observation Error bars represent one standard deviation (n = 3) Figure 2. (A) FO-SPR sandwich bioassay established with different antibody combinations for de- tecting SK-BR-3 EVs in buffer. Bar graphs represent the FO-SPR shifts (obtained after subtracting the negative control, i.e., SPR signal for 0 particles/mL) by combining anti-HER2 as capture an- tibody with different detection antibodies (Banti-CD9, Banti-CD63, Banti-CD81 or Banti-mix) for detecting SK-BR-3 EVs at 1.55 × 108 particles/mL concentration (measurements shown from two independent experiments). (B) FO-SPR-based detection of a series of SK-BR-3 EV concentrations spiked in the detection buffer (50 mM MES pH 6, 0.01% BSA, 0.01% Tween 20) when using the anti-HER2/Banti-CD9 antibody combination (obtained after subtracting the negative control, i.e., SPR signal for 0 particles/mL). Simple linear regression ﬁtting was performed by GraphPad Prism soft- ware (Version 8.0.1, GraphPad Software Inc., MA, USA). g ( 2.1. FO-SPR Surface Functionalization with HER2-Speciﬁc Antibodies Figure 2A depicts the FO-SPR signal shift after subtracting the negative control, the latter being the SPR shift detected in the sample without spiked EVs (i.e., 0 particles/mL). Based on these results, it was observed that using Banti-CD9 or Banti-mix detection antibodies generated higher SPR shifts (around 7 nm) compared to combinations with Banti-CD63 or Banti-CD81 detection antibodies. This suggested that both Banti-CD9 and Banti-mix conditions could be considered for speciﬁcally detecting SK-BR-3 EVs on the FO-SPR platform. However, anti-HER2/Banti- CD9 was selected as the preferred option because of the lower FO-SPR signal shifts (1.21 nm and 0.97 nm) of the negative control compared to 2.14 nm and 3.31 nm obtained when using the combination of anti-HER2/Banti-mix (Supplementary Materials Figure S1). was observed that using Banti-CD9 or Banti-mix detection antibodies gen shifts (around 7 nm) compared to combinations with Banti-CD63 or Ban antibodies. This suggested that both Banti-CD9 and Banti-mix condition ered for specifically detecting SK-BR-3 EVs on the FO-SPR platform HER2/Banti-CD9 was selected as the preferred option because of the low shifts (1.21 nm and 0.97 nm) of the negative control compared to 2.1 Tetraspanins have been described as a superfamily of membrane proteins, including adhesion, signaling and adaptor proteins, that are highly organized and regulate various cell signaling pathways that affect various biological processes [32,33]. This experiment revealed how biomarker selection could affect the ﬁnal signal shift due to the heterogeneity of the tetraspanin expression proﬁle, even originating from the same EV subpopulation [33]. Our results might further build on the previously reported involvement of CD9 tetraspanin in breast cancer invasiveness and metastases in several studies with high CD9 expression levels [34]. Int. J. Mol. Sci. 2023, 24, 3764 l. Sci. 2023, 24, x FOR PEER 6 of 14 Figure 2. (A) FO-SPR sandwich bioassay established with different antibody combinations for d tecting SK-BR-3 EVs in buffer. Bar graphs represent the FO-SPR shifts (obtained after subtracti the negative control, i.e., SPR signal for 0 particles/mL) by combining anti-HER2 as capture antibo with different detection antibodies (Banti-CD9, Banti-CD63, Banti-CD81 or Banti-mix) for detecti SK-BR-3 EVs at 1.55 × 108 particles/mL concentration (measurements shown from two independe experiments). g ( 2.1. FO-SPR Surface Functionalization with HER2-Speciﬁc Antibodies (B) FO-SPR-based detection of a series of SK-BR-3 EV concentrations spiked in t detection buffer (50 mM MES pH 6, 0.01% BSA, 0.01% Tween 20) when using the anti-HER2/Ban CD9 antibody combination (obtained after subtracting the negative control, i.e., SPR signal for particles/mL). Simple linear regression fitting was performed by GraphPad Prism software (Versi 8.0.1, GraphPad Software Inc., MA, USA). The dotted lines indicate the 95% prediction bands fo new observation. Error bars represent one standard deviation (n = 3). The combination of anti-HER2/Banti-CD9 was exploited for building a calibratio curve by spiking in buffer different SK-BR-3 EV concentrations ranging from 9.7 × 106 1.55 × 108 particles/mL and also including a negative control without any EVs—0 par cles/mL. The obtained FO-SPR signal shifts after subtracting the negative control (n = were plotted as a function of SK-BR-3 EV concentration (Figure 2B). The calibration curv showed that the FO-SPR biosensor can detect SK-BR-3 EVs over the entire tested conce tration range with a limit of detection (LOD) of 2.1 × 107 particles/mL. The results indicat that the obtained LOD was 1.5-fold lower than the previously reported LOD value of rEV (3.13 × 107 particles/mL) detected in buffer using FO-SPR technology [24]. This improv ment can be related to several factors, such as (1) strong affinity of selected anti-HER antibody against isolated SK-BR-3 EVs, (2) the number of HER2 oncogenic proteins on t EVs, and (3) the difference in number and distribution of general EV biomarkers, such CD9, CD63, and CD81, between rEVs and SK-BR-3 EVs. 2.3. Development of an FO-SPR Sandwich Bioassay for Detecting SK-BR-3 EVs in Plasma To further test the potential of our FO-SPR anti-HER2/Banti-CD9 bioassay establish in buffer, SK-BR-3 EVs were spiked in 100-fold diluted pooled plasma. In this context, w first reexamined the antibody combinations by using anti-HER2 as the capture antibod together with Banti-CD9, Banti-CD63, Banti-CD81 or Banti-mix as the detection antibod The plasma samples were spiked with an order of magnitude higher EV concentratio (1.55 × 109 particles/mL) compared to the experiments in buffer to ensure EVs will be d tected in such a complex sample. As per Figure 3A, 7 nm signal shifts were obtained aft subtracting the negative control when using Banti-CD9, which matched previous resu obtained in buffer (Figure 2A). Although the combination with Banti-mix gave a high Figure 2. g ( 2.1. FO-SPR Surface Functionalization with HER2-Speciﬁc Antibodies (A) FO-SPR sandwich bioassay established with different antibody combinations for de- tecting SK-BR-3 EVs in buffer. Bar graphs represent the FO-SPR shifts (obtained after subtracting the negative control, i.e., SPR signal for 0 particles/mL) by combining anti-HER2 as capture an- tibody with different detection antibodies (Banti-CD9, Banti-CD63, Banti-CD81 or Banti-mix) for detecting SK-BR-3 EVs at 1.55 × 108 particles/mL concentration (measurements shown from two independent experiments). (B) FO-SPR-based detection of a series of SK-BR-3 EV concentrations spiked in the detection buffer (50 mM MES pH 6, 0.01% BSA, 0.01% Tween 20) when using the anti-HER2/Banti-CD9 antibody combination (obtained after subtracting the negative control, i.e., SPR signal for 0 particles/mL). Simple linear regression ﬁtting was performed by GraphPad Prism soft- ware (Version 8.0.1, GraphPad Software Inc., MA, USA). The dotted lines indicate the 95% prediction bands for a new observation. Error bars represent one standard deviation (n = 3). The combination of anti-HER2/Banti-CD9 was exploited for building a calibration curve by spiking in buffer different SK-BR-3 EV concentrations ranging from 9.7 × 106 to 1.55 × 108 particles/mL and also including a negative control without any EVs—0 particles/mL. The obtained FO-SPR signal shifts after subtracting the negative control (n = 3) were plotted as a function of SK-BR-3 EV concentration (Figure 2B). The calibration curves showed that the FO-SPR biosensor can detect SK-BR-3 EVs over the entire tested concentration range with a limit of detection (LOD) of 2.1 × 107 particles/mL. The results indicated that the obtained LOD was 1.5-fold lower than the previously re- ported LOD value of rEVs (3.13 × 107 particles/mL) detected in buffer using FO-SPR technology [24]. This improvement can be related to several factors, such as (1) strong afﬁnity of selected anti-HER2 antibody against isolated SK-BR-3 EVs, (2) the number of HER2 oncogenic proteins on the EVs, and (3) the difference in number and distribution of general EV biomarkers, such as CD9, CD63, and CD81, between rEVs and SK-BR-3 EVs. 2.3. Development of an FO-SPR Sandwich Bioassay for Detecting SK-BR-3 EVs in Plasma To further test the potential of our FO-SPR anti-HER2/Banti-CD9 bioassay established in buffer, SK-BR-3 EVs were spiked in 100-fold diluted pooled plasma. In this context, we ﬁrst reexamined the antibody combinations by using anti-HER2 as the capture antibody together with Banti-CD9, Banti-CD63, Banti-CD81 or Banti-mix as the detection antibody. g ( 2.1. FO-SPR Surface Functionalization with HER2-Speciﬁc Antibodies The dotted lines indicate the 95% prediction bands for a new observation. Error bars represent one standard deviation (n = 3). ure 2. (A) FO-SPR sandwich bioassay established with different antibody combination Figure 2. (A) FO-SPR sandwich bioassay established with different antibody combinations for de ure 2. (A) FO-SPR sandwich bioassay established with different antibody combinations ing SK-BR-3 EVs in buffer. Bar graphs represent the FO-SPR shifts (obtained after subt negative control, i.e., SPR signal for 0 particles/mL) by combining anti-HER2 as capture an h different detection antibodies (Banti-CD9, Banti-CD63, Banti-CD81 or Banti-mix) for de BR-3 EVs at 1.55 × 108 particles/mL concentration (measurements shown from two indep eriments). (B) FO-SPR-based detection of a series of SK-BR-3 EV concentrations spiked ection buffer (50 mM MES pH 6, 0.01% BSA, 0.01% Tween 20) when using the anti-HER 9 antibody combination (obtained after subtracting the negative control, i.e., SPR sign ticles/mL). Simple linear regression fitting was performed by GraphPad Prism software (V 1, GraphPad Software Inc., MA, USA). The dotted lines indicate the 95% prediction band w observation. Error bars represent one standard deviation (n = 3). Figure 2. (A) FO-SPR sandwich bioassay established with different antibody combinations for de- tecting SK-BR-3 EVs in buffer. Bar graphs represent the FO-SPR shifts (obtained after subtracting the negative control, i.e., SPR signal for 0 particles/mL) by combining anti-HER2 as capture an- tibody with different detection antibodies (Banti-CD9, Banti-CD63, Banti-CD81 or Banti-mix) for detecting SK-BR-3 EVs at 1.55 × 108 particles/mL concentration (measurements shown from two independent experiments). (B) FO-SPR-based detection of a series of SK-BR-3 EV concentrations spiked in the detection buffer (50 mM MES pH 6, 0.01% BSA, 0.01% Tween 20) when using the anti-HER2/Banti-CD9 antibody combination (obtained after subtracting the negative control, i.e., SPR signal for 0 particles/mL). Simple linear regression ﬁtting was performed by GraphPad Prism soft- ware (Version 8.0.1, GraphPad Software Inc., MA, USA). The dotted lines indicate the 95% prediction bands for a new observation. Error bars represent one standard deviation (n = 3). eriments). (B) FO-SPR-based detection of a series of SK-BR-3 EV concentrations spiked ection buffer (50 mM MES pH 6, 0.01% BSA, 0.01% Tween 20) when using the anti-HER 9 antibody combination (obtained after subtracting the negative control, i.e., SPR sign ticles/mL). Simple linear regression fitting was performed by GraphPad Prism software (V 1, GraphPad Software Inc., MA, USA). g ( 2.1. FO-SPR Surface Functionalization with HER2-Speciﬁc Antibodies g p y ( , 1 or Banti-mix) for detecting SK-BR-3 EVs at 1.55 × 109 particles/mL concentra- d detection of a series of SK-BR-3 EV concentrations in 100-fold diluted pooled ti-HER2/Banti-CD9 antibody combination (obtained after subtracting the neg- signal for 0 particles/mL). Simple linear regression fitting was performed by are The dotted lines indicate the 95% prediction bands for a new observation Banti-CD81 or Banti-mix) for detecting SK-BR-3 EVs at 1.55 × 109 particles/mL concentration. (B) FO- SPR-based detection of a series of SK-BR-3 EV concentrations in 100-fold diluted pooled plasma when using anti-HER2/Banti-CD9 antibody combination (obtained after subtracting the negative control, i.e., SPR signal for 0 particles/mL). Simple linear regression ﬁtting was performed by GraphPad Prism software. The dotted lines indicate the 95% prediction bands for a new observation. Error bars represent one standard deviation (n = 3). ne standard deviation (n = 3). we selected the anti-HER2/Banti-CD9 antibody combination to build n 100-fold diluted pooled plasma by spiking a series of SK-BR-3 EVs ging from 9.7 × 107 to 1.55 × 109 particles/mL (including the negative Consequently, we selected the anti-HER2/Banti-CD9 antibody combination to build a calibration curve in 100-fold diluted pooled plasma by spiking a series of SK-BR-3 EVs concentrations, ranging from 9.7 × 107 to 1.55 × 109 particles/mL (including the negative control). As described in the previous section, the calibration curves were ﬁtted, revealing that the FO-SPR biosensor could detect SK-BR-3 EVs throughout the tested concentration range with an LOD value of 7 × 108 particles/mL (Figure 3B). g g p ( g g d in the previous section, the calibration curves were fitted, revealing sensor could detect SK-BR-3 EVs throughout the tested concentration value of 7 × 108 particles/mL (Figure 3B). bioassay decreased by threefold compared to detection in buffer. This effect of the human blood plasma matrix, which contains approxi- mL of proteins [35] and consequently, the aspecific interference of non- with the fiber surface. Nevertheless, the obtained LOD was approxi- er than the reported concentration of EVs in plasma of cancer patients considerable potential of the established FO-SPR bioassay for sensi- oreover, while surpassing the reported LOD values from a number of WB (105-fold), ELISA (104-fold), NTA (103-fold) and μNMR (10-fold), 100 ti l iti th th iMEX t h l [17 18] g p g The LOD of the bioassay decreased by threefold compared to detection in buffer. g ( 2.1. FO-SPR Surface Functionalization with HER2-Speciﬁc Antibodies This improvement can be related to several factors, such as (1) strong afﬁnity of selected anti-HER2 antibody against isolated SK-BR-3 EVs, (2) the number of HER2 oncogenic proteins on the EVs, and (3) the difference in number and distribution of general EV biomarkers, such as CD9, CD63, and CD81, between rEVs and SK-BR-3 EVs. s, and (3) the difference in number and distribution of general EV biomark 2.3. Development of an FO-SPR Sandwich Bioassay for Detecting SK-BR-3 EVs in Plasma 9, CD63, and CD81, between rEVs and SK-BR-3 EVs. Development of an FO-SPR Sandwich Bioassay for Detecting SK-BR-3 EVs in Plasma To further test the potential of our FO-SPR anti-HER2/Banti-CD9 bioassay estab buffer, SK-BR-3 EVs were spiked in 100-fold diluted pooled plasma. In this conte t reexamined the antibody combinations by using anti-HER2 as the capture an ether with Banti-CD9, Banti-CD63, Banti-CD81 or Banti-mix as the detection ant e plasma samples were spiked with an order of magnitude higher EV concen 55 × 109 particles/mL) compared to the experiments in buffer to ensure EVs will ted in such a complex sample. As per Figure 3A, 7 nm signal shifts were obtaine btracting the negative control when using Banti-CD9, which matched previous To further test the potential of our FO-SPR anti-HER2/Banti-CD9 bioassay established in buffer, SK-BR-3 EVs were spiked in 100-fold diluted pooled plasma. In this context, we ﬁrst reexamined the antibody combinations by using anti-HER2 as the capture antibody together with Banti-CD9, Banti-CD63, Banti-CD81 or Banti-mix as the detection antibody. The plasma samples were spiked with an order of magnitude higher EV concentration (1.55 × 109 particles/mL) compared to the experiments in buffer to ensure EVs will be detected in such a complex sample. As per Figure 3A, 7 nm signal shifts were obtained after subtracting the negative control when using Banti-CD9, which matched previous results obtained in buffer (Figure 2A). Although the combination with Banti-mix gave a higher signal shift in one of the repetitions (10.10 nm), the poor reproducibility of this condition (mainly due to the usage of Banti-CD81 antibody) together with larger signal shifts obtained from the negative control (Supplementary Materials Figure S2) made it a less favorable condition, similar to the results obtained in buffer. 7 of 14 of 14 Int. J. Mol. Sci. 2023, 24, 3764 ER REVIEW Figure 3. g ( 2.1. FO-SPR Surface Functionalization with HER2-Speciﬁc Antibodies This might be due to the effect of the human blood plasma matrix, which contains approximately 60 to 80 mg/mL of proteins [35] and consequently, the aspeciﬁc interference of non-EV- related proteins with the ﬁber surface. Nevertheless, the obtained LOD was approximately 103 times lower than the reported concentration of EVs in plasma of cancer patients [16], demonstrating considerable potential of the established FO-SPR bioassay for sensitive EV analysis. Moreover, while surpassing the reported LOD values from a number of technologies, like WB (105-fold), ELISA (104-fold), NTA (103-fold) and µNMR (10-fold), our FO-SPR biosensor was 100 times less sensitive than the iMEX technology [17,18]. However, it should be noted that the iMEX technology, similar to the µNMR, detected only CD63- positive EV subpopulations without prior calibration, while the FO-SPR biosensor was previously calibrated with rEVs for reproducible and reliable detection independent from the targeted EV subpopulation [23]. Furthermore, the established FO-SPR biosensor with HER2-positive EV bioassay can potentially be used to detect EVs directly from diluted human blood plasma samples without prior EV isolation. g ( 2.1. FO-SPR Surface Functionalization with HER2-Speciﬁc Antibodies (A) FO-SPR sandwich bioassay with different antibody combinations for detecting SK-BR- 3 EVs in 100-fold diluted pooled plasma. Bar graphs represent the FO-SPR shifts from two inde- pendent measurements (after subtracting the negative control, i.e., SPR signal for 0 particles/mL) obtained by combining anti-HER2 capture antibody with different detection antibodies (Banti-CD9, Banti-CD63, Banti-CD81 or Banti-mix) for detecting SK-BR-3 EVs at 1.55 × 109 particles/mL concentra- tion. (B) FO-SPR-based detection of a series of SK-BR-3 EV concentrations in 100-fold diluted pooled plasma when using anti-HER2/Banti-CD9 antibody combination (obtained after subtracting the neg- ative control, i.e., SPR signal for 0 particles/mL). Simple linear regression fitting was performed by GraphPad Prism software. The dotted lines indicate the 95% prediction bands for a new observation. Figure 3. (A) FO-SPR sandwich bioassay with different antibody combinations for detecting SK-BR-3 EVs in 100-fold diluted pooled plasma. Bar graphs represent the FO-SPR shifts from two independent measurements (after subtracting the negative control, i.e., SPR signal for 0 particles/mL) obtained by combining anti-HER2 capture antibody with different detection antibodies (Banti-CD9, Banti-CD63, Banti-CD81 or Banti-mix) for detecting SK-BR-3 EVs at 1.55 × 109 particles/mL concentration. (B) FO- SPR-based detection of a series of SK-BR-3 EV concentrations in 100-fold diluted pooled plasma when using anti-HER2/Banti-CD9 antibody combination (obtained after subtracting the negative control, i.e., SPR signal for 0 particles/mL). Simple linear regression ﬁtting was performed by GraphPad Prism software. The dotted lines indicate the 95% prediction bands for a new observation. Error bars represent one standard deviation (n = 3). andwich bioassay with different antibody combinations for detecting SK-BR- ted pooled plasma. Bar graphs represent the FO-SPR shifts from two inde- ts (after subtracting the negative control, i.e., SPR signal for 0 particles/mL) g anti-HER2 capture antibody with different detection antibodies (Banti-CD9, 1 or Banti-mix) for detecting SK-BR-3 EVs at 1.55 × 109 particles/mL concentra- d detection of a series of SK-BR-3 EV concentrations in 100-fold diluted pooled ti-HER2/Banti-CD9 antibody combination (obtained after subtracting the neg- signal for 0 particles/mL). Simple linear regression fitting was performed by are. The dotted lines indicate the 95% prediction bands for a new observation. Figure 3. (A) FO-SPR sandwich bioassay with different antibody combinations for detecting SK-BR-3 EVs in 100-fold diluted pooled plasma. g ( 2.1. FO-SPR Surface Functionalization with HER2-Speciﬁc Antibodies Bar graphs represent the FO-SPR shifts from two independent measurements (after subtracting the negative control, i.e., SPR signal for 0 particles/mL) obtained by combining anti-HER2 capture antibody with different detection antibodies (Banti-CD9, Banti-CD63, Banti-CD81 or Banti-mix) for detecting SK-BR-3 EVs at 1.55 × 109 particles/mL concentration. (B) FO- SPR-based detection of a series of SK-BR-3 EV concentrations in 100-fold diluted pooled plasma when using anti-HER2/Banti-CD9 antibody combination (obtained after subtracting the negative control, i.e., SPR signal for 0 particles/mL). Simple linear regression ﬁtting was performed by GraphPad Prism software. The dotted lines indicate the 95% prediction bands for a new observation. Error bars represent one standard deviation (n = 3). andwich bioassay with different antibody combinations for detecting SK-BR- Figure 3. (A) FO-SPR sandwich bioassay with different antibody combinations for detecting SK-BR-3 andwich bioassay with different antibody combinations for detecting SK-BR- ted pooled plasma. Bar graphs represent the FO-SPR shifts from two inde- ts (after subtracting the negative control, i.e., SPR signal for 0 particles/mL) g anti-HER2 capture antibody with different detection antibodies (Banti-CD9, 1 or Banti-mix) for detecting SK-BR-3 EVs at 1.55 × 109 particles/mL concentra- d detection of a series of SK-BR-3 EV concentrations in 100-fold diluted pooled ti-HER2/Banti-CD9 antibody combination (obtained after subtracting the neg- signal for 0 particles/mL). Simple linear regression fitting was performed by ware. The dotted lines indicate the 95% prediction bands for a new observation. Figure 3. (A) FO-SPR sandwich bioassay with different antibody combinations for detecting SK-BR-3 EVs in 100-fold diluted pooled plasma. Bar graphs represent the FO-SPR shifts from two independent measurements (after subtracting the negative control, i.e., SPR signal for 0 particles/mL) obtained by combining anti-HER2 capture antibody with different detection antibodies (Banti-CD9, Banti-CD63, Banti-CD81 or Banti-mix) for detecting SK-BR-3 EVs at 1.55 × 109 particles/mL concentration. (B) FO- SPR-based detection of a series of SK-BR-3 EV concentrations in 100-fold diluted pooled plasma when using anti-HER2/Banti-CD9 antibody combination (obtained after subtracting the negative control, i.e., SPR signal for 0 particles/mL). Simple linear regression ﬁtting was performed by GraphPad Prism software. The dotted lines indicate the 95% prediction bands for a new observation. Error bars represent one standard deviation (n = 3). sor was 100 times less sensitive than the iMEX technolog be noted that the iMEX technology, similar to the μNMR 2.4. Expanding the Established FO-SPR Sandwich Bioassay towards MCF7 EVs mbining anti-EpCAM capture antibody with different detection antibodies ( a Banti-CD81 or Banti-mix) for detecting MCF7 EVs at 1 × 109 particles/mL con R-based detection of a series of MCF7 EV concentrations when using anti-EpC dy combination in 100-fold diluted pooled plasma (obtained after subtractin l, i.e., SPR signal for 0 particles/mL). Simple linear regression fitting was Pad Prism software The dotted lines indicate the 95% prediction bands for a ne Banti-CD81 or Banti-mix) for detecting MCF7 EVs at 1 × 109 particles/mL concentration. (B) FO-SPR- based detection of a series of MCF7 EV concentrations when using anti-EpCAM/Banti-mix antibody combination in 100-fold diluted pooled plasma (obtained after subtracting the negative control, i.e., SPR signal for 0 particles/mL). Simple linear regression ﬁtting was performed by GraphPad Prism software. The dotted lines indicate the 95% prediction bands for a new observation. Error bars represent one standard deviation (n = 3). p bars represent one standard deviation (n = 3). The combination of anti-EpCAM/Banti-mix was further used to build for MCF7 EVs spiked in 100-fold diluted pooled plasma with a series o ns, ranging from 2 × 107 to 8 × 108 particles/mL (including the negative ed a e a e FO SPR hift ( 3) e e lotted a a fu tio of EV o The combination of anti-EpCAM/Banti-mix was further used to build a calibration curve for MCF7 EVs spiked in 100-fold diluted pooled plasma with a series of EV concen- trations, ranging from 2 × 107 to 8 × 108 particles/mL (including the negative control). The obtained average FO-SPR shifts (n = 3) were plotted as a function of EV concentrations (Figure 4B). The calibration curve showed that the FO-SPR biosensor detected MCF7 EVs with an LOD value of 1.1 × 108 particles/mL, comparable to the LOD obtained from SK-BR-3 EV detection in 100-fold diluted human blood plasma. ned average FO-SPR shifts (n = 3) were plotted as a function of EV co re 4B). The calibration curve showed that the FO-SPR biosensor detecte an LOD value of 1.1 × 108 particles/mL, comparable to the LOD obtain EV detection in 100-fold diluted human blood plasma. sor was 100 times less sensitive than the iMEX technolog be noted that the iMEX technology, similar to the μNMR 2.4. Expanding the Established FO-SPR Sandwich Bioassay towards MCF7 EVs or was 100 times less sensitive than the iMEX technolog be noted that the iMEX technology similar to the μNMR 2.4. Expanding the Established FO-SPR Sandwich Bioassay towards MCF7 EVs be noted that the iMEX technology, similar to the μNMR, detected EV subpopulations without prior calibration, while the FO-SPR bio- ly calibrated with rEVs for reproducible and reliable detection inde- rgeted EV subpopulation [23]. Furthermore, the established FO-SPR 2-positive EV bioassay can potentially be used to detect EVs directly blood plasma samples without prior EV isolation. To test the applicability of the established FO-SPR sandwich bioassay, we selected another breast cancer-speciﬁc EV biomarker, namely EpCAM (epithelial cell adhesion molecule, type I transmembrane glycoprotein), to detect EVs isolated from an MCF7 breast cancer line well known to overexpress EpCAM [36]. Anti-EpCAM (20 µg/mL) was immobilized as capture antibody on the FO-SPR surface using buffers with lower ionic strength, i.e., 10 mM NaAc buffer (pH 5.2, 5.4 and 5.6) and 50 mM MES buffer (pH 6.0) with the aim of obtaining the maximum FO-SPR shift (nm). Based on the results shown in Supplementary Materials Figure S3, 10 mM NaAc buffer pH 5.6 was selected for Int. J. Mol. Sci. 2023, 24, 3764 8 of 14 immobilizing anti-EpCAM since it gave the highest FO-SPR shifts (8.16 nm and 8.28 nm) compared to other conditions. Subsequently, MCF7 EVs (1 × 109 particles/mL) were spiked in 100-fold diluted pooled plasma. Similar to HER2-speciﬁc bioassay, here we also examined different antibody combinations with anti-EpCAM as capture antibody and Banti-CD9, Banti-CD63, Banti-CD81 or Banti-mix as detection antibodies. Based on the results presented in Figure 4A, combinations with Banti-CD9 and Banti-mix gave the overall highest signal shifts after subtracting the negative control (i.e., 0 particles/mL). However, as shown in Supplementary Materials Figure S4, the negative control signal of Banti-mix was between 0.31 nm and 0.88 nm and distinctly lower than when used with anti-HER2 as capture antibody (Supplementary Materials Figure S2). That is why we selected the combination of anti-EpCAM/Banti-mix for building a calibration curve in 100-fold diluted plasma. ent antibody combinations with anti-EpCAM as capture antibody an CD63, Banti-CD81 or Banti-mix as detection antibodies. Based on the d in Figure 4A, combinations with Banti-CD9 and Banti-mix gave the ov shifts after subtracting the negative control (i.e., 0 particles/mL). sor was 100 times less sensitive than the iMEX technolog be noted that the iMEX technology, similar to the μNMR 2.4. Expanding the Established FO-SPR Sandwich Bioassay towards MCF7 EVs H n in Supplementary Materials Figure S4, the negative control signal of B en 0.31 nm and 0.88 nm and distinctly lower than when used with a re antibody (Supplementary Materials Figure S2). That is why we selec on of anti-EpCAM/Banti-mix for building a calibration curve in 100- ma. Figure 4. (A) FO-SPR sandwich bioassay with different antibody combinations for de EVs in 100-fold diluted pooled plasma. Bar graphs represent the FO-SPR shifts (after s negative control, i.e., SPR signal for 0 particles/mL) obtained from four independent m by combining anti-EpCAM capture antibody with different detection antibodies (Ban CD63, Banti-CD81 or Banti-mix) for detecting MCF7 EVs at 1 × 109 particles/mL conc FO-SPR-based detection of a series of MCF7 EV concentrations when using anti-EpC antibody combination in 100-fold diluted pooled plasma (obtained after subtracting control, i.e., SPR signal for 0 particles/mL). Simple linear regression fitting was p GraphPad Prism software The dotted lines indicate the 95% prediction bands for a new Figure 4. (A) FO-SPR sandwich bioassay with different antibody combinations for detecting MCF7 EVs in 100-fold diluted pooled plasma. Bar graphs represent the FO-SPR shifts (after subtracting the negative control, i.e., SPR signal for 0 particles/mL) obtained from four independent measurements by combining anti-EpCAM capture antibody with different detection antibodies (Banti-CD9, Banti-CD63, Banti-CD81 or Banti-mix) for detecting MCF7 EVs at 1 × 109 particles/mL concentration. (B) FO-SPR- based detection of a series of MCF7 EV concentrations when using anti-EpCAM/Banti-mix antibody combination in 100-fold diluted pooled plasma (obtained after subtracting the negative control, i.e., SPR signal for 0 particles/mL). Simple linear regression ﬁtting was performed by GraphPad Prism software. The dotted lines indicate the 95% prediction bands for a new observation. Error bars represent one standard deviation (n = 3). 4. (A) FO-SPR sandwich bioassay with different antibody combinations for d 100-fold diluted pooled plasma. Bar graphs represent the FO-SPR shifts (after s ve control, i.e., SPR signal for 0 particles/mL) obtained from four independent mbining anti-EpCAM capture antibody with different detection antibodies (Ban Banti-CD81 or Banti-mix) for detecting MCF7 EVs at 1 × 109 particles/mL con R-based detection of a series of MCF7 EV concentrations when using anti-EpC dy combination in 100-fold diluted pooled plasma (obtained after subtractin , i.e., SPR signal for 0 particles/mL). Simple linear regression fitting was Pad Prism software The dotted lines indicate the 95% prediction bands for a new Figure 4. sor was 100 times less sensitive than the iMEX technolog be noted that the iMEX technology, similar to the μNMR 2.4. Expanding the Established FO-SPR Sandwich Bioassay towards MCF7 EVs (A) FO-SPR sandwich bioassay with different antibody combinations for detecting MCF7 EVs in 100-fold diluted pooled plasma. Bar graphs represent the FO-SPR shifts (after subtracting the negative control, i.e., SPR signal for 0 particles/mL) obtained from four independent measurements by combining anti-EpCAM capture antibody with different detection antibodies (Banti-CD9, Banti-CD63, Banti-CD81 or Banti-mix) for detecting MCF7 EVs at 1 × 109 particles/mL concentration. (B) FO-SPR- based detection of a series of MCF7 EV concentrations when using anti-EpCAM/Banti-mix antibody combination in 100-fold diluted pooled plasma (obtained after subtracting the negative control, i.e., SPR signal for 0 particles/mL). Simple linear regression ﬁtting was performed by GraphPad Prism software. The dotted lines indicate the 95% prediction bands for a new observation. Error bars represent one standard deviation (n = 3). e 4. (A) FO-SPR sandwich bioassay with different antibody combinations for d Figure 4. (A) FO-SPR sandwich bioassay with different antibody combinations for detecting MCF7 4. (A) FO-SPR sandwich bioassay with different antibody combinations for de 100-fold diluted pooled plasma. Bar graphs represent the FO-SPR shifts (after s ve control, i.e., SPR signal for 0 particles/mL) obtained from four independent m mbining anti-EpCAM capture antibody with different detection antibodies (Ban Banti-CD81 or Banti-mix) for detecting MCF7 EVs at 1 × 109 particles/mL con R-based detection of a series of MCF7 EV concentrations when using anti-EpC dy combination in 100-fold diluted pooled plasma (obtained after subtractin l, i.e., SPR signal for 0 particles/mL). Simple linear regression fitting was Pad Prism software The dotted lines indicate the 95% prediction bands for a new Figure 4. (A) FO-SPR sandwich bioassay with different antibody combinations for detecting MCF7 EVs in 100-fold diluted pooled plasma. Bar graphs represent the FO-SPR shifts (after subtracting the negative control, i.e., SPR signal for 0 particles/mL) obtained from four independent measurements by combining anti-EpCAM capture antibody with different detection antibodies (Banti-CD9, Banti-CD63, Banti-CD81 or Banti-mix) for detecting MCF7 EVs at 1 × 109 particles/mL concentration. (B) FO-SPR- based detection of a series of MCF7 EV concentrations when using anti-EpCAM/Banti-mix antibody combination in 100-fold diluted pooled plasma (obtained after subtracting the negative control, i.e., SPR signal for 0 particles/mL). Simple linear regression ﬁtting was performed by GraphPad Prism software. The dotted lines indicate the 95% prediction bands for a new observation. Error bars represent one standard deviation (n = 3). 2.5. Testing FO-SPR Bioassays’ Speciﬁcity across Plasma Samples from Healthy Donors 9 2.5. Testing FO-SPR Bioassays’ Speciﬁcity across Plasma Samples from Healthy Donors 9 In the ﬁnal step, we evaluated the speciﬁcity of the established FO-SPR anti-HER2/B anti-CD9 and anti-EpCAM/Banti-mix bioassays and their potential to be used for the detection of breast cancer EVs in patient samples. This was done by running the two sandwich bioassays in the 100-fold diluted plasma from 10 individual healthy donors unknown to be diagnosed with breast cancer (P1–P10, Figure 5). As can be seen from Figure 5A,B, the SPR shift remained for all the samples well below 2 nm. Moreover, the obtained signal was similar to the one from 100-fold diluted pooled plasma (control), as well as from rEVs spiked in 100-fold diluted pooled plasma (1 × 109 particles/mL), which do not express any of the breast cancer biomarkers [24]. Contrary to this, shifts of approximately 8 nm were obtained when SK-BR-3 and MCF7 EVs were spiked in 100-fold diluted pooled plasma (1 × 109 particles/mL), demonstrating that this bioassay is speciﬁc to EVs that express HER2 or EpCAM proteins on their surface when anti-HER2 or anti-EpCAM were used as capture antibodies, respectively. These results support our established bioassay being not only sensitive but also highly speciﬁc to the EVs of interest. The low negative control signal detected in healthy donors highlights the potential of reliable direct detection of cancer-speciﬁc EVs in human blood plasma. to the one from 100-fold diluted pooled plasma (control), as well as n 100-fold diluted pooled plasma (1 × 109 particles/mL), which do not reast cancer biomarkers [24]. Contrary to this, shifts of approximately when SK-BR-3 and MCF7 EVs were spiked in 100-fold diluted pooled icles/mL), demonstrating that this bioassay is specific to EVs that ex- AM proteins on their surface when anti-HER2 or anti-EpCAM were ibodies, respectively. These results support our established bioassay itive but also highly specific to the EVs of interest. The low negative ted in healthy donors highlights the potential of reliable direct detec- fic EVs in human blood plasma. Figure 5. Specificity testing of the FO-SPR bioassays in 100-fold diluted plasma using (A) anti- HER2/Banti-CD9 and (B) anti-EpCAM/Banti-mix antibody combinations. P1 to 10 are plasma sam- ples of 10 individual healthy donors unknown to be diagnosed with breast cancer. Control is 100- fold diluted pooled plasma. sor was 100 times less sensitive than the iMEX technolog be noted that the iMEX technology, similar to the μNMR 2.4. Expanding the Established FO-SPR Sandwich Bioassay towards MCF7 EVs Although the human body is abundant with EVs, the quantity of cancer man blood is an undetermined variable that depends on numerous fac ate of the disease, age and gender of the patients, and the applied treatm p Although the human body is abundant with EVs, the quantity of cancer-speciﬁc EVs in human blood is an undetermined variable that depends on numerous factors, such as the state of the disease, age and gender of the patients, and the applied treatment, among others. Even though the achieved LOD values are very promising compared to the conventional techniques, further improvements are required to reach higher sensitivity to utilize FO-SPR biosensor for real clinical settings. Avenues for further research might include investigating alternative antibodies with higher afﬁnities towards EV surface proteins or other surface chemistries, such as NTA-SAM [25], to establish an organized surface through oriented antibody immobilization. Int. J. Mol. Sci. 2023, 24, 3764 9 of 14 2.5. Testing FO-SPR Bioassays’ Speciﬁcity across Plasma Samples from Healthy Donors 9 Two additional controls represent (1) rEVs spiked in 100-fold diluted pooled plasma and (2) SK-BR-3 (in panel (A) or MCF7 EVs (in panel (B) spiked in 100-fold diluted pooled plasma. For panel (A), two independent measurements were performed for all the samples, whereas the number of repetitions was three for panel (B) (although some of the obtained values are close to zero and thus barely visible). Figure 5. Speciﬁcity testing of the FO-SPR bioassays in 100-fold diluted plasma using (A) anti- HER2/Banti-CD9 and (B) anti-EpCAM/Banti-mix antibody combinations. P1 to 10 are plasma samples of 10 individual healthy donors unknown to be diagnosed with breast cancer. Control is 100-fold diluted pooled plasma. Two additional controls represent (1) rEVs spiked in 100-fold diluted pooled plasma and (2) SK-BR-3 (in panel (A) or MCF7 EVs (in panel (B) spiked in 100-fold diluted pooled plasma. For panel (A), two independent measurements were performed for all the samples, whereas the number of repetitions was three for panel (B) (although some of the obtained values are close to zero and thus barely visible). testing of the FO-SPR bioassays in 100-fold diluted plasma using (A) anti- (B) anti-EpCAM/Banti-mix antibody combinations. P1 to 10 are plasma sam- healthy donors unknown to be diagnosed with breast cancer. Control is 100- lasma. Two additional controls represent (1) rEVs spiked in 100-fold diluted 2) SK-BR-3 (in panel (A) or MCF7 EVs (in panel (B) spiked in 100-fold diluted anel (A), two independent measurements were performed for all the samples, of repetitions was three for panel (B) (although some of the obtained values Figure 5. Speciﬁcity testing of the FO-SPR bioassays in 100-fold diluted plasma using (A) anti- HER2/Banti-CD9 and (B) anti-EpCAM/Banti-mix antibody combinations. P1 to 10 are plasma samples of 10 individual healthy donors unknown to be diagnosed with breast cancer. Control is 100-fold diluted pooled plasma. Two additional controls represent (1) rEVs spiked in 100-fold diluted pooled plasma and (2) SK-BR-3 (in panel (A) or MCF7 EVs (in panel (B) spiked in 100-fold diluted pooled plasma. For panel (A), two independent measurements were performed for all the samples, whereas the number of repetitions was three for panel (B) (although some of the obtained values are close to zero and thus barely visible). Int. J. Mol. Sci. 2023, 24, 3764 10 of 14 10 of 14 3.1. Reagents and Antibodies All buffer reagents were obtained from Sigma-Aldrich (Bornem, Belgium), unless stated otherwise, and were made using deionized water puriﬁed with the Milli-Q Plus sys- tem (Millipore, Marlborough, MA, USA). The following suppliers were used for purchasing the buffer reagents: (1) AppliChem GmbH (Darmstadt, Germany) for Tween 20, (2) VWR (Leuven, Belgium) for ethanol, hydrochloric acid and sodium hydroxide, (3) Thermo Fisher Scientific (Erembodegem, Belgium) for superblock buffer, acetone and N-hydroxysuccinimide (NHS), (4) Merck Life Science (Hoeilaart, Belgium) for M N-(3-dimethylaminopropyl)- N′-ethylcarbodiimide hydrochloride (EDC), (5) GERBU Biotechnik GmbH (Heidelberg, Germany) for COOH-SAM and (6) BBI Solutions (Cardiff, UK) for AuNPs, conjugated with goat anti-biotin antibody, of 40 nm diameter and optical density (OD) of 10. g y p y In this study, we used EV-validated antibodies previously characterized through various quality-control tests, such as WB, ﬂow cytometry, ELISA, and FO-SPR technology, as demonstrated in our previous study [23]. Mouse monoclonal EpCAM-speciﬁc antibody (anti-EpCAM, Cat. no: 324202), as well as biotinylated anti-CD63 (Cat. no: 353018), biotinylated anti-CD9 (Cat. no: 312110) and biotinylated anti-CD81 (Cat. no: 349514) antibodies (Banti-CD9, Banti-CD63 and Banti-CD81, respectively) were obtained from Biolegend (ImTec Diagnostics, Antwerp, Belgium). HER2-speciﬁc antibody (anti-HER2, 4D5), which is a murine IgG1 equivalent of trastuzumab, was obtained from PharmAbs, the KU Leuven Antibody Center (Leuven, Belgium) [37]. All antibody concentrations are indicated in Section 3.4. Pooled plasma was obtained from more than 25 healthy donors, and plasma of 10 random healthy donors, unknown to be diagnosed with breast cancer, was recruited at the Laboratory for Thrombosis Research (KU Leuven, Campus Kulak Kortrijk, Belgium) with a signed informed consent form. Six tubes per donor (±35 mL blood per donor) were collected using BD vacutainer trisodium citrate tubes (BD 366575, BD, Temse, Belgium), and all the tubes were pooled together into several 50 mL tubes. Later, they were centrifuged for 15 min at 2200 rpm, and plasma was collected with a sterile pipette (612-1685, VWR, Leuven, Belgium) in a sterile container on ice. After processing all blood samples, the pooled plasma was put in a warm water bath at 37 ◦C for 7 min to be aliquoted afterwards to 10 mL in 15 mL tubes and stored at −80 ◦C. 3.2. Isolation and Characterization of SK-BR-3, MCF7 and rEVs SK-BR-3, MCF7 and rEVs [23,24] were separated from the culture medium of SK-BR-3, MCF7 (both originating from human breast cancer) and HEK293T (human embryonic kidney) cell lines, respectively, using OptiPrep™density gradient (Alere Technologies AS, Oslo, Norway) as previously described [25,38]. A short description of characterization and isolation of rEVs and OptiPrep™density gradient is given in Supplementary Materials. Aliquots of EVs were stored at −80 ◦C until further use and thawed carefully on ice just before the analysis. y The EV solutions were diluted in phosphate-buffered saline (PBS) buffer to a ﬁnal volume of 800 µL for NTA [39]. The concentration and particle size distribution of EV samples were identiﬁed using a NanoSight LM10 (Malvern Instruments, Worcestershire, UK) conﬁgured with a 405 nm laser and connected to an sCMOS camera with the detection threshold set between 4 to 5 for recording. Six videos of 30 s were taken to calculate particle size and concentration distributions using NanoSight NTA analytical software (version 2.3, Nanosight Ltd., Wiltshire, UK) as presented in Supplementary Figure S5. 3.4. FO-SPR Sandwich Bioassay for Detection of EVs in Buffer and Plasma After the functionalization of the FO-SPR probes with COOH SAM, they were im- mersed in 0.4 EDC/0.1 M NHS solution dissolved in 50 mM MES buffer at pH 6 for 15 min for the activation of COOH groups on the ﬁber surface. Then, breast cancer EV-speciﬁc capture antibodies (i.e., anti-HER2 or anti-EpCAM) diluted in different buffers as speciﬁed throughout this study (i.e., 10 mM NaAc buffer at pH 5.2, 5.4 and 5.6, and 50 mM MES buffer at pH 6) were bound covalently to the activated COOH groups at a concentration of 20 µg/mL with shaking at 200 rpm, for 900 s. Afterwards, the FO-SPR probes were immersed consecutively in blocking buffers: superblock (300 s), 1 M ethanolamine (600 s) at pH 8, and again in superblock (300 s) to minimize the aspeciﬁc binding. The biofunctionalized FO-SPR probes were then introduced to the detection buffer (50 mM MES pH 6 supplemented with 0.01% BSA and 0.01% Tween 20) or plasma samples diluted 100-fold in the detection buffer. Next, they were dipped into samples containing SK-BR-3 (9.7 × 106 to 1.55 × 108 particles/mL spiked in detection buffer or 9.7 × 107 to 1.55 × 109 particles/mL spiked in 100-fold diluted plasma) or MCF7 EVs (2 × 107 to 8 × 108 particles/mL spiked in 100-fold diluted plasma), including 0 particles/mL as a negative control for 20 min to record the real-time, label-free binding of EVs. In order to achieve signal ampliﬁcation, the FO-SPR probes were subsequently immersed in the same detection buffer or 100-fold diluted plasma with 10 µg/mL concentration of biotinylated detection antibodies (Banti-CD9, Banti-CD63, Banti-CD81 or Banti-mix) for 900 s at 200 rpm. p Finally, the FO-SPR probes were reintroduced into the detection buffer or 100-fold diluted plasma, followed by immersion into the PBS with 0.5% of BSA to obtain a baseline signal for the next step with AuNPs functionalized with goat anti-biotin antibody. These AuNPs were prepared, prior to their use in the FO-SPR bioassay, by centrifugation at 5000 rpm for 30 min at 4 ◦C. After removing the supernatant, AuNPs were resuspended in PBS with 0.5% of BSA with 1:10 dilution ratio to obtain an OD of 1. FO-SPR probes were immersed in 150 µL solution of AuNPs for 1 h. All the steps of EV detection bioassay were performed at room temperature. 3.5. Data Analysis The obtained data were recorded and further processed using custom-built software developed by FOx Biosystems Ltd. (Diepenbeek, Belgium). The calibration curves were obtained by plotting the obtained FO-SPR shifts—after subtracting the negative control sig- nal at 0 particles/mL—as a function of the different EV concentrations. A linear regression curve was ﬁtted using GraphPad Prism software. The LOD values were calculated as EV concentrations (particles/mL) corresponding to the sum of the respective blank signal and three times the standard deviation of the blank signal. 3.3. FO-SPR Biosensor and Manufacturing of FO-SPR Probes A benchtop FO-SPR biosensor, introduced by our group and commercialized by FOx Biosystems (Diepenbeek, Belgium), was used to perform FO-SPR bioassays for EV detection. FO-SPR probes were prepared manually for each experiment as previously described [40,41]. In summary, a ﬁnal length of 4.3 cm of FO-SPR probes was cut from Int. J. Mol. Sci. 2023, 24, 3764 11 of 14 11 of 14 a multimode optical ﬁber (TEQS, Thorlabs, Munich, Germany) with a core diameter of 400 µm and endings were stripped and cleaned, leaving 0.6 cm on the sensor side. Later, a thin layer (~50 nm) of gold was sputtered using a sputter coater (Quorum Q150T ES, Quorum Technologies, East Sussex, UK). Gold-coated FO-SPR probes were functionalized at 4 ◦C in a 0.1 mM ethanol/COOH SAM solution (volume ratio of 9:1) for 2 days. Finally, just before the experiment, the probes were washed with ethanol to remove any unbound material and used immediately. 4. Conclusions This study reported on the development of a breast cancer-speciﬁc EV detection bioas- say directly in blood plasma using the FO-SPR biosensor, which was previously calibrated with rEVs as a reference material [23]. For the ﬁrst time, full calibration curves were estab- lished for detecting EVs with the FO-SPR bioassay, which allowed us to determine the LODs of 7 × 108 particles/mL and 1.1 × 108 particles/mL when using anti-HER2/Banti-CD9 and anti-EpCAM/Banti-mix as antibody combinations, respectively. Moreover, because of the implemented sandwich bioassay format, information regarding the colocalization of EV Int. J. Mol. Sci. 2023, 24, 3764 12 of 14 12 of 14 surface proteins could be obtained using FO-SPR biosensor. Cancer-speciﬁc EVs were dif- ferentiated from those originating from healthy cells as long as a cancer-speciﬁc biomarker was present. Previously, the colocalization of EV surface biomarkers was obtained either by (1) imaging strategies that require expensive equipment, while being restricted by the availability of the ﬂuorescent labels/imaging channels or (2) conventional ELISA that re- quires lengthy preparation steps and processes that are highly dependent on sample purity for EV analysis. However, the FO-SPR biosensor offers a possibility to study colocalization of proteins on EVs while enabling real-time detection directly in the crude samples in a short time-to-result manner. Finally, the time to result of FO-SPR detection bioassay was further shortened for cancer-speciﬁc EV detection (compared to the previous work [23]), resulting in 2 h and 40 min, leaving additional room for improvements. These capabilities of the bioassay combined with essential features of the FO-SPR biosensor reveal the great potential of this technology to be used as a standardized diagnostic tool and signiﬁcantly contributing to the EV research ﬁeld. Supplementary Materials: The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/ijms24043764/s1. Author Contributions: Conceptualization, D.S. and J.L.; methodology, Y.Y. and D.S.; software, Y.Y.; validation, Y.Y.; formal analysis, Y.Y.; investigation, Y.Y., K.D. and H.S.K.T.; resources, R.B., D.T., N.G., A.H. and J.L.; data curation, Y.Y.; writing—original draft preparation, Y.Y. and D.S.; writing—review and editing, K.D., H.S.K.T., R.B., D.T., N.G., A.H. and J.L.; visualization, Y.Y.; supervision, J.L. and D.S.; project administration, J.L. and D.S.; funding acquisition, A.H., J.L. and D.S. All authors have read and agreed to the published version of the manuscript. Data Availability Statement: Not applicable. Acknowledgments: We are grateful to the Laboratory for Thrombosis Research (KU Leuven, Campus Kulak Kortrijk, Belgium) for providing us with pooled plasma samples from healthy donors and plasma samples from 10 healthy individuals. Conﬂicts of Interest: Jeroen Lammertyn is a board member of FOx Biosystems, a spin-off com- pany of KU Leuven commercializing FO-SPR platforms, next to the principal investigator of the Biosensors group. Biosensors group. 4. Conclusions Funding: This research was funded by Research Foundation-Flanders (FWO SBO/S006319N), VLK (C8744), Kom op tegen Kanker (Stand up to Cancer, the Flemish cancer society, projectID: 13050) and the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska- Curie grant agreement 764281 (H2020-MSCA-ITN-AiPBAND). Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of KU Leuven (protocol code S66725 and date of approval 18 November 2017). Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of KU Leuven (protocol code S66725 and date of approval 18 November 2017). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Not applicable. , , [ ] [ ] 6. Liu, S.Y.; Liao, Y.; Hosseinifard, H.; Imani, S.; Wen, Q.L. Diagnostic Role of Extracellular Vesicles in Cancer: A Comprehensive Systematic Review and Meta-Analysis. Front. Cell Dev. Biol. 2021, 9, 2749. [CrossRef] References Shao, H.; Chung, J.; Balaj, L.; Charest, A.; Bigner, D.D.; Carter, B.S.; Hochberg, F.H.; Breakeﬁeld, X.O.; Weissleder, R.; Lee, H. Protein Typing of Circulating Microvesicles Allows Real-Time Monitoring of Glioblastoma Therapy. Nat. Med. 2012, 18, 1835–1840. [CrossRef] [ ] 18. Jeong, S.; Park, J.; Pathania, D.; Castro, C.M.; Weissleder, R.; Lee, H. Integrated Magneto-Electrochemical Sensor for Exosome Analysis. ACS Nano 2016, 10, 1802. [CrossRef] 19. Park, J.; Park, J.S.; Huang, C.H.; Jo, A.; Cook, K.; Wang, R.; Lin, H.Y.; van Deun, J.; Li, H.; Min, J.; et al. A High-Throughput Magneto-Electrochemical Array for the Integrated Isolation and Proﬁling of Extracellular Vesicles from Plasma. Nat. Biomed. Eng. 2021, 5, 678. [CrossRef] 20. Wang, Q.; Zou, L.; Yang, X.; Liu, X.; Nie, W.; Zheng, Y.; Cheng, Q.; Wang, K. Direct Quantiﬁcation of Cancerous Exosomes via Surface Plasmon Resonance with Dual Gold Nanoparticle-Assisted Signal Ampliﬁcation. Biosens. Bioelectron. 2019, 135, 129–136. [CrossRef] 21. Fan, Y.; Duan, X.; Zhao, M.; Wei, X.; Wu, J.; Chen, W.; Liu, P.; Cheng, W.; Cheng, Q.; Ding, S. High-Sensitive and Multiplex Biosensing Assay of NSCLC-Derived Exosomes via Different Recognition Sites Based on SPRi Array. Biosens. Bioelectron. 2020, 154, 112066. [CrossRef] 22. Yang, Y.; Zhai, C.; Zeng, Q.; Khan, A.L.; Yu, H. Multifunctional Detection of Extracellular Vesicles with Su Microscopy. Anal. Chem. 2020, 92, 4884–4890. [CrossRef] [PubMed] 23. Yildizhan, Y.; Vajrala, V.S.; Geeurickx, E.; Declerck, C.; Duskunovic, N.; de Sutter, D.; Noppen, S.; Delport, F.; Schols, D.; Swinnen, J.V.; et al. FO-SPR Biosensor Calibrated with Recombinant Extracellular Vesicles Enables Speciﬁc and Sensitive Detection Directly in Complex Matrices. J. Extracell. Vesicles 2021, 10, e12059. [CrossRef] [PubMed] p Directly in Complex Matrices. J. Extracell. Vesicles 2021, 10, e12059. [CrossRef] [PubMed] 24. Geeurickx, E.; Tulkens, J.; Dhondt, B.; Van Deun, J.; Lippens, L.; Vergauwen, G.; Heyrman, E.; De Sutter, D.; Gevaert, K.; Impens, F.; et al. The Generation and Use of Recombinant Extracellular Vesicles as Biological Reference Material. Nat. Commun. 2019, 10, 3288. [CrossRef] [PubMed] 25. Qu, J.H.; Peeters, B.; Delport, F.; Vanhoorelbeke, K.; Lammertyn, J.; Spasic, D. Gold Nanoparticle Enhanced Multiplexed Biosensing on a Fiber Optic Surface Plasmon Resonance Probe. Biosens. Bioelectron. 2021, 192, 113549. [CrossRef] [PubMed] 26. Horta, S.; Qu, J.H.; Dekimpe, C.; Bonnez, Q.; Vandenbulcke, A.; Tellier, E.; Kaplanski, G.; Delport, F.; Geukens, N.; Lammertyn, J.; et al. References The Biology, Function, and Biomedical Applications of Exosomes. Science 2020, 367, eaau6977. [CrossRef] 7. Kalluri, R.; LeBleu, V.S. The Biology, Function, and Biomedical Applications of Exosomes. Science 2020, 367, eaau6977. [CrossRef] 8. Gandham, S.; Su, X.; Wood, J.; Nocera, A.L.; Alli, S.C.; Milane, L.; Zimmerman, A.; Amiji, M.; Ivanov, A.R. Technologies and Standardization in Research on Extracellular Vesicles. Trends Biotechnol. 2020, 38, 1066–1098. [CrossRef] [PubMed] 9. Shao, H.; Im, H.; Castro, C.M.; Breakeﬁeld, X.; Weissleder, R.; Lee, H. New Technologies for Analysis of Extracellular Vesicles. j g Standardization in Research on Extracellular Vesicles. Trends Biotechnol. 2020, 38, 1066–1098. [CrossRef] [PubMed] 9. Shao, H.; Im, H.; Castro, C.M.; Breakeﬁeld, X.; Weissleder, R.; Lee, H. New Technologies for Analysis of Extracellular Vesicles. Chem. Rev. 2018, 118, 1917–1950. [CrossRef] 10. Théry, C.; Witwer, K.W.; Aikawa, E.; Alcaraz, M.J.; Anderson, J.D.; Andriantsitohaina, R.; Antoniou, A.; Arab, T.; Archer, F.; Atkin-Smith, G.K.; et al. Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018): A Position Statement of the International Society for Extracellular Vesicles and Update of the MISEV2014 Guidelines. J. Extracell. Vesicles 2018, 7, 1535750. [CrossRef] [ ] 11. Coumans, F.A.W.; Gool, E.L.; Nieuwland, R. Bulk Immunoassays for Analysis of Extracellular Vesicles. Platelets 2017, 28, 242–248. [CrossRef] [PubMed] 12. Welsh, J.A.; van der Pol, E.; Bettin, B.A.; Carter, D.R.F.; Hendrix, A.; Lenassi, M.; Langlois, M.A.; Llorente, A.; van de Nes, A.S.; Nieuwland, R.; et al. Towards Deﬁning Reference Materials for Measuring Extracellular Vesicle Refractive Index, Epitope Abundance, Size and Concentration. J. Extracell. Vesicles 2020, 9, 1816641. [CrossRef] [PubMed] 13. Bandu, R.; Oh, J.W.; Kim, K.P. Mass Spectrometry-Based Proteome Proﬁling of Extracellular Vesicles and Their Roles in Cancer Biology. Exp. Mol. Med. 2019, 51, 1–10. [CrossRef] [PubMed] 14. Nishida-Aoki, N.; Izumi, Y.; Takeda, H.; Takahashi, M.; Ochiya, T.; Bamba, T. Lipidomic Analysis of Cells and Extracellular Vesicles from High- and Low-Metastatic Triple-Negative Breast Cancer. Metabolites 2020, 10, 67. [CrossRef] 15. Jalaludin, I.; Lubman, D.M.; Kim, J. A Guide to Mass Spectrometric Analysis of Extracellular Vesicle Proteins for Biomarker Discovery. Mass Spectrom. Rev. 2023, 42, 844–872. [CrossRef] y p 16. Yekula, A.; Minciacchi, V.R.; Morello, M.; Shao, H.; Park, Y.; Zhang, X.; Muralidharan, K.; Freeman, M.R.; Weissleder, R.; Lee, H.; et al. Large and Small Extracellular Vesicles Released by Glioma Cells In Vitro and In Vivo. J. Extracell. Vesicles 2020, 9, 1689784. [CrossRef] , [ ] 17. References G.; Raposo, G. Shedding Light on the Cell Biology of Extracellular Vesicles. Nat. Rev. Mol. Cell Biol. 2018, [PubMed] 1. Van Niel, G.; D’Angelo, G.; Raposo, G. Shedding Light on the Cell Biology of Extracellular Vesicles. Nat 19, 213–228. [CrossRef] [PubMed] 2. Ma, Y.; Dong, S.; Li, X.; Kim, B.Y.S.; Yang, Z.; Jiang, W. Extracellular Vesicles: An Emerging Nanoplatform for Cancer Therapy. Front. Oncol. 2021, 10, 3340. [CrossRef] [PubMed] 3. Xu, R.; Rai, A.; Chen, M.; Suwakulsiri, W.; Greening, D.W.; Simpson, R.J. Extracellular Vesicles in Cancer—Implications for Future Improvements in Cancer Care. Nat. Rev. Clin. Oncol. 2018, 15, 617–638. [CrossRef] 4. Becker, A.; Thakur, B.K.; Weiss, J.M.; Kim, H.S.; Peinado, H.; Lyden, D. Extracellular Vesicles in Cancer: Cell-to-Cell Mediators of Metastasis. Cancer Cell 2016, 30, 836–848. [CrossRef] [PubMed] 4. Becker, A.; Thakur, B.K.; Weiss, J.M.; Kim, H.S.; Peinado, H.; Lyden, D. Extracellular Vesicles in Cancer: Cell-to-Cell Mediators of Metastasis. Cancer Cell 2016, 30, 836–848. [CrossRef] [PubMed] 5. Simeone, P.; Bologna, G.; Lanuti, P.; Pierdomenico, L.; Guagnano, M.T.; Pieragostino, D.; del Boccio, P.; Vergara, D.; Marchisio, M.; Miscia, S.; et al. Extracellular Vesicles as Signaling Mediators and Disease Biomarkers across Biological Barriers. Int. J. Mol. Sci. 2020, 21, 2514. [CrossRef] [PubMed] 5. Simeone, P.; Bologna, G.; Lanuti, P.; Pierdomenico, L.; Guagnano, M.T.; Pieragostino, D.; del Boccio, P.; Vergara, D.; Marchisio, M.; Miscia, S.; et al. Extracellular Vesicles as Signaling Mediators and Disease Biomarkers across Biological Barriers. Int. J. Mol. Sci. 2020, 21, 2514. [CrossRef] [PubMed] [ ] [ ] 6. Liu, S.Y.; Liao, Y.; Hosseinifard, H.; Imani, S.; Wen, Q.L. Diagnostic Role of Extracellular Vesicles in Cancer: A Comprehensive Systematic Review and Meta-Analysis. Front. Cell Dev. Biol. 2021, 9, 2749. [CrossRef] 13 of 14 13 of 14 Int. J. Mol. Sci. 2023, 24, 3764 7. Kalluri, R.; LeBleu, V.S. The Biology, Function, and Biomedical Applications of Exosomes. Science 2020, 367, eaau6977. [CrossRef] 8. Gandham, S.; Su, X.; Wood, J.; Nocera, A.L.; Alli, S.C.; Milane, L.; Zimmerman, A.; Amiji, M.; Ivanov, A.R. Technologies and Standardization in Research on Extracellular Vesicles. Trends Biotechnol. 2020, 38, 1066–1098. [CrossRef] [PubMed] 7. Kalluri, R.; LeBleu, V.S. The Biology, Function, and Biomedical Applications of Exosomes. Science 2020, 367, eaau6977. [CrossRef] 8 Gandham S ; Su X ; Wood J ; Nocera A L ; Alli S C ; Milane L ; Zimmerman A ; Amiji M ; Ivanov A R Technologies and 7. Kalluri, R.; LeBleu, V.S. 29. Lu, J.; van Stappen, T.; Spasic, D.; Delport, F.; Vermeire, S.; Gils, A.; Lammertyn, J. Fiber Optic-SPR Platform for Fast and Sensitive Inﬂiximab Detection in Serum of Inﬂammatory Bowel Disease Patients. Biosens. Bioelectron. 2016, 79, 173–179. [CrossRef] References Co(III)-NTA Mediated Antigen Immobilization on a Fiber Optic-SPR Biosensor for Detection of Autoanti- bodies in Autoimmune Diseases: Application in Immune-Mediated Thrombotic Thrombocytopenic Purpura. Anal. Chem. 2020, 92, 13880–13887. [CrossRef] 27. Ren, W.; Liu, Y.; Wan, S.; Fei, C.; Wang, W.; Chen, Y.; Zhang, Z.; Wang, T.; Wang, J.; Zhou, L.; et al. BMP9 Inhibits Proliferation and Metastasis of HER2-Positive SK-BR-3 Breast Cancer Cells through ERK1/2 and PI3K/AKT Pathways. PLoS ONE 2014, 9, e96816. [CrossRef] 28. Li, Z.; Zhuo, W.; Chen, L.; Zhang, X.; Chen, C.; Hu, D.; Chen, Y.; Yang, J.; Zhou, Y.; Mao, M.; et al. Establishment and Characterization of a HER2-Positive Cell Line Derived from the Pleural Effusion of a Drug-Resistant Breast Cancer Patient. Front. Cell Dev. Biol. 2021, 9, 1349. [CrossRef] 29. Lu, J.; van Stappen, T.; Spasic, D.; Delport, F.; Vermeire, S.; Gils, A.; Lammertyn, J. Fiber Optic-SPR Platform for Fast and Sensitive Inﬂiximab Detection in Serum of Inﬂammatory Bowel Disease Patients. Biosens. Bioelectron. 2016, 79, 173–179. [CrossRef] Int. J. Mol. Sci. 2023, 24, 3764 14 of 14 14 of 14 30. Lu, J.; Spasic, D.; Delport, F.; van Stappen, T.; Detrez, I.; Daems, D.; Vermeire, S.; Gils, A.; Lammertyn, J. Immunoassay for Detection of Inﬂiximab in Whole Blood Using a Fiber-Optic Surface Plasmon Resonance Biosensor. Anal. Chem. 2017, 89, 3664–3671. [CrossRef] [ ] 31. Zhang, J.; Shi, J.; Zhang, H.; Zhu, Y.; Liu, W.; Zhang, K.; Zhang, Z. Localized Fluorescent Imaging Individual Extracellular Vesicles Using Rolling Circle Ampliﬁcation for Cancer Diagnosis. J. Extracell. 31. Zhang, J.; Shi, J.; Zhang, H.; Zhu, Y.; Liu, W.; Zhang, K.; Zhang, Z. Localized Fluorescent Imaging of Multiple Proteins on Individual Extracellular Vesicles Using Rolling Circle Ampliﬁcation for Cancer Diagnosis. J. Extracell. Vesicles 2020, 10, 12025. 32. Andreu, Z.; Yáñez-Mó, M. Tetraspanins in Extracellular Vesicle Formation and Function. Front. Immunol. 2014, 5, 442. [CrossRef] [PubMed] g g p g J , , 32. Andreu, Z.; Yáñez-Mó, M. Tetraspanins in Extracellular Vesicle Formation and Function. Front. Immunol. 2014, 5, 442. [CrossRef] [PubMed] 32. Andreu, Z.; Yáñez-Mó, M. Tetraspanins in Extracellular Vesicle Formation and Function. Front. Immuno [PubMed] [ ] 33. Termini, C.M.; Gillette, J.M. Tetraspanins Function as Regulators of Cellular Signaling. Front. Cell Dev. Biol. 2017, 5, 34. [CrossRef] [PubMed] 34. Rappa, G.; Green, T.M.; Karbanová, J.; Corbeil, D.; Lorico, A.; Rappa, G.; Green, T.M.; Karbanová, J.; Corbeil, D.; Lorico, A. References Tetraspanin CD9 Determines Invasiveness and Tumorigenicity of Human Breast Cancer Cells. Oncotarget 2015, 6, 7970–7991. [CrossRef] [ ] 35. Leeman, M.; Choi, J.; Hansson, S.; Storm, M.U.; Nilsson, L. Proteins and Antibodies in Serum, Plasma, and Whole Blood—Size Characterization Using Asymmetrical Flow Field-Flow Fractionation (AF4). Anal. Bioanal. Chem. 2018, 410, 4867–4873. [CrossRef] 36. Gostner, J.M.; Fong, D.; Wrulich, O.A.; Lehne, F.; Zitt, M.; Hermann, M.; Krobitsch, S.; Martowicz, A.; Gastl, G.; Spizzo, G. Effects of EpCAM Overexpression on Human Breast Cancer Cell Lines. BMC Cancer 2011, 11, 45. [CrossRef] 37. Hollevoet, K.; de Smidt, E.; Geukens, N.; Declerck, P. Prolonged in Vivo Expression and Anti-Tumor Response of DNA-Based Anti-HER2 Antibodies. Oncotarget 2018, 9, 13623–13636. [CrossRef] g 38. Van Deun, J.; Mestdagh, P.; Sormunen, R.; Cocquyt, V.; Vermaelen, K.; Vandesompele, J.; Bracke, M.; de Wever, O.; Hendrix, A. The Impact of Disparate Isolation Methods for Extracellular Vesicles on Downstream RNA Proﬁling. J. Extracell. Vesicles 2014, 3, 24858. [CrossRef] [ ] 39. Szatanek, R.; Baj-Krzyworzeka, M.; Zimoch, J.; Lekka, M.; Siedlar, M.; Baran, J. The Methods of Choice for Extracellular Vesicles (EVs) Characterization. Int. J. Mol. Sci. 2017, 18, 1153. [CrossRef] 40. Pollet, J.; Delport, F.; Janssen, K.P.F.; Jans, K.; Maes, G.; Pfeiffer, H.; Wevers, M.; Lammertyn, J. Fiber Optic SPR Biosensing of DNA Hybridization and DNA–Protein Interactions. Biosens. Bioelectron. 2009, 25, 864–869. [CrossRef] y 41. Arghir, I.; Spasic, D.; Verlinden, B.E.; Delport, F.; Lammertyn, J. Improved Surface Plasmon Resonance Biosensing Using Silanized Optical Fibers. Sens. Actuators B Chem. 2015, 216, 518–526. [CrossRef] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
https://openalex.org/W4252897481	https://www.erudit.org/fr/revues/irrodl/2014-v15-n2-irrodl04928/1065282ar.pdf	English	null	Editorial: Volume 15, Number 2	International review of research in open and distance learning	2,014	cc-by	760	Découvrir la revue Copyright (c) Rory McGreal, 2014 Ce document est protégé par la loi sur le droit d’auteur. L’utilisation des services d’Érudit (y compris la reproduction) est assujettie à sa politique d’utilisation que vous pouvez consulter en ligne. https://apropos.erudit.org/fr/usagers/politique-dutilisation/ Cet article est diffusé et préservé par Érudit. Érudit est un consortium interuniversitaire sans but lucratif composé de l’Université de Montréal, l’Université Laval et l’Université du Québec à Montréal. Il a pour mission la promotion et la valorisation de la recherche. https://www.erudit.org/fr/ Copyright (c) Rory McGreal, 2014 Copyright (c) Rory McGreal, 2014 Ce document est protégé par la loi sur le droit d’auteur. L’utilisation des services d’Érudit (y compris la reproduction) est assujettie à sa politique d’utilisation que vous pouvez consulter en ligne. https://apropos.erudit.org/fr/usagers/politique-dutilisation/ Editorial – Volume 15, Issue Number 2 Rory McGreal Volume 15, numéro 2, avril 2014 Document généré le 24 oct. 2024 00:43 Document généré le 24 oct. 2024 00:43 International Review of Research in Open and Distributed Learning Editorial – Volume 15, Issue Number 2 Rory McGreal Cet article est diffusé et préservé par Érudit. Érudit est un consortium interuniversitaire sans but lucratif composé de l’Université de Montréal, l’Université Laval et l’Université du Québec à Montréal. Il a pour mission la promotion et la valorisation de la recherche. Editorial – Volume 15, Issue Number 2 N Rory McGreal Co-Editor, IRRODL N Rory McGreal Co-Editor, IRRODL Rory McGreal Co-Editor, IRRODL This spring edition of IRRODL begins with several articles describing open educational resources (OER) followed by some financial considerations and a MOOC investigation. These papers will also be included in the OERKnowledgeCloud, which is supported by the UNESCO/Commonwealth of Learning/International Council for Open and Distance Education Chairs in three countries. (I am one of them.) For those readers, who are interested in OER and MOOCs, I would recommend that they visit this repository of more than 600 research articles and reports on issues of relevance to researchers in the field. These are followed by papers on student interaction and support as well as synchronous and asynchronous learning. The later articles investigate blended learning, educational research, and the mobile cloud. Schuwer and Kusters lead off the OER topic with an investigation into mass customization in industry and how it can help address individual learner needs in open content development. Using the concepts of “self-efficacy” and “outcome judgment”, Kelly, in the next article, analyses educator perceptions of OER and makes recommendations on “easy to use” designs to improve the effectiveness of OER. Mtebe and Raisamo expose several “barriers” to implementing OER in Tanzania providing us with a new understanding of how OER initiatives might be implemented in Sub- Saharan Africa. Hilton et al. return to IRRODL with another analysis of how OER can reduce the cost of textbooks, reporting on open textbook initiatives in eight US colleges. MOOCs can be seen as a development emerging from the OER movement. In his blog mining analysis of MOOCs, Chen highlights some of the challenges that need to be addressed to ensure sustainability. In contrast, Marty, focuses on monetizing distance education, with fieldwork analyzing the cultural evolution of a French educational institution from a “public good” mandate to a commercial orientation. The next topic includes the themes of interactivity and student support. Wang et al. provide us with a framework for analyzing interaction within a connectivist paradigm with four levels (operation, wayfinding, sensemaking, and innovation). Barberà et al. Editorial : Editorial : 15(2) McGreal provide us with a tri-country, tri-discipline study on how faculty define competencies and how they design for competency development. Jung and Hong identify the key concerns about student support as expressed by Asian DE students in 10 jurisdictions, noting gender differences. Editorial – Volume 15, Issue Number 2 They propose a list of supporting strategies. In a qualitative, self study, Yamagata-Lynch investigates synchronous and asynchronous approaches focusing on how best to provide support services. In a high school environment Chang et al. compare and contrast blended and traditional classroom environments. As expected, this investigation can be added to Tom Russell’s list of more than 350 “no significant difference” articles. This edition is rounded off with two articles. Teräs and Herrington, using an iterative design and rapid prototyping, show how this helps to “refine design principles” for an authentic elearning programme. Wang et al. provide us with a case study of mobile learning using cloud computing in a higher education institution. The notes sections include a critique of MOOCs by V. Dolan followed by a book review by T. Anderson. by T. Anderson. Vol 15 \| No 2 April/14
https://openalex.org/W3155654874	https://www.researchsquare.com/article/rs-400738/latest.pdf	English	null	Socioecological factors' effect on the subjective experience of COVID-19 quarantine policy in Korea: Consensual qualitative research	Research Square (Research Square)	2,021	cc-by	8,647	Socioecological factors' effect on the subjective experience of COVID-19 quarantine policy in Korea: Consensual qualitative research Socioecological factors effect on the subjective experience of COVID-19 quarantine policy in Korea: Consensual qualitative research Gee Yeon Ro Seoul National University Yun-Jeong Shin (  yj.shin@snu.ac.kr ) Seoul National University https://orcid.org/0000-0002-1236-1781 Sunghyuck Mah Seoul National University Yeseul Min Seoul National University Hyunjung Park Seoul National University Sodam Jeong Seoul National University Research Keywords: COVID-19, quarantine, subjective experience, ecological influence, CQR Posted Date: April 9th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-400738/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Gee Yeon Ro Seoul National University Yun-Jeong Shin (  yj.shin@snu.ac.kr ) Seoul National University https://orcid.org/0000-000 Sunghyuck Mah Seoul National University Yeseul Min Seoul National University Hyunjung Park Seoul National University Sodam Jeong Seoul National University Research Keywords: COVID-19, quarantine, subjective experience, ecological influence, CQR Posted Date: April 9th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-400738/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 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. R d F ll Li Page 1/19 Page 1/19 Page 1/19 Abstract Background: In face of the COVID-19, South Korea has provided the ‘Global Golden Standard’ of containment effort. Mandatory quarantine, one of the core policies in place, has proven its efficacy in ensuring public health. Nonetheless, no previous study has examined the policy’s comprehensive impact on its subjects. In addition to providing an account of holistic subjective experience of quarantine, this study also examines the socioecological factors’ influence on the subjective experience by applying the Bronfenbrenner’s model. In order to gather unconstrained information in relation to the contextual background, Consensual Qualitative Research method was used. 17 adults of Korean nationality were interviewed about their experience. Results: 10 categories within four domains of Subjective experience were found: (a) changed life style because of quarantine, continued pre-quarantine life, lasting effect of quarantine in Lifestyle domain; (b) Physical health domain with no subcategory; (c) discomfort, infection anxiety, accepting, satisfying, and gratitude within Psychological Experience domain; and (d) suggestion and change of perspective within Reflection domain. Next, 13 categories of socioecological factors belonged to four different levels of domain: (a) personality and belief in Within-individual domain; (b) quarantine space, personal relationship, coresident, student status, and employment status in Microsystem; (c) Korea’s quarantine policy, maintenance, resources, and abroad’s preventive measure against COVID in Exosystem; and (d) stigma and social responsibility in Macrosystem. Conclusions: The reported subjective experiences of self-quarantine were not uniformly negative or positive, which is unlike previous research findings that were dominantly negative. Identifying socioecological factors that shape an individual’s quarantine experience shed light onto how the government can protect its people from the potential threats of quarantine. The examples include promoting sense of safety through clear and coherent communication about the disease and the measures that are being placed, maximizing opportunities for the subjects to exert control over their lives during quarantine, devising ways to make virtual social connection accessible, and etc. Background COVID-19 has been one of the biggest and most widespread pandemics of human history. While it was first identified in Wuhan, China in December 2019, it soon became a worldwide concern due to its highly contagious nature. On January 30th of 2020, World Health Organization (WHO) declared it to be a Public Health Emergency of International Concern [1]. The uncontrollable spread of the disease has caused enormously serious harm to the medical, social, and economic ecosystems of the world. Consequently, governments have implemented various policies to minimize further calamities. While the policies around the world are vastly different, South Korea has provided the ‘Global Golden Standard’ in responding to COVID [2]. While many aspects of Korean response to COVID contributed to this unique success, mandatory quarantine is more noteworthy than others. This is because detection of Page 2/19 the infected and prevention of contact between the infected and the noninfected are essential in successful management of the disease, as COVID is transmitted through human contact. Currently in Korea, anyone who may have been in contact with infected individuals, and those entering from abroad must quarantine for two weeks. Of course, those traveling for national or public interest are exempt from this duty. Korean Center for Disease Control (KCDC) set various rules that the subjects need to abide by for 14 days [3]. They cannot make any direct or indirect (sharing food or objects) contact with another person, and must maintain personal sanitation. Furthermore, they must download the ‘Safe Quarantine Application’ on their phones and record their temperatures into the app twice a day. Lastly, when government workers make their daily contacts, they must report any potential symptoms of COVID. If one resists these rules, they must wear location-tracking bracelets [4]. While quarantine has proven effective in promoting public health [5], no previous study has closely examined the policy’s comprehensive impact on its subjects. Prior studies have each reported on quarantine’s negative impact on the subjects’ psychological, behavioral, physical health. Psychologically, subjects experience fear about contracting COVID, feeling of being stigmatized, and etc [6]. Furthermore, quarantined youths experience greater psychological distress, such as worry, helplessness, and fear, when compared to non-quarantined groups [7]. Quarantine, usually characterized by increase in consumption of alcohol and food with low nutrition [8], can have long-term effects on cardiovascular disease [9]. Background Since it is clear that quarantine can have permeating effect on an individual’s overall health, it is necessary to explore its holistic impact. While observing the subjective experience itself does bring insight to the quarantine experience, the individuals’ experiences are always under the influence of relevant ecological factors. For example, external support such as family, friends, health professionals, and a comfortable living environment can alleviate the negative mood that arise during quarantine [6]. Unfortunately, while researchers so far were able to touch upon the interplay between ecological factors and one’s experience, their scope was limited to the ‘external support to cope with emotional changes during quarantine’ [6]. However, external factors that impact an individual’s experience can exist in a non-supportive context too, and quarantine can bring more than emotional changes. Thus, the current study will thoroughly dissect the influences of various systems that surround an individual in addition to delineating the individual’s experiences. Bronfenbrenner’s socioecological model will be applied to analyze the exchange between the socioecological factors and the subjective experience [10]. According to Bronfenbrenner, an individual continuously influences, and is influenced by, the following systems [10]. Microsystem refers to one’s most immediate environment such as family, school, and friends. Mesosystem is the interaction between multiple microsystems, such as interaction between an individual’s parents and friends. Exosystem is a system that indirectly influences an individual; media, public policies, and etc. Macrosystem refers to attitudes, ideologies, and values of the culture that an individual is part of. Lastly, Chronosystem is the changes that occur over time that influence development, such as marriage, divorce, retirement, and etc. For the purpose of this research, only the influences of one’s microsystem, exosystem, and macrosystem Page 3/19 Page 3/19 will be analyzed, as the rest are polydimensional. Mesosystem requires an interaction, and chronosystem is inclusive of all other systems. This polydimensional nature could hinder the analysis of a system’s unique influence over an individual. will be analyzed, as the rest are polydimensional. Mesosystem requires an interaction, and chronosystem is inclusive of all other systems. This polydimensional nature could hinder the analysis of a system’s unique influence over an individual. For the purpose of this study, Consensual Qualitative Research (CQR) was utilized [11]. CQR is the most fitting research method for this study for two reasons. CQR allows the researchers to gather unconstrained responses of participants [11]. Background This trait is necessary to capture the comprehensive experience of the participants. Secondly, CQR provides understanding of the participants’ experiences in relation to the context through inductive and rigorous analysis, and the infusion of multiple viewpoints through consensus building [11-13]. This trait is crucial to extract the contextual influence of various systems on the subjective experience. Thus, by using the CQR, we examined the holistic experience of quarantine by asking the following two questions: (a) What is the subjective experience of quarantine like? and (b) What ecological factors influence the subjective experience of quarantine? Research Team As Hill et al. recommended, we created a research team consisting of five graduate students - four doctoral students and one master student - with training experience in CQR, and one auditor who is a professor and licensed psychologist with extensive experience with conducting CQR research [11]. All researchers are pursuing counseling psychology, except for the one doctoral student who is majoring in organizational psychology in the business department. As a research team, we have experience working in agency, school, and hospital settings. Participants We recruited 17 participants (four males and 13 females) ranging in age from 20 to 47 (M = 29.88, SD = 6.83). 13 participants were students, and four were working professionals. 15 participants experienced self-quarantine due to their entrance from another country, and two did so because they shared space with a COVID-19 patient.12 out of 17 participants had co-residents in the quarantine space. 13 participants engaged in mandatory quarantine while four participants quarantined voluntarily (Table 1). Table 1 Participant characteristics Table 1 Participant characteristics Page 4/19 Characteristics N % Age 20-29 30-39 40-49 11 4 2 64.7% 23.5% 11.8% Gender Female Male 13 4 76.5% 23.5% Occupation Student Working professional 14 3 82.4% 17.6% Reason for quarantine Entered Korea from another country Indirect contact with a COVID-19 patient 15 2 88.2% 11.8% Coresident in quarantine space Yes No 12 5 70.6% 29.4% Type of quarantine Obligatory Voluntary 13 4 76.5% 23.5% Interviewing and transcription Once deemed eligible, participants gave their consents by signing the consent form. We then conducted semi-structured, one-on-one, in-depth interviews that lasted around 45–80 minute via ZOOM or in person. We strictly adhered to the sanitary guidelines to prevent COVID-19 in case of in-person interviews. The interview environments were quiet, and interruptions were minimal. We created an interview protocol that we designed (Appendix A). With the permission of the participants, we audio recorded the interview to boost the accuracy of transcription. The interviews were transcribed and assigned a code number. Recruitment After getting approval from the Institutional Review Board (IRB) at authors’ affiliation, we distributed recruitment advertisements to bloggers who shared that they were in quarantine, and used snowball sampling through personal contacts. Those who were interested in participating contacted the researchers. Once connected, researchers explained the purpose and procedures of the study, and screened their eligibility to participate. The inclusion criteria were as follows: (1) experienced self- quarantine due to entrance from abroad or close contacts with COVID-19 patients; (2) voluntarily participated in the study; and (3) a Korean citizen over 18 years old. The exclusion criteria were: (1) did not complete self-quarantine or (2) unable to communicate effectively. Among 18 potential participants, one was excluded from the final participant pool due to unresponsiveness. Page 5/19 Page 5/19 Procedures for Data Analysis Researchers engaged in continuous data analysis and frequent discussions with each other to identify the themes and subthemes. First, we individually read the transcripts for emergent domains - topic areas that accurately capture the participants’ experiences -, and converted the raw data into core ideas [11, 14]. We then cross-analyzed the representativeness of domains and core ideas. Following the initial analysis, the auditor reviewed the work to ensure that the core ideas fit well into the domains, and to confirm that the labels accurately capture the nature of the categories. Using the auditor’s feedback, the team modified the list of domain and core ideas once a consensus was reached that they are still reflective of the raw data. Finally, we coded the raw data into corresponding core ideas and assigned frequency labels. Results As a result of analyzing the data to investigate the subjective experience of the participants’ self- quarantine, four domains were found; Lifestyle, Physical health, Psychological experience, and Reflection. Next, exploring the multiple layers of contextual influences on the subjective experiences of quarantine in South Korea revealed four domains with 13 categories, (a) personality and belief in Within-individual domain, (b) quarantine space, personal relationship, coresident, student status, and employment status in Microsystem, (c) Korea’s quarantine policy, maintenance, resources, and abroad’s preventive measure against COVID in Exosystem, and (d) stigma and social responsibility in Macrosystem. Frequency labels, General, Typical, and Variant, are based on a total of 17 cases. General describes 16 or 17 cases, Typical describes 9 to 15 cases, and Variant describes 8 or less cases. Subjective Experience of Self-Quarantine The consensus meaning of four domains of Korean participants’ subjective experience of quarantine is as follows. Lifestyle refers to the activities and behaviors that the participants engaged in as a result of, and during, quarantine. Physical health refers to the phenomenon that manifested itself in the physical body as a result of, and during, quarantine. Psychological experience encompasses various internal experiences that arose within the participants, and Reflection presents the participants’ afterthoughts of quarantine (Table 2). Table 2 Subjective Experience Table 2 Subjective Experience Page 6/19 Domain Category Frequency Lifestyle Changed lifestyle because of quarantine 11/Typical Continued pre-quarantine life 16/General Lasting effect of quarantine 5/Variant Physical health 10/Typical Psychological experience Discomfort 13/Typical Infection Anxiety 6/Variant Accepting 14/Typical Satisfying 6/Variant Gratitude 8/Typical Reflection Suggestion 7/Variant Change of Perspective 4/Variant Lifestyle On the other hand, quarantine left opposite lasting effects on others. These participants reported that they became more comfortable with staying at home as they adapted to their quarantine experience. They felt it was safer to stay at home due to the still-standing COVID-19 situation. “Even though the quarantine was over, I decided not to go outside because I felt okay and safer to be at home”. Some participants grew a tendency to stay inside because they felt that keeping their masks on outside felt just as stuffy. “In Korea, I have to wear a mask when I go outside. But when I wear it, it makes me stuffy and out of breath. So, I’d rather not go out”. Physical Health The second domain of subjective experience of quarantine was Physical health. Participants typically reported that quarantine had physical consequences. Concerns surrounding actual or the potential of weight gain were the most prevalent. Due to quarantine, they could not engage in much physical activity. On the contrary, their food intake remained the same or increased. These factors together resulted in concerns of weight gain. Furthermore, few participants shared that quarantine took quite a toll on the body. They struggled from jet lag, which made them “fall asleep when I shouldn’t, and stay awake at night. Not having enough sleep made my mental state worse”. Lifestyle The domain Lifestyle consisted of three different core ideas: changed lifestyle because of quarantine, continued pre-quarantine life, lasting effect of quarantine. Of the 17 participants, 11 reported that strictly following the quarantine regulations typically changed their lifestyles. The more strictly a participant followed the rules, the greater was the change in one’s lifestyle. One participant used the kitchen only in the middle of the night, when “no one else would be awake”, to minimize contact with the family members. Another participant had to “hand-wash the laundry every morning” to not use the same washing machine as the family. On the other hand, not all participants experienced changes in lifestyle due to quarantine. In fact, participants generally continued their pre-quarantine lives without detecting much quarantine-specific differences. They continued their schoolwork and work through online mediums. Some participants continued their pre-quarantine lives, but with more emphasis on recharging over responsibility. For example, they “rested and got enough sleep”, “focused on my hobby like making Gundam figures and puzzles. Time flies when I play with these things. So I just did those things freely without much concern”. Regardless of the degree of changes in lifestyle, quarantine had variantly lasting effects on some participants’ post-quarantine lives. Those who felt unprecedentedly constrained and trapped during quarantine had a harder time staying indoors than ever before. These participants felt that they “cannot stand being at home more than before”, and were “sick and tired of being home”. They would rather be outside with a mask than stay inside without one, and tried to stay indoors as little as possible. Some participants even wanted to distance themselves from their quarantine spaces. Page 7/19 Page 7/19 On the other hand, quarantine left opposite lasting effects on others. These participants reported that they became more comfortable with staying at home as they adapted to their quarantine experience. They felt it was safer to stay at home due to the still-standing COVID-19 situation. “Even though the quarantine was over, I decided not to go outside because I felt okay and safer to be at home”. Some participants grew a tendency to stay inside because they felt that keeping their masks on outside felt just as stuffy. “In Korea, I have to wear a mask when I go outside. But when I wear it, it makes me stuffy and out of breath. So, I’d rather not go out”. Reflection One participant said “Ever since I went through quarantine, I have more understanding towards the people who unfortunately spread the disease by moving around while they were infected. I used to blame them for being selfish, but I now realize that they were just living their lives. They just didn’t know that they had the virus. So now, I feel like it’s not really their fault. Or actually, anyone’s fault”. services...kitchenware...and human connection are essential to surviving”. Quarantine also had the effect of expanding one’s perspective towards the pandemic situation itself. One participant said “Ever since I went through quarantine, I have more understanding towards the people who unfortunately spread the disease by moving around while they were infected. I used to blame them for being selfish, but I now realize that they were just living their lives. They just didn’t know that they had the virus. So now, I feel like it’s not really their fault. Or actually, anyone’s fault”. Psychological Experience Furthermore, participants who “genuinely enjoy being home…[I] was really happy that I had to be home”. Lastly, gratitude towards the situation was typically observed among participants. These participants viewed quarantine as a tool that the government was utilizing “to contain COVID effectively” for public health promotion, and felt “especially safe” to abide by the rules. In addition, they saw quarantine as an opportunity to be “home with…mom’s home cooked meals”. In fact, participants typically remained in Korea even after the quarantine. Psychological Experience Alongside Lifestyle and Physical health, Psychological experience was the third domain, and could be categorized into five categories: discomfort, infection anxiety, accepting, satisfying, gratitude. First, participants typically identified discomfort as the challenging psychological experience of quarantine. Reports of feeling constrained were the most common, while the type and intensity of discomfort that participants experienced ranged from mere boredom to depressive symptoms. Few were tempted to “go outside…I felt healthy and my COVID screening result was negative”. Some also “felt completely alone. And I could see that feeling of solitude intensifying into depression”. Participants variantly reported infection anxiety. All participants were obligated to quarantine because the possibility of becoming a COVID patient could not be ruled out. This possibility triggered infection anxiety, as they started to see normal physical phenomena such as “coughing here and there” as potential COVID symptoms. “I heard on the news that some people tested positive on their second test even if they already tested negative on their first one. So, I was anxious that my result was a false negative”. Unfortunately, infection anxiety remained high until they “got a negative at that second screening”. Nonetheless, not all psychological experiences were negative. Typically, participants accepted the situation as a necessary public health policy instead of as an unnecessary enforcement. One participant shared, “I said earlier that I felt shunned when I saw my mom disinfecting the stuff I touched. But after one day, I started to think, ‘Of course she should’. I got used to it quickly, and my parents and I prioritized COVID prevention”. Some participants “controlled emotions by changing my thoughts. If you think being in quarantine is tough, the struggle will never diminish”. Page 8/19 Page 8/19 Furthermore, participants variantly experienced satisfaction during quarantine, as they utilized the 2-week period as a very concentrated time for their needs and desires. Participants who needed to fulfill their academic responsibilities such as “TAing…papers, researches, and final” experienced quarantine as an “undisturbed time that increased my productivity and concentration”. Furthermore, participants who “genuinely enjoy being home…[I] was really happy that I had to be home”. Furthermore, participants variantly experienced satisfaction during quarantine, as they utilized the 2-week period as a very concentrated time for their needs and desires. Participants who needed to fulfill their academic responsibilities such as “TAing…papers, researches, and final” experienced quarantine as an “undisturbed time that increased my productivity and concentration”. Reflection The last domain of subjective experience of quarantine was participants’ Reflection of their experiences, and mainly involved suggestions for the future and changed perspectives. First, suggestions for the future quarantine experience included “bringing some kind of entertainment to make time go by faster”. Suggestions for the systematic structure that runs the quarantine policy were targeted towards the government workers. One participant stated, “They should give the workers more incentives to increase their motivation. Because COVID is here for the long run, there is always a possibility for the situation to get worse and for them to be busier. They probably already feel like their work is worthy, but giving them more practical affirmations can’t hurt. A raise would be a good idea”. In addition, as quarantine inevitably created distance between the participants and their ordinary lives, they were able to view their lives with a new perspective. More specifically, they were able to realize that “routine is not boring, but is a source of calmness”, and that ordinary things such as “delivery services...kitchenware...and human connection are essential to surviving”. Quarantine also had the effect of expanding one’s perspective towards the pandemic situation itself. One participant said “Ever since I went through quarantine, I have more understanding towards the people who unfortunately spread the disease by moving around while they were infected. I used to blame them for being selfish, but I now realize that they were just living their lives. They just didn’t know that they had the virus. So now, I feel like it’s not really their fault. Or actually, anyone’s fault”. In addition, as quarantine inevitably created distance between the participants and their ordinary lives, they were able to view their lives with a new perspective. More specifically, they were able to realize that “routine is not boring, but is a source of calmness”, and that ordinary things such as “delivery In addition, as quarantine inevitably created distance between the participants and their ordinary lives, they were able to view their lives with a new perspective. More specifically, they were able to realize that “routine is not boring, but is a source of calmness”, and that ordinary things such as “delivery services...kitchenware...and human connection are essential to surviving”. Quarantine also had the effect of expanding one’s perspective towards the pandemic situation itself. Ecological Factors Influencing the Subjective Experience of Quarantine Bronfenbrenner’s socioecological model was used to examine the effects of the various layers of one’s surrounding on subjective experiences of self-quarantine. Four domains of ecological factors were found: Within-individual, Microsystem, Exosystem, and Macrosystem (Table 3). Table 3 Ecological factors that affect subjective experience of quarantine Page 9/19 Page 9/19 Domain Category Frequency Within-Individual Personality 14/Typical Belief 10/Typical Microsystem Quarantine space 9/Typical Personal Relationships 12/Typical Coresident 10/Typical Student status 9/Typical Employment Status 4/Variant Exosystem Korea's quarantine policy 10/Typical Korea's quarantine maintenance 16/General Korea's quarantine resources 14/Typical Abroad's preventive measures against COVID 14/Typical Macrosystem Stigma 10/Typical Social Responsibility 9/Typical Within-Individual: Personality and Belief Within-Individual: Personality and Belief First domain was Within-individual, which contained two categories, personality and belief. Even though all the participants quarantined for the first time and only once, their subjective experiences varied significantly due to their personality and beliefs. Typically, participants reported how their personality impacted their quarantine experience. For example, introverted participants who described themselves as “homebody” were less impacted by the quarantine. These individuals were already having a significant amount of alone time before COVID, quarantine did not clash with their innate tendencies. In fact, one participant said, “I enjoyed the quarantine period because I didn’t have to go anywhere”. On the contrary, extroverts who normally “feel cramped if I stay at home alone for more than a day” felt “suffocated” because they tend to gain energy from being with others. Personal beliefs also typically affected the subjective experience of the quarantine. Some participants believed that, if infected, they could be most properly treated in their mother country. They believed that having access to a medical system that they are well-acquainted with was an advantage. Consequently, they chose to come to Korea, making themselves subjects of quarantine. One participant delineated how such belief helped her remain grateful even in the face of the compulsory quarantine. She stated, “As soon as I landed in Korea, I felt less scared because I knew I would be treated well even if I got sick”. Page 10/19 Microsystem: Space, Relationship, Coresident and Employment status Microsystem: Space, Relationship, Coresident and Employment status Page 10/19 Page 10/19 The second domain Microsystem included quarantine space, personal relationships, coresident, student status, and employment status. It was typical for participants to identify their quarantine space as one of the factors that shaped their quarantine experience. One participant noted, “I quarantined at a house with a front yard. I spent a lot of time in the yard, which made my quarantine favorable compared to others”. On the other hand, some felt frustrated due to restricted spaces. “I felt suffocated when I looked around the tiny room. I almost had a panic attack because suddenly, I felt so cramped. I questioned how I’m going to survive in this small space”. Also, participants typically reported that connecting with their personal relationships during the quarantine worked against the feeling of isolation. “My grandmother called me every morning to ask, ‘How are you? Are you sick?’ This was great psychological support”. Connecting with others who were also quarantining provided “an unparalleled amount of connectedness”. One participant emphasized, “We contacted each other about our own quarantine experience. It was such a great feeling of homogeneity”. Moreover, the existence of a coresident typically altered the participants’ quarantine experience. It sometimes functioned as practical aid (e.g., preparing a meal), as well as psychological support. One participant said, “The presence of somebody put me at ease”. However, some participants felt hurt when coresidents expressed the anxiety of contagion. One said, “My mom forced me to strictly follow the quarantine rules as if she was a government authority. And she got scared whenever I had to pass through the living room. I ended up telling her that I feel unappreciated”. Last two categories in the Microsystem were related to the social status of individuals, such as student status and employment status. First, although the medium to conduct and evaluate academic performance was changed, nine participants who self-identified as students reported that their self- quarantine revolved around school. One participant reported, “I had to continue my academic duties through remote schooling”. We separated the employment status with student status since one’s employment condition variantly impacted the participants’ quarantine experience. One self-employed participant recounted, “I had to quarantine in Korea while...there was no one to check on the office space (which was abroad). But I still had to pay the rent. So I was feeling quite anxious and helpless”. Page 10/19 Microsystem: Space, Relationship, Coresident and Employment status On the other hand, others continued to work smoothly as they “could do my work on a computer. So my geographic location was not a concern as long as I could work”. Exosystem: Policy, Maintenance, Resources, and Aborad’s COVID Measures The third domain was Exosystem, as nationwide quarantine measures influenced the subjective experience of quarantine. Korea’s quarantine policy, maintenance, and resources, as well as abroad’s preventive measures against COVID fell under this domain. First, Korea’s quarantine regulations typically dictated the structural aspects of the participants’ subjective experience of quarantine. The regulations instructed the participants on how, when, and where to perform all quarantine related tasks. As a result, Page 11/19 one participant had to “quarantine in a fire-service academy dormitory because quarantining with a coresident (sister) who works closely with a high-risk population (elementary school students) was forbidden”. Secondly, the scrutiny of quarantine adherence monitoring, which differed by the government worker in charge, still generally impacted many aspects of the quarantine experience. One participant had the police on her front door after “forgetting to report my daily temperature and COVID symptoms”. Some implied that the monitoring was loose, and that “anyone could fool it” if they were determined to do so. Furthermore, the uncommon experience of having their moves tracked and monitored generated various reactions. Some participants viewed the close monitoring as “troublesome”, but more “felt rather protected as...it enables immediate detection of the symptoms...and early response”. In addition, Korea’s quarantine resources typically played a significant role in the participants’ subjective experience. Number of participants expressed relief in being able to get tested for COVID-19 free of charge, and in being safely escorted to their quarantine space. Quarantine kit was one of the most apparent and tangible resources that influenced the subjective experience. According to the participants, all quarantine kits included essentials such as food supplies, sanitizers, thermometers, garbage bags, and etc. In some cases, the kit included non-essentials too: exercise equipment and a flower pot. Many participants were touched at what their country was doing for them. “It’s just protocol, but it was still so touching to receive the aid kit. How caring and thoughtful they are!”. Lastly, participants typically mentioned that the abroad’s Exosystem, such as preventive measures against COVID-19, was what drove them to quarantine, as it increased their desire to come to Korea. In participants’ residing countries, securing food supplies was difficult, frenzy rumors about martial law were going around, many were dying due to lack of proper medical care, and etc. The unfolding “was so scary… That’s why I packed all my things and came to Korea”. Exosystem: Policy, Maintenance, Resources, and Aborad’s COVID Measures Many also pointed out that they were likely to receive little support if they actually caught COVID-19 abroad. The difference is captured in the following words: “In Korea, it’s like, ‘You just abide by our rules, and we will take full care of you. Food, medical service… It’s all on us. You just do your job of quarantining’. But there (U.S), it’s more like, ‘You do everything on your own. You could get infected, so take good care of yourself’”. Macrosystem: Stigma and Social Responsibility Page 12/19 Last domain that interacted with participant’s quarantine experience was Macrosystem, within which belonged two cultural categories: stigma and social responsibility. First, 10 out of 17 participants typically expressed concerns of being stigmatized. Although all participants were COVID-negative, they feared being labeled as the potential virus-holder. In the beginning of the outbreak, crowds did not hesitate to express rage towards confirmed patients who had unintentionally spread the virus to their communities. Subsequently, the possibility of being stigmatized as a ‘spreader’ motivated the participants to adhere to the quarantine requirements more. “What if my story got on the news? That was my greatest fear. What if I was broadcasted as a Ms. Something living in Pohang (city in Korea) who went out during quarantine and all of my family was exposed?”. Another participant commented, “If my behaviors were identified as the cause that the virus spread, everyone would blame and curse me for it. That’s why I was even more cautious”. Lastly, participants’ social responsibility was another factor that typically enhanced quarantine compliance. They not only wanted to avoid getting reprimanded by their communities, but also wanted to fulfill their responsibility as good standing individuals within their communities. These participants “saw it (quarantine) as something I simply had to do. There was no doubt about it. I have been overseas, and I had symptoms of a cold. It was obvious that I would do this for our country and our people”. Some participants were especially considerate of the more vulnerable. For example, one chose to self- quarantine to “make sure to keep my elderly parents safe”. Another said, “I work in an academy with young kids. Spreading the virus there would be really terrible. We all know kids’ immune systems aren’t as strong”. Discussion The reported subjective experiences of self-quarantine were not uniformly negative or positive, but rather varied in terms of lifestyle, physical health, psychological experience, and reflection. The findings shed light onto the factors and actions that could maximize adherence to quarantine rules and positive experience of quarantine. In addition, it was revealed that multiple levels of socioecological variables influence the subjective experience of quarantine: from individual traits, social relations and status, social infrastructure, to culturally shared beliefs. The results inform that interventions on various socioecological levels can improve and worsen the subjective quarantine experiences. Subjective experience One factor that increased quarantine adherence was the participants’ infection anxiety. Being categorized as the subjects of self-quarantine increased their perceived likelihood of being infected even though they took the necessary measures to protect themselves from COVID. This increase in perceived possibility of being infected triggered their infection anxiety, causing them to adhere to the quarantine regulations more strictly. In fact, the quarantined experience significantly higher levels of anxiety and depression compared to non-quarantined [15], and anxiety about infection lead to safety actions [16]. Given these findings, following the self-quarantine rules can be interpreted as a safety action against infection anxiety. As this study’s findings make it possible to deduce that infection anxiety functioned as a catalyst to strengthen one’s quarantine adherence, future research should explore whether or not the infection anxiety persists even after the completion of quarantine. Additionally, having accurate knowledge about the quarantine protocol was a critical factor that improved adherence to quarantine guidelines. This finding is consistent with the findings of a literature review of 14 studies related to quarantine adherence during infectious disease outbreaks such as Ebola, SARS, and Swine flu, and etc [17]. Consequently, it can be inferred that the strategic and transparent information Page 13/19 Page 13/19 sharing of the Korean government contributed to the high adherence rate of current study’s participants. Conversely, a participant who voluntarily started quarantine before the policy was fully in place violated quarantine by leaving the house to get snacks. This echoes the aforementioned review's finding that lack of clear quarantine instruction can cause one to invent their own quarantine rules and acceptable degree of contact with others [17]. In conclusion, current study’s findings emphasize the need of assuring the clarity and correctness of self-quarantine guidelines, and of correcting any harmful, false, and misleading information that are being broadcasted [18]. Continuing pre-quarantine lifestyles during quarantine acted as a protective factor for many participants. Quarantine robs people of their freedom to choose what activities to engage in and when, and who to be with. Therefore, being in quarantine can feel like they have no control over their lives. Meanwhile, continuing their pre-quarantine routine enables them to gain a sense of control, as they are able to decide on what to do at which moment. In fact, people's perceived sense of control mediates the effects of positive mental health on stress [19]. Subjective experience Consequently, future research on self-quarantine and sense of control should aim to identify guidelines or activities that could increase people’s sense of control in the midst of quarantine. Generally, self-quarantine is known as an experience that causes psychological distress [20]. Our research findings echo this finding, as participants’ psychological experiences included feeling constrained, bored and depressed. Conversely, a new-found phenomenon such as gratitude was also reported. Those who felt gratitude thought highly of Korea’s measures against COVID. They decided to come to Korea because they thought that the benefits of coming to Korea overrode the risks of traveling in midst of COVID and the inconvenience of quarantining. They felt safe not only because they were coming to their home country, but also because they could be under Korea’s care. In fact, Korea’s response to COVID has been heralded as one of the more exemplary models, since the government recognized the need to engage in preventive measures, such as travel restrictions, health screening, quarantine and testing, and public awareness, early on [21]. Subsequently, Korea's response to COVID gave the participants a sense of safety, which resulted in their feelings of gratitude despite being in distressful self-quarantine situations. These results highlight the fact that a country’s response to a pandemic has grave implications. Overall, while this study corroborated previous studies’ findings that quarantine leads to psychological difficulties or trauma [17, 20], it also shed light into the neutral and positive quarantine experiences such as acceptance, gratitude, and satisfaction. Therefore, it is necessary to acknowledge these positive experiences and conduct various studies on how to maximize the potential protective factors of one’s quarantine. Ecological factors that affect self-quarantine experience Unique within-individual factors influenced the self-quarantine experience. As personality refers to unique traits behaving in identical ways across diverse circumstances [22], participants’ response to quarantine, a uniformly structured policy, differed because of their distinctive personalities. This is in line with Page 14/19 Page 14/19 findings of a recent study on personality: extraversion, openness, agreeableness, or conscientiousness did not significantly fluctuate during the acute phase of the COVID pandemic [23]. At the same time, a myriad of microsystems such as quarantine space, social status, and relationships constantly shaped the subjective experience. Findings particularly highlight the importance of relationships, even if they are virtual or indirect. Participants appreciated and actively engaged in phone calls and text messaging with friends and family members. As quarantine goes against humans’ basic needs of belongings or connectedness [24, 25], alternative fulfilment of such needs could strongly mitigate the psychological threat from being quarantined [26, 27]. To extend this benefit of relational support, policymakers could consider creating an online community for quarantined people to come together to share their experiences, feelings, or tips during and after the quarantine. Participants’ exosystem influenced the subjective experience the most. Although aggressive contact tracing could have raised privacy infringement concerns, many felt protected through their interactions with the Korean government [21]. This can be explained by Korea’s ‘trace, test, and treat strategy’, as this approach traces specifically to enable safe testing and seamless treatment [28]. Resources that existed specifically for the subjects of quarantine also augmented their feelings of being cared for instead of being locked in. However, there is also room for improvement. Since scrutiny of participant monitoring differed by each government official in charge, there was incoherence and confusion regarding the importance of adhering to the rules. In addition, it is worth noting that the resources that participants identified as conducive to the quarantine experience already existed in Korea before COVID. Affordable and universal health insurance, effective and fast delivery systems, and advanced Internet and communication network services are such resources [29]. Since there is no guarantee that COVID will come to an end, it is imperative for governments to build infrastructures that could ensure convenient and healthy daily lives in the midst of life-threatening pandemic. Lastly, cultural values such as stigma towards the confirmed patients and social responsibility also played a decisive role in the subjective quarantine experience. Ecological factors that affect self-quarantine experience In cultures that value collectivism, the self is constructed within a fundamental relatedness to each other [30]. Since South Korea holds collective values such as group harmony and collaboration, becoming the target of stigma is a significant threat to an individual. Moreover, the participants viewed quarantine as an act of taking social responsibility for the community. However, these phenomena cannot be solely attributed to the collective aspect of Korean culture. ‘Dataveillant collectivism’, ongoing exposure to existential threats and technological development, alongside neo-Confucian traditions, provided a context in which COVID-19 containment was favorable in Korea [31]. Growing literature also acknowledges the interplay of cultural values and jurisdictional responses to the pandemic, social responsibility, and compliance with social distancing and quarantine [17, 32]. Subsequently, future studies will benefit from taking the complex cultural factors into consideration when exploring how individuals perceive and adhere to quarantine measures. Page 15/19 Our research findings should be interpreted in light of some limitations. First, our results may reflect the experience of the socio-economically privileged population, thus confining generalization of the results. It is still a meaningful finding given that COVID-19 is equally threatening across all social classes. Furthermore, lack of external validity is an inherent shortcoming of a qualitative study, as it aims to attain Our research findings should be interpreted in light of some limitations. First, our results may reflect the experience of the socio-economically privileged population, thus confining generalization of the results. It is still a meaningful finding given that COVID-19 is equally threatening across all social classes. Furthermore, lack of external validity is an inherent shortcoming of a qualitative study, as it aims to attain an in-depth understanding of a subgroup of the population rather than to achieve generalization [33]. Nevertheless, underprivileged populations are likely to suffer varying degrees of additional difficulties from quarantine measures. Hence, future studies should expand on the range of samples with respect to socio-economic backgrounds, occupations, and etc. Secondly, our qualitative approach to quarantine makes it difficult to produce a more computable interpretation of findings. For example, capturing the participants’ psychological experiences during quarantine is also possible by utilizing well-established measurements in psychology. Such quantitative approach is also valuable because computing the subjective experience and systematic influences could provide opportunities to empirically verify the effectiveness of individually-used coping strategies and public health policies. Conclusion This study unveils the subjective experience of quarantine implemented as a measure to cope with COVID-19, and the influencing ecological factors of it. Unlike earlier researches that focused on field- specific phenomena such as psychology [7], public policy [36], or nutrition [37], our approach was holistic and comprehensive. The findings resonate the need to implement preventive measures against a disease while successfully ensuring the individuals’ wellbeing. Ecological factors that affect self-quarantine experience Lastly, although the study contained some practical implications for future quarantine measures, we focused on individual experiences, not the governmental involvements, of quarantines. This was an intentional effort to bring balance to the current literature that has been focusing on governance of COVID-19 at national levels [34, 35]. Thus, there was a need to highlight the subjective experience of the individuals who forfeited their freedom for common good. As our findings identify several approaches that governments can take on to improve the individuals’ quarantine experience, future studies should closely examine the corresponding results of such public policy modification. Funding: None Authors’ contributions: GR made substantial contribution to the acquisition of data, interpretation of data, and drafted the work, and substantively revised it. YS made substantial contribution to the design of the work, interpretation of data, and substantively revised it. SM, YM, HP, and SJ equally made substantial contribution to the acquisition of data, interpretation of data, and drafted the work. All authors read and approved the submitted version of the manuscript. All authors have agreed both to be personally accountable for the author’s own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature. Acknowledgements: Not applicable. Declarations Ethics approval and consent to participate: Seoul National University’s IRB approved this study. IRB No. 2007/003-026. Consent for publication: Not applicable. Availability for data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Page 16/19 Competing interests: The authors declare that they have no competing interests. Competing interests: The authors declare that they have no competing interests. Funding: None References 1. COVID-19 Public Health Emergency of International Concern (PHEIC) Global research and innovation forum [Internet]. Who.int. 2021 [cited 5 January 2021]. Available from: https://www.who.int/publications/m/item/covid-19-public-health-emergency-of-international- concern-(pheic)-global-research-and-innovation-forum 2. Rubin T. Coronavirus lessons: How South Korea got face masks for everyone and Germany kept death rate down \| Trudy Rubin [Internet]. https://www.inquirer.com. 2021 [cited 4 January 2021]. Available from: https://www.inquirer.com/health/coronavirus/coronavirus-masks-shortage-south- korea-germany-fatality-rates-trump-20200407.html 3. Korean Disease Control and Prevention Agency [Internet]. Korean Disease Control and Prevention Agency. 2021 [cited 3 February 2021]. Available from: http://www.kdca.go.kr/gallery.es? mid=a20503020000&bid=0003&act=view&list_no=144811 3. Korean Disease Control and Prevention Agency [Internet]. Korean Disease Control and Prevention Agency. 2021 [cited 3 February 2021]. Available from: http://www.kdca.go.kr/gallery.es? mid=a20503020000&bid=0003&act=view&list_no=144811 4. Choi Y. The Power of Collaborative Governance: The Case of South Korea Responding to COVID‐19 Pandemic. World Medical & Health Policy. 2020;12(4):430-442. 4. Choi Y. The Power of Collaborative Governance: The Case of South Korea Responding to COVID‐19 Pandemic. World Medical & Health Policy. 2020;12(4):430-442. 5. Brooks S, Webster R, Smith L, Woodland L, Wessely S, Greenberg N et al. The psychological impact of quarantine and how to reduce it: rapid review of the evidence. The Lancet. 2020;395(10227):912- 920. 5. Brooks S, Webster R, Smith L, Woodland L, Wessely S, Greenberg N et al. The psychological impact of quarantine and how to reduce it: rapid review of the evidence. The Lancet. 2020;395(10227):912- 920. 6. Chen D, Song F, Tang L, Zhang H, Shao J, Qiu R et al. Quarantine experience of close contacts of COVID-19 patients in China: A qualitative descriptive study. General Hospital Psychiatry. 2020;66:81- 88. 7. Saurabh K, Ranjan S. Compliance and Psychological Impact of Quarantine in Children and Adolescents due to Covid-19 Pandemic. The Indian Journal of Pediatrics. 2020;87(7):532-536. Page 17/19 Page 17/19 8. Sidor A, Rzymski P. Dietary Choices and Habits during COVID-19 Lockdown: Experience from Poland. Nutrients. 2020;12(6):1657. 9. Mattioli A, Sciomer S, Cocchi C, Maffei S, Gallina S. Quarantine during COVID-19 outbreak: Changes in diet and physical activity increase the risk of cardiovascular disease. Nutrition, Metabolism and Cardiovascular Diseases. 2020;30(9):1409-1417. 10. Bronfenbrenner U. Ecology of Human Development: Experiments by Nature and Design. Harvard University Press; 1979. 11. Hill C, Thompson B, Williams E. A Guide to Conducting Consensual Qualitative Research. The Counseling Psychologist. 1997;25(4):517-572. 12. Hill C, Knox S, Thompson B, Williams E, Hess S, Ladany N. Consensual qualitative research: An update. Journal of Counseling Psychology. 2005;52(2):196-205. 13. Hill C. Consensual qualitative research. Washington, DC: American Psychological Assoc.; 2012. References 14. Thompson BJ, Vivino BL, Hill CE. Coding the data: Domains and core ideas. In Hill CE, editor. Consensual qualitative research: A practical resource for investigating social science phenomena. American Psychological Association; 2011: p. 103–116. Consensual qualitative research: A practical resource for investigating social science phenomena. American Psychological Association; 2011: p. 103–116. 15. Tang F, Liang J, Zhang H, Kelifa M, He Q, Wang P. COVID-19 related depression and anxiety among quarantined respondents. Psychology & Health. 2020;36(2):164-178. 16. Reiss S, Franchina V, Jutzi C, Willardt R, Jonas E. From anxiety to action—Experience of threat, emotional states, reactance, and action preferences in the early days of COVID-19 self-isolation in Germany and Austria. PLOS ONE. 2020;15(12):e0243193. 16. Reiss S, Franchina V, Jutzi C, Willardt R, Jonas E. From anxiety to action—Experience of threat, emotional states, reactance, and action preferences in the early days of COVID-19 self-isolation in Germany and Austria. PLOS ONE. 2020;15(12):e0243193. 17. Webster R, Brooks S, Smith L, Woodland L, Wessely S, Rubin G. How to improve adherence with quarantine: rapid review of the evidence. Public Health. 2020;182:163-169. 17. Webster R, Brooks S, Smith L, Woodland L, Wessely S, Rubin G. How to improve adherence with quarantine: rapid review of the evidence. Public Health. 2020;182:163-169. 18. Transparency, communication and trust: The role of public communication in responding to the wave of disinformation about the new Coronavirus [Internet]. OECD. 2021 [cited 21 February 2021]. Available from: https://www.oecd.org/coronavirus/policy-responses/transparency- %20communication%20-and-trust-bef7ad6e/ 19. Brailovskaia J, Margraf J. Predicting adaptive and maladaptive responses to the Coronavirus (COVID-19) outbreak: A prospective longitudinal study. International Journal of Clinical and Health Psychology. 2020;20(3):183-191. 20. Serafini G, Parmigiani B, Amerio A, Aguglia A, Sher L, Amore M. The psychological impact of COVID- 19 on the mental health in the general population. QJM: An International Journal of Medicine. 2020;113(8):531-537. 20. Serafini G, Parmigiani B, Amerio A, Aguglia A, Sher L, Amore M. The psychological impact of COVID- 19 on the mental health in the general population. QJM: An International Journal of Medicine. 2020;113(8):531-537. 21. Kim M, Cho W, Choi H, Hur J. Assessing the South Korean Model of Emergency Management during the COVID-19 Pandemic. Asian Studies Review. 2020;44(4):567-578. 21. Kim M, Cho W, Choi H, Hur J. Assessing the South Korean Model of Emergency Management during the COVID-19 Pandemic. Asian Studies Review. 2020;44(4):567-578. 22. Roberts B. Back to the future: Personality and Assessment and personality development. Journal of Research in Personality. References 2020;74(6):850-851. 37. Muscogiuri G, Barrea L, Savastano S, Colao A. Nutritional recommendations for CoVID-19 quarantine. European Journal of Clinical Nutrition. 2020;74(6):850-851. References 2009;43(2):137-145. Page 18/19 23. Sutin A, Luchetti M, Aschwanden D, Lee J, Sesker A, Strickhouser J et al. Change in five-factor model personality traits during the acute phase of the coronavirus pandemic. PLOS ONE. 2020;15(8):e0237056. 24. Maslow A. A theory of human motivation. Psychological Review. 1943;50(4):370-396. 25. Frey L, Wilhite K. Our Five Basic Needs. Intervention in School and Clinic. 2005;40(3):156-160. 26. Hagerty S, Williams L. The impact of COVID-19 on mental health: The interactive roles of brain biotypes and human connection. Brain, Behavior, & Immunity - Health. 2020;5:100078. 27. Kuwahara K, Kuroda A, Fukuda Y. COVID-19: Active measures to support community-dwelling older adults. Travel Medicine and Infectious Disease. 2020;36:101638. 27. Kuwahara K, Kuroda A, Fukuda Y. COVID-19: Active measures to support community-dwelling older adults. Travel Medicine and Infectious Disease. 2020;36:101638. 28. Tackling COVID-19 Health, Quarantine and Economic Measures: Korean Experience [Internet]. 2020 [cited 5 January 2021]. Available from: https://ecck.or.kr/wp-content/uploads/2020/03/Tackling- COVID-19-Health-Quarantine-and-Economic-Measures-of-South-Korea.pdf 29. Shin Y, Lee J. South Korea’s proactive approach to the COVID-19 global crisis. Psychological Trauma: Theory, Research, Practice, and Policy. 2020;12(5):475-477. 29. Shin Y, Lee J. South Korea’s proactive approach to the COVID-19 global crisis. Psychological Trauma: Theory, Research, Practice, and Policy. 2020;12(5):475-477. 30. Markus H, Kitayama S. Culture and the self: Implications for cognition, emotion, and motivation. Psychological Review. 1991;98(2):224-253. 30. Markus H, Kitayama S. Culture and the self: Implications for cognition, emotion, and motivation. Psychological Review. 1991;98(2):224-253. 31. 31. Kasdan D, Campbell J. Dataveillant Collectivism and the Coronavirus in Korea: Values, Biases, and Socio-Cultural Foundations of Containment Efforts. Administrative Theory & Praxis. 2020;42(4):604-613. 32. Velamoor V, Persad E. Covid-19: Cultural perspectives. Asian Journal of Psychiatry. 2020;53:102439. 32. Velamoor V, Persad E. Covid-19: Cultural perspectives. Asian Journal of Psychiatry. 2020;53:102439. 33. Morrow S. Qualitative Research in Counseling Psychology. The Counseling Psychologist. 2007;35(2):209-235. 33. Morrow S. Qualitative Research in Counseling Psychology. The Counseling Psychologist. 2007;35(2):209-235. 34. Shah J, Shah J, Shah J. Quarantine, isolation and lockdown: in context of COVID-19. Journal of Patan Academy of Health Sciences. 2020;7(1):48-57. 35. Moon M. Fighting COVID ‐19 with Agility, Transparency, and Participation: Wicked Policy Problems and New Governance Challenges. Public Administration Review. 2020;80(4):651-656. 36. Anderson M, Mckee M, Mossialos E. Developing a sustainable exit strategy for COVID-19: health, economic and public policy implications. Journal of the Royal Society of Medicine. 2020;113(5):176- 178. 37. Muscogiuri G, Barrea L, Savastano S, Colao A. Nutritional recommendations for CoVID-19 quarantine. European Journal of Clinical Nutrition. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Appendixglobalizationandhealth.docx Appendixglobalizationandhealth.docx Page 19/19 Page 19/19 Page 19/19
https://openalex.org/W4241594290	https://newprairiepress.org/cgi/viewcontent.cgi?article=1391&context=gdr	English	null	Conferences	GDR bulletin	1,984	cc-by-sa	930	Conferences Conferences various authors Follow this and additional works at: https://newprairiepress.org/gdr s work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. GDR Bulletin GDR Bulletin GDR Bulletin GDR Bulletin Volume 10 Issue 1 Spring Article 2 Recommended Citation Recommended Citation authors, various (1984) "Conferences," GDR Bulletin: Vol. 10: Iss. 1. https://doi.org/10.4148/ gdrb v10i1 694 authors, various (1984) "Conferences," GDR Bulletin: Vol. 10: Iss. 1. https://doi.org/10.4148/ gdrb.v10i1.694 This Announcement is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in GDR Bulletin by an authorized administrator of New Prairie Press. For more information, please contact cads@k-state.edu. JOURNAL NOTES Abstracts of papers and/or propo- sals far panels and workshops should be submitted by May 1 ,1984 to Dr. Peter Suzuki, Department of Pu- b l i c Administration / Urban Studies, or Dr. Patricia Kolasa, Department of Education Foundations* University of Nebraska at Gmaha, Gmaha, NE 68182. • Sinn und Form . Beiträge zur Litera- tur. Hrsg. v. d. Akademie der Künste der DDR. 35. Jahr (1983), Heft 2-3. VOL. X , No. 1 VOL. X , No. 1 SPRING 1984 CONFERENCES The Ninth Annual European Studies Conference i s an interdisciplinary meeting with sessions devoted to current research, research techni- ques, and teaching methodologies, as well as traditional topics. The Con- ference stresses the i n t e r d i s c i p l i - nary theme focusing on Europe from the Atlantic to the Urals. The Johns Hopkins University Baltimore, MD 21219 Tel. (301) 338-8068 RESEARCH IN PROGRESS INDEX page Research in Progress 1 Conferences 1 Notes in Brief 1 Journal Notes 1 Recent Criticism 6 Recent Literature 6 Book Reviews 7 Visiting Lecturers 13 Book Reviews cont'd 14 INDEX page Research in Progress 1 Conferences 1 Notes in Brief 1 Journal Notes 1 Recent Criticism 6 Recent Literature 6 Book Reviews 7 Visiting Lecturers 13 Book Reviews cont'd 14 RESEARCH IN PROGRESS f i l i a t e i n s t i t u t e , the American In- s t i t u t e for Contemporary German Studies. The Institute w i l l have i t s own Board of Trustees chaired by Donald Rumsfeld, former US Secretary of Defense and US Ambassador to NATO. Dr. Steven Muller, President of Johns Hopkins, w i l l serve as Vice Chairman. States, Canada, Australia, New Zea- land, the United Kingdom, Israel, India, and Ireland w i l l be included in the survey. A survey of English-language re- searchers who have published studies about the GDR i s being conducted. The information gathered w i l l be publis- hed in a monograph,scheduled for au- tumn 1984 publication. It w i l l i n - clude the names of scholars working in the field of GDR studies and their areas of specialization so that others engaged in GDR research w i l l know where to contact collegues with whom one might exchange information and ideas. Scholars in the United If you have published contributions to the growing body of GDR research and have not yet received a question- naire from Dr. Anita M. Mallinckrodt, please contact her at once (2937 Macomb St. NW, Washington, DC 20CO8) so that she can send you the survey material. • The Institute w i l l sponsor re- search projects in various f i e l d s including p o l i t i c s , foreign policy, economics, history since 1945, and German culture in i t s social and p o l i t i c a l context. In 1984, experts w i l l be invited from American and German universities and research i n - stitutions to serve a3 v i s i t i n g scholars at the Institute. A library on post-1945 Germany, and a program of lectures and conferences focusing primarily on the FRG, but including the GDR as well, w i l l be initiated. GERMAN DEMOCRATIC REPUBLIC VOL. X , No. 1 GDR BULLETIN Newsletter for Literature and Culture in the authors: Conferences GERMAN DEMOCRATIC REPUBLIC NOTES IN BRIEF Der 1982 verstorbene Filmregisseur und Präsident der Akademie der Künste Konrad Wolf war seit 1942 Angehöriger der Roten Armee. Am Anfang des 2. Heftes 1983 i s t zum ersten Mal eine Auswahl seiner "Briefe von der Front" an die Familie abgedruckt. Zum 80. Geburtstag von Erich Arendt steuert Hubert Witt einen einfühlsamen Auf- satz bei, der besonders die zentrale Rolle des Traum-Motivs in Arendts Werk beleuchtet und die Entwicklung dieses "Klassikers der s o z i a l i s t i - schen Moderne" (S. 283) von den frü- hen expressionistischen Gedichten bis Amnesty International has recently published two flyers on freedom of expression in the GDR, with a third flyer forthcoming. A longer booklet on the same topic w i l l also appear shortly. The Departments of MFLL and Drama at Kenyon College are j o i n t l y sponsoring a symposium on contempor- ary German language drama from A p r i l 16-21, 1984. One of the featured speakers w i l l be Professor Heinz-Uwe Haus (Institut für Schauspielregie, Berlin). For details contact Prof. Edmund P. - Hecht, Kenyon Collage, Gambier, Ohio 43022. • Interested parties may inquire: Amnesty International USA 304 West 58th Street New York, NY 10019 Tel. (212) 582-4440 Johns Hopkins University is spon- soring, as of October 1983, an af- 1 Published by New Prairie Press, 1984
https://openalex.org/W4213302720	https://www.researchsquare.com/article/rs-1274934/latest.pdf	English	null	Underlying the mechanisms of changes in cancer prevalence among the U.S. Medicare beneficiaries: contributions of incidence, survival, and ascertainment at early stages.	Research Square (Research Square)	2,022	cc-by	6,793	Underlying the mechanisms of changes in cancer prevalence among the U.S. Medicare bene¦ciaries: contributions of incidence, survival, and ascertainment at early stages. Igor Akushevich (  igor.akushevich@duke.edu ) Duke University https://orcid.org/0000-0003-3471-7846 Arseniy Yashkin Duke University Social Science Research Institute Mikhail Kovtun Medanta Duke Research Institute Anatoliy I. Yashin Medanta Duke Research Institute Julia Kravchenko Duke University School of Medicine Underlying the mechanisms of changes in cancer prevalence among the U.S. Medicare beneficiaries: contributions of incidence, survival, and ascertainment at early stages. Akushevich1, A.Yashkin1, M. Kovtun1, A. I. Yashin1, J Kravchenko2 1Center for Population Health and Aging, Duke University, Durham, NC, USA 2Department of Surgery, Duke University School of Medicine, Duke University, Durham, NC, USA 1Center for Population Health and Aging, Duke University, Durham, NC, USA 2Department of Surgery, Duke University School of Medicine, Duke University, Durham, NC, USA Corresponding author: Igor Akushevich, email: igor.akushevich@duke.edu Research Article Keywords: Time trends, Decomposition, Partitioning, Incidence, Survival, Stage ascertainement Posted Date: February 15th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1274934/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Keywords: Time trends, Decomposition, Partitioning, Incidence, Survival, Stage ascertainement Abstract: Purpose: To quantitatively evaluate contributions of trends in incidence, relative survival, and stage at diagnosis to the dynamics in the prevalence of major cancers (lung, prostate, colon, breast, urinary bladder, ovaries, stomach, pancreas, esophagus, kidney, liver, and skin melanoma) among older U.S. adults age 65+. Methods: Trend partitioning was applied to the Surveillance, Epidemiology, and End Results Program data for 1973-2016. Results: Growth of cancer prevalence in older adults decelerated or even decreased over time for all studied cancers due to decreasing incidence and improving survival for most of cancers, with a smaller contribution of the stage at cancer diagnosis. Changes in prevalence of cancers of lung, colon, stomach, and breast were predominantly due to decreasing incidence, increasing survival and more frequent diagnoses at earlier stages. Changes in prevalence of some other cancers demonstrated adverse trends such as decreasing survival in localized and regional stages (urinary bladder and ovarian) and growing impact of late-stage diagnoses (esophageal cancer). Conclusion: While decelerating or decreasing prevalence of many cancers were due to a beneficial combination of decreasing incidence and increasing survival, there are cancers for which decelerating prevalence is due to the lack of improvement in their stage-specific survival and/or increasing frequency of diagnosis at advanced stages. Overall, if the observed trends persist, it is likely that cancer burden due to its prevalence in older U.S. population will be lower than it was projected in earlier studies. Keywords: Time trends, Decomposition, Partitioning, Incidence, Survival, Stage ascertainement 1 Introduction More than half of all cancer survivors in the United States are older than 65 years and the number of older survivors is rising [1] thus posing a challenge to the delivery of quality cancer care [2]. Changes in cancer prevalence result from the interplay of the dynamics of cancer incidence, survival, and stage ascertainment; therefore, evaluation of the mechanisms of changes in prevalence over time provides valuable information on the effectiveness of cancer prevention, early diagnosis, and treatment. Furthermore, prevalence is a measure of disease burden in the population that allows for the planning of health care needs [3]. Information on cancer burden in the older U.S. population can be used in analyses of health expenditures, morbidity, quality of life, and life expectancy. It is increasingly recognized that older survivors have complex healthcare needs, but these are poorly understood [4]. At present, little is known about the burden of cancer and comorbidity among the older adults (i.e., especially those aged 85+) and about the dynamics of this burden over time [5]. 2 While studies on prevalence are common for chronic diseases with a long duration (e.g., diabetes [6], Alzheimer’s disease [7], or osteoarthritis [8]), studies on cancer prevalence are less common with more emphasis placed on cancer incidence or mortality, especially in older U.S. adults. This could be partially due to the complexity of interpretating prevalence trends which result from three distinct interrelated processes: dynamics in cancer incidence, survival, and the average stage at time of diagnosis. The partitioning analysis [9-12] used in this study, generates models of cancer prevalence, incidence, survival and stage ascertainment and then provides quantitative estimates of the relative fractions of the contributions of the above components to the total change in cancer prevalence. Together, changes in the relative magnitudes of these components provide a quantitative picture describing the epidemiologic causes of prevailing prevalence trends and how these have changed over time in response to changes in screening, treatment and exposure to risk-related factors (e.g., smoking, asbestos exposure etc.). This allows researchers to identify both positive trajectories in the epidemiology of a specific cancer (e.g., falling incidence, increasing survival, higher proportion of cases diagnosed a lower stages) as well as potential areas of concern requiring urgent attention from the medical and health policy communities. In a time when improved cancer survivorship is becoming a prominent challenge 2 to the U.S. Introduction healthcare system, such estimates can provide information invaluable in preparing for the expected increase in cancer survivors [13, 14]. Our study focuses on the U.S. population of older adults age 65+ because this population has the highest cancer prevalence among all age groups and, due to the high rates of chronic morbidities in this age group, represents the highest potential challenge for maintaining the health and wellbeing of cancer survivors. Data and Methods Data. The SEER data for 1973-2016 were used in this study. Data before 1992 were used as a look-back period to calibrate the model. SEERStat software was used to evaluate age-adjusted prevalence that was used for comparisons with prevalence proportions calculated using the partitioning approach. The following codes of SEER site records ICD-O-3 were used for analysis: cancer of lung and bronchus (C340-C349), breast (C500-509), prostate (C619), colon (C180, C182-C189, C199), pancreas (C250-C259), urinary bladder (C670-C679), esophagus (C150-C159), liver (C220), kidney (C649, C659), stomach (C160-C169), ovarian (C569), and skin melanoma (C440-C449). Partitioning Analysis The partitioning approach [9-12] is based on an explicit representation of disease prevalence with no simplifying assumptions. The full mathematical derivation of the partitioning approach and its application to lung cancer is available in the study by Akushevich et al. [11]. In brief, the method predicts cancer prevalence and decomposes (or partitions) their time trends into their constituent components by calculating the relative impact each component has on the overall trend as well as inter-temporal changes in the strength and direction of these impacts. Specific outcome measures are age-specific and age-adjusted prevalence; the constituent components are incidence, relative survival, and frequency of cancer stage at diagnoses. The model of cancer prevalence is based on the idea that the probability of being prevalent at a given age requires being incident before and survival after that age. Time trends in age-adjusted prevalence are defined as their derivatives with respect to time y . Explicit calculation of time 3 trends in age-adjusted prevalence results in ( ) / ( ) ( ) ( ) ( ) inc S P y P y T y T y T y   = + + , representing changes in incidence, cancer stage at the time of diagnosis, and relative survival, respectively. Three stages (localized regional and distant) are defined by SEER or modeled from AJCC stages (for prostate cancer). Explicit expressions for partitioning components of cancer age-adjusted prevalence are given in Equations 5 in the study by Akushevich et al. [11]. Each component is to be interpreted as the rate of change at any point in time (increasing if >0, and decreasing if <0) with the magnitude of the effect indicating the speed of the change. The sum of the contributions adds to +100% if the decomposed prevalence is increasing and to -100% if the decomposed prevalence is decreasing at a given point in time. Model Estimation The quantities of interest (i.e., ( ) inc T y , ( ) T y  , and ( ) S T y ) are expressed in terms of the derivatives of the respective functions with respect to time. In our approach, we used explicit analytic parameterizations for all functions for which derivatives are needed. Therefore, we calculated the derivatives analytically. This allowed us to avoid dealing with possible numeric instabilities occurring when derivatives are evaluated numerically. For any function, we used a two-stage approach: i) we parameterized the function and fitted data for each year of diagnosis and ii) we used B-splines to model relationships between year-specific model parameters and evaluate y-dependence of the function. B-splines allow explicit calculation of derivatives without requiring additional simplifying assumptions. The analysis involved the design and estimation of three models: i) for the incidence rate, ii) for frequencies of stage at diagnosis, and iii) for the probability of relative survival after cancer diagnosis. The distribution of age and time at cancer onset was modeled using the Armitage-Doll model with additional individual predisposition modeled by gamma or inverse Gaussian distributions (for incidence) [15], the Weibull model for time after disease onset (for relative survival), and the quadratic function of age dependence (for frequencies of stage at diagnosis). Time trends were modeled by estimating model parameters for each year and applying B-splines to fit the time patterns of obtained parameters. We note that the Weibull model for relative survival was parameterized in terms of  and  exactly as described in refs. [11, 12] for all cancer sites except of cancers of prostate and breast, and skin melanoma 4 for which we used the model in terms of  and . The latter model allows for relative survival for certain cancer sites exceeding 100% (or >1.00): i.e., when survival of patients with certain early-stage cancers was better than survival of individuals in general U.S. population aged 65+, that is observed for these cancer sites. All three models (incidence, stage frequency, and relative survival) were estimated separately and obtained fits were used to construct the model for cancer prevalence for each cancer site. To assess the goodness-of-fit of our model, age-patterns of cancer incidence (averaged over 1992- 2016) and time patterns of age-adjusted measures for incidence, stage frequencies, and relative survival are shown in the 12 cancer-specific panels of Supplementary Figure 1. Model Estimation The predicted prevalence model calculated based on partitioning approach was compared to the estimates provided by SEER (available through SEERStat software). The SEERStat estimates for cancer prevalence were generated using two alternative lookback periods: 18 years, the longest period available for all years of cancer initial diagnosis; and up to 23 years, the longest period available in the data, although not for all years of cancer initial diagnosis. Results The trends of cancer-specific prevalence were calculated using the partitioning approach and compared to corresponding estimates provided by the SEERStat tool (Figure 1). Adding the look-back years in SEERStat estimates increases the prevalence by identifying additional patients who were still surviving persons after being diagnosed with a cancer over the look-back period. Our model, which calculates cancer prevalence outside the bounds of the look-back periods, yielded the highest prevalence levels. The agreement between empirical data and our model was good for all cancers: the shape of the model reflected respective empirical patterns for 18 years, with differences in magnitude reflecting the effect of accumulated prevalence from earlier years as expected [12]. Cancer prevalence estimated from our model monotonically increased for all cancer sites with exceptions for four cancers for which a maximum in prevalence was reached with subsequent decline. These were: cancers of colon (with maximum in 1999), urinary bladder (2008), ovarian 5 5 (2008), and prostate (2010) (Figure 1). Cancers of pancreas, esophagus, liver, kidney, and melanoma had the most pronounced increases in prevalence over time. The relative rate of change (i.e., ( ) / ( ) P y P y  ) is shown in Figure 2 together with three components, incidence, relative survival, and frequencies of stage at diagnoses which, together, add up to the total. The magnitude of the increase in prevalence goes down for all cancers with two evident exceptions, pancreas and kidney cancer, for which there exist periods of acceleration in prevalence growth: 1992-2008 for kidney and 1992-2013 for pancreas cancer. Supportive information on explicit trends of partitioning components obtained empirically and in the model is shown in Supplementary Figure 1. Empirical trends showed decreasing age-adjusted (65+) incidence rates for cancers of lung, colon, stomach, and ovarian over the entire study period. Only skin melanoma had increasing incidence rates during entire study period. Other cancers had periods of both increasing and decreasing incidence rates (Supplemental Figure 1). The results of partitioning analysis (Figure 2) show that changes in incidence rates were the main contributors to the deceleration of prevalence growth. Most substantially incidence contributed to prevalence deceleration for cancers of lung, colon, urinary bladder (since 2005), stomach, ovaries, and breast (since 2010). Decline in the prevalence for several cancers, colon, bladder (since 2005), ovarian (since 2008) and, prostate (since 2011) was the result of the effect of incidence trends. Results Although trends in incidence differ, the rate of change in the incidence effect goes down for most of the studied cancers (Figure 2), although for cancers of kidney, liver, and pancreas the decline starts on or about 2005. 6 Empirical trends of 1- and 3-year survival showed gradual improvements in relative survival among older adults age 65+ for all cancers except urinary bladder and ovarian (for local and regional stages), liver (for regional and distant stages), and prostate (local and distant) (Supplemental Table 1). Partitioning analysis showed that the relative survival from all cancers had a beneficial trend and pushed cancer prevalence up, though improvements in relative 6 6 survival (Figure 2). However, the impacts of better survival on the prevalence trend were slowing down over time for all cancers except kidney cancer. Most rapid decline in the effect of survival was detected for urinary bladder and ovarian cancers. Empirical trends showed that over time, in older adults age 65+, cancers of breast, prostate, skin melanoma, colon, urinary bladder, liver, and kidney were more often diagnosed at early (i.e., localized) stages (Supplemental Table 1). However, cancers of lung, pancreas, stomach, ovaries and esophagus were more often diagnosed at distant rather than in localized or regional stages, and this trend was growing for lung, stomach and especially esophageal cancers (Supplemental Table 1). Compared to the contributions of incidence and relative survival, the contribution of stage at ascertainment to the dynamics of cancer prevalence were less pronounced for most studied cancers. This contribution was minimal for cancers of colon, urinary bladder, prostate, and skin melanoma, but the effect of stage at ascertainment on dynamics of cancer prevalence overcame the impacts of incidence for pancreatic cancer (since 2013) and was comparable to impact of relative survival for kidney cancer (Figure 2). A disadvantageous and relatively strong in magnitude effect of stage ascertainment was detected for esophageal cancer (i.e., the impacts of cancer being diagnosed at advanced stage) (Figure 2). Discussion Using partitioning approach [11, 12], we quantitatively evaluated contributions of cancer incidence, relative survival, and early-stage diagnoses to the changes in cancer prevalence in older U.S. population aged 65+. In this study, partitioning of prevalence was analyzed together with empiric patterns of cancer incidence and stage-specific relative survival thus providing more detailed information on the mechanisms of changes in prevalence of various cancers in the U.S. For all studied cancers, growth of prevalence decelerated over time and for cancers of colon, urinary bladder, ovaries, and prostate, the prevalence passed its respective maximum resulting in declines over recent years. Trends in cancers of lung, colon, stomach, and breast reflect positive reasons for the observed prevalence change: decreasing incidence and increasing 7 7 survival together with increases in diagnoses at earlier stages over time. However, prevalence trends of certain cancers reflect adverse and sometimes alarming patterns. Specifically, cancer of urinary bladder shows decreasing survival in localized and regional stages, ovarian cancer shows decreasing survival in localized and regional stages, although with an increase in early- stage diagnoses, and esophageal cancer shows an increasing impact of late-stage diagnoses. Why it is important to understand underlying mechanisms of cancer trends in older adults. Aging of the U.S. population is a well-recognized factor contributing to cancer burden [16, 17]; therefore, timely information on cancer burden in older U.S. adults can be used for further optimization of cancer prevention, early diagnosis, and treatment strategies in this growing sector of the population. At present, information on cancer site-specific epidemiologic components (such as incidence, survival, and diagnosis at early vs. advanced stage) that define the dynamic of cancer prevalence is sparse. However, this information is important for understanding the mechanisms behind changes in cancer prevalence, evaluation of health expenditures and cancer burden among older U.S. adults. In addition to higher cancer prevalence, older patients often have functional impairment, multiple coexisting diseases, cognitive impairment, and a higher risk of treatment side effects that could lead to the choices toward less aggressive/effective treatment [18]. Despite their numbers, older adults continue to be understudied, a point emphasized by the Institute of Medicine in addressing the challenges in delivery of a quality cancer care [19]. Cancer patients age 65+ were largely excluded from clinical trials, precluding opportunities to develop a greater insight about the unique needs of older patients, including long-term survivors [2, 19, 20]. Discussion Therefore, it is crucial to investigate cancer outcomes and their recent trends in older patients to obtain an important information on this growing group of population that remains at increased risk of social, physical, and mental vulnerability. 8 Dynamics of cancer prevalence among older U.S. adults. The main driving forces underlying the prevalence trends observed in our study were a deceleration in incidence increase and improved relative survival for the majority of cancers studied. If current trends persist, we expect a substantial deceleration in the prevalence of many cancers in the U.S. population of older adults age 65+ within the next decades. For many cancers dynamics of prevalence have slowed 8 8 down (lung, stomach, and breast) or even started to decline (colon, prostate, ovarian, and urinary bladder). These trends are not in a full agreement with a recent projections suggesting that the prevalence of cancer survivors will increase from 15.5 million to 26.1 million (across all age groups) from 2016 to 2040, with the largest growth of cancer survivors in the older population [2]. The same study estimated that compared to 1975, the 2040 projected cancer prevalence will be 6-fold higher for those aged 65-74 years, 10-fold higher for ages 75-84 years, and 17-fold higher for ages 85+ [2]. However, this study held the rates of cancer incidence and survival constant for the whole period of the projection, while in our approach we incorporated the dynamics of cancer incidence, relative survival, and stage at cancer diagnosis when analyzing changes of prevalence of each cancer. Dynamics of cancer incidence and its role in cancer prevalence trends. Deceleration of growth of cancer prevalence obtained in our study can be explained, to a great extent, by the deceleration of the increase or actual decrease of cancer incidence over time such as observed for cancers of lung, colon, stomach, ovaries, urinary bladder, and breast. Effectiveness of ongoing preventive strategies substantially contributed to the deceleration of incidence observed over recent decade. Discussion They include, but not are limited to, decreasing smoking prevalence (affects risk of lung, urinary bladder, kidney, liver, colon, and stomach cancers, with the respective percent of smoking- attributable deaths estimated at 80.2%, 44.8%, 16.8%, 23.6%, 9.7%, and 19.6%, respectively [21]), implementation of gastric cancer prevention programs that includes screening for Helicobacter pylori infection and its treatment [22], and decreasing prevalence of hepatitis B infection and its increasing vaccination rate (for liver cancer [23]). However, persisting or even increasing prevalence of metabolic syndrome, obesity, and hepatitis C, as well as a poor diet, low physical activity, and other risk factors still slow the deceleration of cancer incidence. 9 Dynamics of relative survival and its role in cancer prevalence trends. The trends of relative survival obtained in our study were beneficial for all studied cancers, though improvement in relative survival has slowed down within recent decade for many cancers. The beneficial trends of relative survival could decline over time because of accumulation of cancer patients who benefited from increased survival in earlier years and, therefore, died later [12]. For example, it 9 9 has been shown that the number of cancer survivals among older U.S. adults grew over time, with gender-specific differences widening at older ages: there was 5%-8% more male than female cancer survivors at ages 70–79, 11% more at ages 80–89, and 12% more at age 90+ (with 37% of men aged 90+ being cancer survivors [2]. Slowing improvement or even decline in relative survival detected for several cancers (e.g., bladder, liver, ovarian) in our study can be the result of the accumulation of delayed mortality in individuals diagnosed early in the study time- period and benefiting from improved survival at the time of their diagnosis. Targeted partitioning of cancer mortality [12] would be required to further analyze this hypothesis. The contributions of slowing improvements in survival to dynamics of prevalence were higher for cancers of prostate, ovaries, and urinary bladder. These effects were also observed for empiric trends of stage-specific survival of these cancers: e.g., decreasing trends of prostate cancer survival in epidemiologic analysis for localized and distant stages (Supplemental Figure 1). Another study showed decreases in survival (non-stage specific) among patients with prostate cancer from 2006/2010 to 2011/2016: a 5-year survival decreased for 1.3% for ages 65-79 and for 7.2% for patients aged 80+ [24]. Discussion It has been suggested that lower survival at older ages might be due to a more rapid development of resistant prostate cancer, reduced ability to receive cancer therapies, and the impact of comorbidities [24, 25]. However, that cannot explain the changes in prostate cancer survival among older patients during the recent decade, as well as the negative dynamics of survival at localized stages observed in our study. The trends of ovarian cancer survival among females age 65+ show that while there are positive contributions of the decreasing role of incidence and the increasing frequency of earlier stage diagnoses of ovarian cancer, the relative survival of patients with localized and regional tumors actually decreased since 2003. While publications on survival trends among older patients with ovarian cancer remain sparse, a recent study showed that females age 75+ with ovarian cancer had stage-appropriate cancer surgery (OR=0.58, 95%CI-0.40-0.83) and got multi-agent chemotherapy (OR=0.27, 95%CI=0.17-0.41) [26] less often than patients younger than 50 years old. 10 We also observed decreasing 3-year survival among older patients with localized or regional cancer of urinary bladder. This is in agreement with a study showing that treatment developments over the recent three decades did not result in improvements in mortality from localized or regional cancers of urinary bladder, with this negative trend being especially pronounced among older patients [27]. Better management algorithms and more effective treatment strategies are necessary to improve outcomes for such individuals. Stage at cancer diagnosis and its role in the dynamics of cancer prevalence. Compared to contributions of relative survival and incidence, changes in stage at cancer diagnoses contributed less to the dynamics of cancer prevalence for most of cancers in our study except for cancers of pancreas; kidney and stomach (through increasing frequency of early-stage diagnoses); and esophagus (through increasing frequency of late-stage malignancies). Information on trends of stage at diagnosis for these cancers is sparse, especially for older patients. Cancers of pancreas and kidney are rarely diagnosed at early stages and screening for these cancers is not feasible for the general population [28, 29]. Discussion Screening for pancreatic cancer is recommended for high-risk groups starting from age 50 (with the use of endoscopic ultrasound and/or magnetic resonance imaging) [28, 30], and screening for kidney cancer (with the use of computer tomography or magnetic resonance imaging) is recommended for individuals with a known heritable syndrome associated with the development of renal cell carcinoma [31, 32]. It has been speculated that more frequent use of cross-section imaging has resulted in these malignancies being diagnosed at earlier stages [33]. In contrast, screening for stomach cancer is better established; however, among older adults testing for Helicobacter pylori infection remains low (7.7%), even among those who have risk factors associated with this cancer (11.6%) [34]. While the impacts of screening on the prevalence of the abovementioned cancers is low for all ages [35-37], our study shows that increased diagnoses at early stages impacted both the dynamics of respective cancer prevalences and the trends in relative survival among older patients. Specifically, increasing frequencies of early-stage diagnoses have been shown for cancers of pancreas, kidney, and stomach and 3-year relative survival increased for localized and regional cancers (in 2016 the survival was 20%, 75%, and 45% for pancreatic, kidney, and stomach cancers, respectively; Supplemental Figure 1). On the other hand, for cancers with well recognized screening 11 strategies (e.g., for cancers of prostate, breast, and skin melanoma) the contribution of overtime changes in the frequency of early-stage diagnoses to dynamics of prevalence was minimal because such increases over the recent decades were themselves small when compared to the already high rates of early-stage diagnosis for these tumors. A concerning trend was observed for esophageal cancer which demonstrated a slowing improvement in survival and increasing levels of diagnoses at advanced stages. Both effects substantially contributed to deceleration in cancer prevalence among older U.S. adults. Although recent analysis of SEER data showed that 5-year survival rates for all-age patients with esophageal cancer increased from 1973 to 2010 (for both localized and regional stages), information on survival among older patients remains sparse [38]. It has been speculated that survival of patients with esophageal cancer was slowly improving due to better surgical techniques, adjuvant therapy, and increasing use of upper gastrointestinal endoscopy for early diagnosis [38, 39]. Discussion However, it seems that this may not be the case for older patients for whom a deceleration in survival could be associated with changing patterns of histotype-specific incidence with esophageal adenocarcinoma rates surpassing the rates of squamous cell carcinoma [38, 40]. These patterns could contribute to the observed dynamics of early-stage diagnoses. Future studies should address potential causes of changes in survival among older patients with histotype-specific esophageal cancer. 12 Why are the studies on cancer prevalence trends relatively rare compared to the studies on incidence or mortality? Although many studies focus on cancer mortality trends in the U.S. [41- 44], studies on cancer prevalence including analyses of dynamics of epidemiologic characteristics that impact prevalence trends are less common [2, 45]. In part, this is due to a lower availability of data on disease prevalence, while mortality data are widely available (e.g., in the Multiple Cause of Death datafiles). Even when the data on cancer prevalence can be obtained (e.g., from SEER-Medicare data), the results requires complex interpretation compared to the results on incidence or mortality [10] because prevalence is based on both disease incidence and its duration, and a high prevalence of disease in a population could reflect high incidence, or better survival, or both (and a low prevalence could indicate low disease incidence, 12 12 or worth survival, or both). Partitioning analysis we used provides: i) high-precision estimates of time trends of cancer prevalence; ii) explanations of these trends based on the trends of cancer incidence, stage-specific relative survival, and ascertainment of cancer at early stages; and iii) interpretation of the obtained results and identification of the groups of cancers with similar patterns. This method reveals the mechanisms of interrelations between cancer incidence, prevalence, and relative survival, thus being an important step in assessing current and future cancer burden, identification of potential problem areas that requires more attention, and evaluation of how these important epidemiological characteristics respond to implemented health interventions over time. Study limitations. The SEER database used in this study represents approximately 28% of the U.S. population and underrepresents certain racial/ethnic groups and geographic regions. Competing Interests The authors have no relevant financial or non-financial interests to disclose. All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by [Igor Akushevich], [Arseniy Yashkin] and [Mikhail Kovtun]. The first draft of the manuscript was written by [Igor Akushevich] and [Julia Kravchenko] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Funding This study was supported by the National Institute on Aging (R01-AG066133, RF1-AG046860, R01-AG057801). The sponsors had no role in the design and conduct of this study. Data Availability The datasets generated during and/or analyzed during the current study are available in the National Cancer Institute repository, https://seer.cancer.gov/data/. Ethics approval This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Institutional Review Board of Duke University (FWA00009025), IRB ID Pro00101359. Consent to participate HIPAA Waiver for Informed Consent was approved by the Institutional Review Board of Duke University (FWA00009025). HIPAA Waiver for Informed Consent was approved by the Institutional Review Board of Duke University (FWA00009025). Discussion 14 14 Statements and Declarations Statements and Declarations Discussion In this study we investigated cancer sites without further detailing by histotypes; however, cancers of different histotypes (e.g., adenocarcinomas and squamous cell carcinomas, subtypes of ovarian or breast cancers, etc.) are distinct by their risk factors, biology, time trends, populations at risk, treatment, and survival; therefore, their contributions to prevalence trends also differ. Further, our calculations were based on empiric parametric models for partitioning components, and although the models were constructed based on accepted models some statistical uncertainties still remain. In addition, the model for cancer survival in the most recent years can be less precise because of a limited survival time available. SEER data do not provide individual-level variables on cancer risk factors (e.g., smoking and other); therefore, the dynamics of these factors were not integrated into partitioning analysis. Finally, we did not perform gender- and race/ethnicity-specific partitioning analysis in this study to keep it focused on various cancer sites; further studies will include more detailed analyses for these populations subgroups. Summary. Our analysis shows that the slowing in the increases in the prevalence of many cancers among older U.S. adults over recent decades can be explained by decreasing trends of incidence and improving survival for most cancers, with smaller contributions of the stage at cancer diagnosis. If these dynamics persist, it is likely that cancer burden in older U.S. population of older adults will be lower than projected previously. While decelerating or 13 decreasing prevalence of many cancers are due to a beneficial combination of decreasing incidence and increasing survival, there are cancers for which decelerating prevalence is due to the lack of improvement in their stage-specific survival and/or increasing frequency of diagnosis at advanced stages. Overall, if the observed trends persist, it is likely that cancer burden due to its prevalence in older U.S. population will be lower than it was projected in earlier studies. Although the literature on cancer burden and its trends in older populations remains sparse, clinicians are increasingly recognizing the benefits of a geriatric assessment framework in oncology [5, 46], with the complex health needs of older patients and finding efficient ways to meet the medical surveillance needs of older survivors will become increasingly important [2, 47]. Although a number of interventions have been developed to help survivors cope with cancer, few have targeted older adults and this remains an area of critical need in survivorship science [2, 47]. Consent to publish Not Applicable. Not Applicable. 15 References Evaluating the Number of Stages in Development of Squamous Cell and Adenocarcinomas across Cancer Sites Using Human Population-Based Cancer Modeling. PloS one. 2012;7(5):e37430. 16. Cho H, Mariotto AB, Mann BS, Klabunde CN, Feuer EJ. Assessing Non–cancer-related health status of US cancer patients: other-cause survival and comorbidity prevalence. American journal of epidemiology. 2013;178(3):339-349. 17. Manton KG, Akushevich I, Kravchenko J. Cancer mortality and morbidity patterns in the US population: an interdisciplinary approach. Springer New York:; 2009. 18. Foster JA, Salinas GD, Mansell D, Williamson JC, Casebeer LL. How does older age influence oncologists' cancer management? The oncologist. 2010;15(6):584-592. 19. Levit LA, Balogh E, Nass SJ, Ganz P. Delivering high-quality cancer care: charting a new course for a system in crisis. National Academies Press Washington, DC; 2013. 20. Unger JM, Coltman Jr CA, Crowley JJ, et al. Impact of the year 2000 Medicare policy change on older patient enrollment to cancer clinical trials. Journal of clinical oncology. 2006;24(1):141-144. 21. Siegel RL, Jacobs EJ, Newton CC, et al. Deaths due to cigarette smoking for 12 smoking- related cancers in the United States. JAMA internal medicine. 2015;175(9):1574-1576. 22. Thrift AP, El-Serag HB. Burden of gastric cancer. Clinical Gastroenterology and Hepatology. 2020;18(3):534-542. 23. Kruszon-Moran D, Paulose-Ram R, Martin CB, Barker LK, McQuillan G. Prevalence and trends in hepatitis B virus infection in the United States, 2015–2018. 2020; 24. Siegel DA, O’Neil ME, Richards TB, Dowling NF, Weir HK. Prostate cancer incidence and survival, by stage and race/ethnicity—United States, 2001–2017. Morbidity and Mortality Weekly Report. 2020;69(41):1473-1480. 25. Bernard B, Burnett C, Sweeney CJ, Rider JR, Sridhar SS. Impact of age at diagnosis of de novo metastatic prostate cancer on survival. Cancer. 2020;126(5):986-993. 26. Warren JL, Harlan LC, Trimble EL, Stevens J, Grimes M, Cronin KA. Trends in the receipt of guideline care and survival for women with ovarian cancer: a population-based study. Gynecologic oncology. 2017;145(3):486-492. 27. Abdollah F, Gandaglia G, Thuret R, et al. Incidence, survival and mortality rates of stage- specific bladder cancer in United States: a trend analysis. Cancer epidemiology. 2013;37(3):219-225. 28. Zhang L, Sanagapalli S, Stoita A. Challenges in diagnosis of pancreatic cancer. World journal of gastroenterology. 2018;24(19):2047-2060. 29. de Leon AD, Pedrosa I. Imaging and screening of kidney cancer. Radiologic Clinics. 2017;55(6):1235-1250. 30. Canto MI, Harinck F, Hruban RH, et al. International Cancer of the Pancreas Screening (CAPS) Consortium summit on the management of patients with increased risk for familial pancreatic cancer. References 1. De Moor JS, Mariotto AB, Parry C, et al. Cancer survivors in the United States: prevalence across the survivorship trajectory and implications for care. Cancer Epidemiology and Prevention Biomarkers. 2013;22(4):561-570. 2. Bluethmann SM, Mariotto AB, Rowland JH. Anticipating the “silver tsunami”: prevalence trajectories and comorbidity burden among older cancer survivors in the United States. Cancer Epidemiology and Prevention Biomarkers. 2016;25(7):1029-1036. 3. Ward MM. Estimating disease prevalence and incidence using administrative data: some assembly required. The Journal of Rheumatology; 2013. p. 1241-1243. 4. Bellizzi KM, Aziz NM, Rowland JH, et al. Double jeopardy? Age, race, and HRQOL in older adults with cancer. Journal of cancer epidemiology. 2012;2012:Article ID 478642. 5. Hurria A, Levit LA, Dale W, et al. Improving the evidence base for treating older adults with cancer: American Society of Clinical Oncology statement. J Clin Oncol. 2015;33(32):3826- 3833. 6. Andes LJ, Li Y, Srinivasan M, Benoit SR, Gregg E, Rolka DB. Diabetes prevalence and incidence among Medicare beneficiaries—United States, 2001–2015. Morbidity and Mortality Weekly Report. 2019;68(43):961-966. 7. Association As. 2019 Alzheimer's disease facts and figures. Alzheimer's & dementia. 2019;15(3):321-387. 8. O'Neill TW, McCabe PS, McBeth J. Update on the epidemiology, risk factors and disease outcomes of osteoarthritis. Best practice & research Clinical rheumatology. 2018;32(2):312- 326. 9. Akushevich I, Yashkin AP, Kravchenko J, et al. Theory of partitioning of disease prevalence and mortality in observational data. Theoretical population biology. 2017;114:117-127. 10. Akushevich I, Yashkin AP, Kravchenko J, et al. Identifying the causes of the changes in the prevalence patterns of diabetes in older US adults: A new trend partitioning approach. J Diabetes Complicat. 2018;32(4):362-367. 11. Akushevich I, Kravchenko J, Yashkin AP, Fang F, Yashin AI. Partitioning of time trends in prevalence and mortality of lung cancer. Statistics in medicine. 2019;38(17):3184-3203. 12. Akushevich I, Yashkin AP, Inman BA, Sloan F. Partitioning of time trends in prevalence and mortality of bladder cancer in the United States. Annals of Epidemiology. 2020;47:25-29. 13. Yabroff KR, Gansler T, Wender RC, Cullen KJ, Brawley OW. Minimizing the burden of cancer in the United States: Goals for a high‐performing health care system. CA: a cancer journal for clinicians. 2019;69(3):166-183. 14. Ellis L, Canchola AJ, Spiegel D, Ladabaum U, Haile R, Gomez SL. Trends in cancer survival by health insurance status in California from 1997 to 2014. JAMA oncology. 2018;4(3):317-323. 16 15. Kravchenko J, Akushevich I, Abernethy AP, Lyerly HK. References Gut. 2013;62(3):339-347. 17 31. Society AC. American Cancer Society. Cancer Facts & Figures 2016. 2016. Accessed August 13, 2021. https://www.cancer.org/content/dam/cancer-org/research/cancer-facts-and- statistics/annual-cancer-facts-and-figures/2016/cancer-facts-and-figures-2016.pdf 32. Gray RE, Harris GT. Renal cell carcinoma: diagnosis and management. American family physician. 2019;99(3):179-184. 33. Hollingsworth JM, Miller DC, Daignault S, Hollenbeck BK. Rising incidence of small renal masses: a need to reassess treatment effect. Journal of the National Cancer Institute. 2006;98(18):1331-1334. 34. Florea A, Brown HE, Harris RB, Oren E. Ethnic disparities in gastric cancer presentation and screening practice in the United States: analysis of 1997–2010 surveillance, epidemiology, and end results-medicare data. Cancer Epidemiology and Prevention Biomarkers. 2019;28(4):659-665. 35. Fogel EL, Shahda S, Sandrasegaran K, et al. A multidisciplinary approach to pancreas cancer in 2016: a review. The American journal of gastroenterology. 2017;112(4):537-554. 36. Sung W-W, Ko P-Y, Chen W-J, Wang S-C, Chen S-L. Trends in the kidney cancer mortality-to-incidence ratios according to health care expenditures of 56 countries. Scientific Reports. 2021;11(1):1-8. 37. Kim GH, Liang PS, Bang SJ, Hwang JH. Screening and surveillance for gastric cancer in the United States: is it needed? Gastrointestinal endoscopy. 2016;84(1):18-28. 38. He H, Chen N, Hou Y, et al. Trends in the incidence and survival of patients with esophageal cancer: a SEER database analysis. Thoracic cancer. 2020;11(5):1121-1128. 39. Macías-García F, Domínguez-Muñoz JE. Update on management of Barrett's esophagus. World journal of gastrointestinal pharmacology and therapeutics. 2016;7(2):227-234. 40. Arnal MJD, Arenas ÁF, Arbeloa ÁL. Esophageal cancer: Risk factors, screening and endoscopic treatment in Western and Eastern countries. World journal of gastroenterology: WJG. 2015;21(26):7933-7943. 41. Zahnd WE, James AS, Jenkins WD, et al. Rural–urban differences in cancer incidence and trends in the United States. Cancer Epidemiology and Prevention Biomarkers. 2018. 27(11): p. 1265-1274. 42. Henley SJ. Invasive cancer incidence, 2004–2013, and deaths, 2006–2015, in nonmetropolitan and metropolitan counties—United States. MMWR Surveillance Summaries. 2017;66(14):1-13. 43. O’Keefe EB, Meltzer JP, Bethea TN. Health disparities and cancer: racial disparities in cancer mortality in the United States, 2000–2010. Frontiers in public health. 2015;3:51. 44. Singh GK, Jemal A. Socioeconomic and racial/ethnic disparities in cancer mortality, incidence, and survival in the United States, 1950–2014: over six decades of changing patterns and widening inequalities. Journal of environmental and public health. 2017;2017:Article ID 2819372. 18 45. Phillips SM, Padgett LS, Leisenring WM, et al. Survivors of childhood cancer in the United States: prevalence and burden of morbidity. Cancer Epidemiology and Prevention Biomarkers. 2015;24(4):653-663. 46. Walko CM, McLeod HL. 47. Rowland JH, Bellizzi KM. Cancer survivorship issues: life after treatment and implications for an aging population. Journal of Clinical Oncology. 2014;32(24):2662-2668. 45. Phillips SM, Padgett LS, Leisenring WM, et al. Survivors of childhood cancer in the United States: prevalence and burden of morbidity. Cancer Epidemiology and Prevention Biomarkers. 2015;24(4):653-663. References Personalizing medicine in geriatric oncology. Journal of clinical oncology. 2014;32(24):2581-2586. 47. Rowland JH, Bellizzi KM. Cancer survivorship issues: life after treatment and implications for an aging population. Journal of Clinical Oncology. 2014;32(24):2662-2668. 19 Fig. 1. Estimates of cancer prevalence. Age-adjusted prevalence using our model (solid b line), age-adjusted prevalence using SEERStat using an 18-y look-back period (red line filled circles), and age-adjusted prevalence using SEERStat using an up to 23-y look- period (blue line with filled squares). The value on each plot is a rescaling factor. A Fig. 1. Estimates of cancer prevalence. Age-adjusted prevalence using our model (solid black line), age-adjusted prevalence using SEERStat using an 18-y look-back period (red line with filled circles), and age-adjusted prevalence using SEER*Stat using an up to 23-y look-back period (blue line with filled squares). The value on each plot is a rescaling factor. Actual 20 prevalence is obtained by multiplication of this factor and the value taken from Y-axis of the plot. Fig. 2 Partitioning of main cancer age-adjusted prevalence. Color curves represent contributions of incidence (green), survival (red), and frequency at diagnosis (blue). Fig. 2 Partitioning of main cancer age-adjusted prevalence. Color curves represent the contributions of incidence (green), survival (red), and frequency at diagnosis (blue). 21 Supplementary Figure 1. Empiric patterns for cancers of the study: age-adjusted incidence 65+ (upper left), age-specific incidence averaged over study period (upper right), age-adjusted frequencies of localized, regional, and distant stages (middle left), one- and three-year relative survival for localized (middle right), regional (bottom left) and distant (bottom right) stages. 22 22 Supplementary.pdf Supplementary Files This is a list of supplementary ¦les associated with this preprint. Click to download. Supplementary.pdf
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If the publication is distributed under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license above, please follow below link for the End User Agreement: Document license: CC BY DOI: 10.1016/j.nanoen.2017.09.043 Document status and date: Published: 01/12/2017 Electrochemical reduction of CO2 on compositionally variant Au-Pt bimetallic thin films Citation for published version (APA): Ma, M., Hansen, H. A., Valenti, M., Wang, Z., Cao, A., Dong, M., & Smith, W. A. (2017). Electrochemical reduction of CO2 on compositionally variant Au-Pt bimetallic thin films. Nano Energy, 42(December 2017), 51- 57. https://doi.org/10.1016/j.nanoen.2017.09.043 Citation for published version (APA): Ma, M., Hansen, H. A., Valenti, M., Wang, Z., Cao, A., Dong, M., & Smith, W. A. (2017). Electrochemical reduction of CO2 on compositionally variant Au-Pt bimetallic thin films. Nano Energy, 42(December 2017), 51- 57. https://doi.org/10.1016/j.nanoen.2017.09.043 Document license: CC BY DOI: 10.1016/j.nanoen.2017.09.043 Document status and date: Published: 01/12/2017 Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Please check the document version of this publication: • A submitted manuscript is the version of the article upon submission and before peer-review. There ca important differences between the submitted version and the official published version of record. People interested in the research are advised to contact the author for the final version of the publication, or vis DOI to the publisher's website. • The final author version and the galley proof are versions of the publication after peer review. • The final published version features the final layout of the paper including the volume, issue and page numbers. Link to publication Download date: 24. Oct. 2024 A R T I C L E I N F O Keywords: CO2 conversion Bimetallic catalysts Au-Pt alloy thin ﬁlms Electrocatalysis Keywords: CO2 conversion Bimetallic catalysts Au-Pt alloy thin ﬁlms Electrocatalysis The electrocatalytic reduction of CO2 on Au-Pt bimetallic catalysts with diﬀerent compositions was evaluated, oﬀering a platform for uncovering the correlation between the catalytic activity and the surface composition of bimetallic electrocatalysts. The Au-Pt alloy ﬁlms were synthesized by a magnetron sputtering co-deposition technique with tunable composition. It was found that the syngas ratio (CO:H2) on the Au-Pt ﬁlms is able to be tuned by systematically controlling the binary composition. This tunable catalytic selectivity is attributed to the variation of binding strength of COOH and CO intermediates, inﬂuenced by the surface electronic structure (d- band center energy) which is linked to the surface composition of the bimetallic ﬁlms. Notably, a gradual shift of the d-band center away from the Fermi level was observed with increasing Au content, which correspondingly reduces the binding strength of the COOH and CO intermediates, leading to the distinct catalytic activity for the reduction of CO2 on the compositionally variant Au-Pt bimetallic ﬁlms. In addition, the formation of formic acid in the bimetallic systems at reduced overpotentials and higher yield indicates that synergistic eﬀects can fa- cilitate reaction pathways for products that are not accessible with the individual components. Electrochemical reduction of CO2 on compositionally variant Au-Pt bimetallic thin ﬁlms Ming Maa, Heine A. Hansenb, Marco Valentia, Zegao Wangc, Anping Caod, Mingdong Dongc, Wilson A. Smitha,⁎ a Materials for Energy Conversion and Storage (MECS), Department of Chemical Engineering, Faculty of Applied Sciences, Delft University of Technology, Van d Maasweg 9, 2629 HZ Delft, The Netherlands b Department of Energy Conversion and Storage, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark c Interdisciplinary Nanoscience Center (iNANO), Aarhus University, DK 8000 Aarhus City, Denmark sion and Storage (MECS), Department of Chemical Engineering, Faculty of Applied Sciences, Delft University of Technology, Van der he Netherlands a Materials for Energy Conversion and Storage (MECS), Department of Chemical Engineering, Faculty of Applied Sciences, Delft University of Technology, Van de Maasweg 9, 2629 HZ Delft, The Netherlands b Department of Energy Conversion and Storage Technical University of Denmark 2800 Kgs Lyngby Denmark a Materials for Energy Conversion and Storage (MECS), Department of Chemical Engineering, Faculty of Applied Sciences, Delft University of Technology, Maasweg 9, 2629 HZ Delft, The Netherlands b Department of Energy Conversion and Storage, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark c Interdisciplinary Nanoscience Center (iNANO), Aarhus University, DK 8000 Aarhus City, Denmark d Department of Chemical Engineering and Chemistry, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands b Department of Energy Conversion and Storage, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark c Interdisciplinary Nanoscience Center (iNANO), Aarhus University, DK 8000 Aarhus City, Denmark d Department of Chemical Engineering and Chemistry, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands ⁎ Corresponding author. E-mail address: W.Smith@tudelft.nl (W.A. Smith). www.tue.nl/taverne Take down policy If you believe that this document breaches copyright please contact us at: openaccess@tue.nl providing details and we will investigate your claim. Download date: 24. Oct. 2024 Nano Energy 42 (2017) 51–57 http://dx.doi.org/10.1016/j.nanoen.2017.09.043 Received 23 May 2017; Received in revised form 25 August 2017; Accepted 20 September 2017 ⁎ Corresponding author. E-mail address: W.Smith@tudelft.nl (W.A. Smith). Available online 10 October 2017 2211-2855/ © 2017 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY 1. Introduction In addition, the thickness of the Au-Pt ﬁlms were controlled by deposition time, and the cross-sectional scanning electron microscopy (SEM) image (Fig. S3) indicates that the Au-Pt binary electrocatalyst was comprised of ~ 60–70 nm thick ﬁlm. To conﬁrm the surface composition of the deposited Au-Pt bime- tallic ﬁlms, X-ray photoelectron spectroscopy (XPS) measurements were performed. It was found that the surface atomic ratio of Au to Pt in the Au-Pt bimetallic ﬁlm was tuned by altering the deposition rate of magnetron sputtering (Table S1). It has been reported that Au could segregate to the surface of binary ﬁlms, which is caused by the low surface free energy of Au, resulting in slightly enriched Au in binary ﬁlm [29]. Here, the bulk composition (Table S2) of the deposited Au-Pt bimetallic ﬁlm were characterized by Energy-dispersive X-ray spectro- scopy (EDS). The comparison of the bulk and surface composition of binary ﬁlms is presented in Fig. 1a, which reveals that the bulk com- position of the Au-Pt binary ﬁlms was consistently identical to the surface composition of the ﬁlms, indicating no obvious Au segregation at the surface of the binary ﬁlms prepared by magnetron sputtering. The observation of no Au segregation at the surface is consistent with the previous work on Au-Pt bimetallic ﬁlms fabricated using pulsed laser deposition [30]. Importantly, the surface composition of the Au-Pt ﬁlms was maintained even after 2 h of electrolysis (Table S3). Au is the most active metal electrocatalysts for the reduction of CO2 to CO, however, the catalytic activity on Au is still limited by the ac- tivation of CO2 to stabilize COOH [17]. In contrast, Pt provides favor- able activation and conversion of CO2 to adsorbed CO (limiting step is CO desorption) [17]. Herein, we present the ﬁrst exploration of the electrocatalytic reduction of CO2 on Au-Pt bimetallic thin ﬁlms with controllable compositions and planar morphology. These bimetallic planar ﬁlms provide an ideal platform for investigating the electronic and synergistic eﬀects on the binding strength of intermediates and the formation of ﬁnal products by tuning the composition of bimetallic catalysts while keeping the surface morphology consistent. 1. Introduction (b) XRD patterns of pure Au (orange line), pure Pt (gray line) and Au100−nPtn bimetallic ﬁlms. (c) Variation of the lattice parameter of the Au-Pt bimetallic alloys as a function of the Pt content. M. Ma et al. Nano Energy 42 (2017) 5157 Fig. 1. Characterization of Au-Pt bimetallic ﬁlms. (a) A comparison of surface composition with bulk composition of Au-Pt ﬁlm measured by XPS and EDS, respectively. (b) XRD patterns of pure Au (orange line), pure Pt (gray line) and Au100−nPtn bimetallic ﬁlms. (c) Variation of the lattice parameter of the Au-Pt bimetallic alloys as a function of the Pt content. to HCOOH of ~ 0 V vs. the reversible hydrogen electrode (RHE) and a high faradaic eﬃciency (FE) of 88% for HCOOH formation at −0.4 V vs. RHE. In addition, Takanabe et al. [18] discovered that a nanos- tructured Cu-In bimetallic alloy prepared by the in situ electrochemical reduction of Cu2O in InSO4 electrolytes is able to reduce CO2 to CO with a FE of 85% at −0.6 V vs. RHE. Furthermore, the dramatically im- proved FE for the reduction of CO2 to CO has also been achieved on nanostructured Cu-Sn and Cu-Pd bimetallic catalysts at reduced over- potentials [20,28]. While impressive progress has been made on the improvement of the catalytic activity and selectivity for the reduction of CO2 on alloy catalysts, the fundamental understanding of the correla- tion of the composition of bimetallic catalysts with the catalytic activity remains unclear. This discrepancy largely comes from the fact that many of the studied catalysts for CO2 reduction are nanostructured materials, which make it diﬃcult to distinguish the eﬀects of bimetallic composition and surface morphology (each of which can contribute to altered CO2 reduction performance). Furthermore, synergistic eﬀects of bimetallic systems have not been explained by existing models due to the complexity of the nanostructured surfaces and compositional var- iation. deposited by a magnetron sputtering co-deposition technique (sepa- rated targets) at an argon pressure of 0.3 Pa, as shown in Fig. S1. In this co-deposition process, the deposition rates of the two target materials (Au and Pt) were manipulated by adjusting the deposition power, re- spectively, for synthesizing the controllable compositions of binary metals. The composition of binary ﬁlms will be hereafter expressed by the atomic ratio of Au and Pt (Au100−nPtn, 0 ≤n ≤100). 1. Introduction the catalyst surface. The binding strength of these intermediates plays a key role in determining the selectivity to form speciﬁc reaction pro- ducts [14–17]. Recently, bimetallic electrocatalysts have attracted considerable attention as a promising approach to improve the catalytic activity and selectivity of CO2 reduction [18–24]. Alloying two metals can alter the electronic structure of a catalyst, which in turn alters the binding strength of intermediates due to both electronic and geometric eﬀects [19,25]. Furthermore, the electronic properties of alloys could be due to the combination of the individual contribution of each metal, or a rehybridization of the two metals’ orbitals. Density functional theory (DFT) has been utilized for calculating the binding energy of multiple intermediates on various alloys, giving a theoretical basis for developing suitable bimetallic alloy catalysts with high catalytic ac- tivity for a desired product [14–16,26]. Converting CO2 electrochemically into fuels and valuable chemicals has great potential for the utilization of captured CO2 [1–6]. For a sustainable production of fuels and value-added chemicals with zero carbon emission, renewable electricity sources can be used to power the electrocatalytic reduction of CO2 [1,7]. One of the main challenges for achieving this goal is to develop a cost-eﬀective electrocatalysts, which is capable of reducing CO2 eﬃciently with controllable selectivity and long-term stability. Primary investigations studied various polycrystal- line metallic materials for the electrochemical reduction of CO2 in aqueous electrolyte [2,3,8–11]. While several polycrystalline metal catalysts have been identiﬁed with the capability to electrocatalytically reduce CO2, the high overpotential required for driving selective CO2 reduction with suppressed H2 evolution and the fast catalytic deacti- vation in favor of H2O reduction signiﬁcantly limits the potential for large-scale applications [12,13]. Experimentally, it has been demonstrated that the interaction of the two diﬀerent metal atoms in a bimetallic alloy could signiﬁcantly in- ﬂuence the catalytic activity and selectivity in the electroreduction of CO2. Recently, Koper et al. [27] reported that bimetallic Pd-Pt alloy nanoparticles exhibit a very low onset potential for the reduction of CO2 The electrocatalytic reduction of CO2 is a multi-step reaction with many reaction intermediates (such as COOH and CO) that are bound to Nano Energy 42 (2017) 51–57 M. Ma et al. Fig. 1. Characterization of Au-Pt bimetallic ﬁlms. (a) A comparison of surface composition with bulk composition of Au-Pt ﬁlm measured by XPS and EDS, respectively. 1. Introduction A systematic experimental and theoretical investigation elaborates the mechanism of the eﬀect of binary catalyst composition on the catalytic activity and selectivity of CO2 reduction, revealing that major products formation (H2 and CO) closely follows the surface compositional change, while the formation of HCOOH on Au-Pt ﬁlms was found to occur at lower overpotentials and with signiﬁcantly improved amounts. Thus, the surface composition and bimetallic synergy of two metals both con- tribute to the overall CO2 reduction performance of Au-Pt electro- catalysts. X-ray diﬀraction (XRD) measurements were conducted for verifying the formation of the Au-Pt alloys. As noted in the XRD patterns (Fig. 1b), all compositions of the samples exhibited only one dominant diﬀraction peak which can be assigned to the diﬀraction of the (111) plane from the fcc crystal structure and the (111) peak position gra- dually shifted to larger 2θ with an increase of Pt content, indicating the formation of the Au-Pt bimetallic alloys [29]. The shift of the (111) peak towards larger 2θ with increasing Pt content is attributed to the reduced lattice parameter of Au-Pt alloy caused by the incorporation of Pt (the lattice parameter of the alloy is between the pure Au and Pure Pt). Here, the lattice parameter of Au-Pt alloys (Table S4) was extracted according to the XRD patterns and Bragg's law (Eqs. S1). A plot of the lattice parameter as a function of Pt content in the Au-Pt bimetallic ﬁlms is displayed in Fig. 1c, which includes the lattice parameter of pure Au (0.40736 nm) and pure Pt (0.39290 nm) for comparison. A linear relationship between lattice parameter and the Pt content of Au- Pt ﬁlms was observed (Fig. 1c), which is the typical characteristic of 2. Results and discussion 2.1. Fabrication and characterization of bimetallic Au-Pt ﬁlms 2.2. Electrocatalytic CO2 reduction activity CO and H2 were detected as the major products of CO2 reduction on these Au-Pt bimetallic alloys (Fig. 2). At a ﬁxed potential of −0.65 V vs. RHE, CO formation was not detected on pure Pt, and the highest CO faradaic eﬃciency of ~ 77% was observed on pure Au. Interestingly, the Au-Pt bimetallic alloys exhibited a gradually enhanced FE for CO formation as the Au content increased, simultaneously accompanying with decreased FE for H2 evolution, which reveals that the syngas ratio (CO:H2) could be tailored in the electroreduction of CO2 at a ﬁxed potential by sys- tematically tuning the composition of the Au-Pt bimetallic ﬁlms. To gain further insights into the inﬂuence of the binary composition on the catalytic activity of CO2 reduction, it is critical to distinguish the electronic eﬀect and the geometric eﬀect [19,25]. The electronic eﬀect is linked to the change of electronic structure that is tuned with surface composition of bimetallic catalysts, which results in the variation of the binding strength of intermediates. In addition, the geometric eﬀect that is correlated with the atomic arrangement at the active site which has a signiﬁcant inﬂuence on the interaction between adsorbed species and the surface atoms [19]. In our study, the surface roughness was ana- lyzed by atomic force microscopy (Fig. S5, all ﬁlms on polished Si substrates had a roughness of 0.3–0.5 nm), showing magnetron sput- tering is able to deposit uniform ﬂat surface layer for all diﬀerent compositional alloys, which implies that the variation of the catalytic activity with changing the bimetallic composition should be mainly related to the surface electronic eﬀect. To gain a deeper understanding of the trend of the electrocatalytic activity of CO2 reduction on Au-Pt alloys with diﬀerent compositions, the FE for the major products was plotted at various potentials (Fig. 3). With increasing overpotentials, the Au-Pt bimetallic catalysts with various compositions all experienced a gradually increased FE for the reduction of CO2 to CO, while the peak FE for CO on pure Au was achieved at −0.6 V vs. RHE and then a gradual decrease in the FE for CO was detected with further increasing overpotentials. In addition, the opposite tendency in FE for CO and H2 was also observed on the Au-Pt bimetallic ﬁlms with diﬀerent compositions at various potentials. As displayed in Fig. 2.2. Electrocatalytic CO2 reduction activity The electrochemical reduction of CO2 on the Au-Pt bimetallic ﬁlms with diﬀerent compositions were performed in a CO2-saturated 0.1 M KHCO3 (99.95%) electrolyte (pH = 6.83) at room temperature and atmospheric pressure. The CO2 electroreduction was conducted in an electrochemical cell, which is separated into working and counter electrode compartments by a Naﬁon-115 proton exchange membrane for preventing the oxidation of the CO2 reduction products. The cathodic compartment was continuously purged with CO2 at a constant ﬂow rate, venting directly into the gas-sampling loop of a gas chro- matograph (GC) for periodically quantifying the gas-phase products. To analyze liquid products produced in the CO2 reduction, the CO2 re- duction electrolyte was collected after completion of the electrolysis experiments and then detected by 1H nuclear magnetic resonance (NMR) [32]. In addition to the observed major products, formate was detected as a liquid product. As presented in Fig. 4, the formation of HCOOH (FE of 1.4%) started to be detected on pure Au at −0.6 V vs. RHE, and at a less negative potential of −0.55 V vs. RHE, a FE of 2% for HCOOH was observed on Au78Pt22 (Fig. 4). Notably, for driving the electrocatalytic reduction of CO2 to HCOOH, a potential of −0.5 vs. RHE was required on the Au57Pt43, which is a positive shift of 100 mV and 50 mV in comparison with pure Au and Au78Pt22, respectively. These ﬁndings imply that the Pt content (< 50 at%) in Au-Pt bimetallic catalysts may facilitate the electrocatalytic reduction of CO2 to HCOOH formation at a reduced overpotential. With further increasing the Pt composition, the potential required for driving the reduction of CO2 to HCOOH forma- tion shifted back to a more negative potential (−0.6 V and −0.65 V vs. RHE were required for Au32Pt68 and Pt, respectively). This observation may be due to the synergy of having Au and Pt atoms next to each other, which provide binding sites for reaction intermediates which favor the formation of HCOOH. All the above results indicate that the catalytic activity and selectivity for major products formation in CO2 reduction is strongly linked to the surface composition of the Au-Pt bimetallic alloys, while the synergy of the two metals allows the in- creased production of minor products at lower overpotentials. The comparison of the electrocatalytic activity of the Au-Pt bime- tallic alloys with diﬀerent compositions is presented in Fig. 2. 2.2. Electrocatalytic CO2 reduction activity 3a and b, the increase in the FE for CO formation followed the correspondingly reduced FE for H2 evolution with in- creasing Au incorporation at various potentials. In this study, during the various potential tests for CO2 reduction, CO formation on Pt was only detected at −0.7 V vs. RHE with a very low FE of 1%, and the CO formation on Au32Pt68, Au57Pt43 and pure Au was able to be detected at −0.4 V, −0.35 V and −0.3 V vs. RHE, respectively, which implies the decreased overpotential for the reduction of CO2 to CO with increasing For the eﬀect of the electronic properties on the catalytic activity and selectivity, d-states (that are close to the Fermi level) of transition metals play an important role in the binding strength of adsorbed species on the surface [25,31,33]. The higher energy of d-states leads to the stronger interaction with adsorbed intermediates [33]. Thus, al- loying two metals may alter the average energy of the d-electrons of the catalyst surface, which is correlated with the variation of the binding strength of intermediates, leading to the change of catalytic activity and selectivity. Of particular note, it has been reported that value of d-band center can be used to represent the average energy of the d-electrons [33]. Fig. 2. Faradaic eﬃciency for CO on Au-Pt bimetallic alloys with diﬀerent compositions at −0.65 V versus RHE in CO2-saturated 0.1 M KHCO3 electrolyte (pH = 6.8). In order to reveal the variation of the average energy of the d- electrons with the bimetallic composition, high-resolution surface va- lence band XPS measurements were performed on Au-Pt bimetallic ﬁlms. The surface valence band XPS spectra as a function of composi- tion of the Au-Pt bimetallic ﬁlms is presented in Fig. 5a (the observed surface valence bands could be from the combination of Au and Pt, or a rehybridization of the two metals’ orbitals). The position of the d-band center of Au-Pt bimetallic ﬁlms was determined according to Equation S (2), and was labeled with colored vertical lines in Fig. 5a, which in- dicates an obvious change of the d-band center with diﬀerent atomic ratios of Au/Pt. The energy of the d-band center as a function of Pt content shown in Fig. 5b implies that pure Pt has the closest d-band Fig. 2. 2.1. Fabrication and characterization of bimetallic Au-Pt ﬁlms For obtaining high purity Au-Pt bimetallic ﬁlms, Au-Pt ﬁlms were 52 M. Ma et al. Nano Energy 42 (2017) 51–57 alloying based on Vegard's law [30,31]. These results indicate that Au- Pt alloys were formed over the whole composition range without Au segregation at the surface. Au content of Au-Pt bimetallic catalysts (Fig. 3a). Furthermore, a plot of the partial current density for CO formation as a function of potential in Fig. 3c also reveals a positive shift for the onset potential in the re- duction of CO2 to CO with incorporating more Au into binary ﬁlms. These results indicate that the catalytic activity for the reduction of CO2 to CO and the competing H2 evolution can be tuned on the Au-Pt bi- metallic ﬁlms by systematically controlling the atomic ratio of Au/Pt. 2.2. Electrocatalytic CO2 reduction activity The color line in the center represents the position of d-band center of the corresponding Au-Pt ﬁlm. (b) Energy of d-band center as a function of Pt content. The inset shows the d-band center (Ed) position of Au and Au-Pt alloys with respect to the Fermi level. Fig. 4. Faradaic eﬃciency for HCOOH formation at various potentials in CO2-saturated 0.1 M KHCO3 electrolyte. center (Ed = 3.67 eV) relative to the Fermi level (EF). Notably, the d- band center of Au-Pt bimetallic ﬁlms was shifted away from the Fermi level with gradually increasing Au content, correspondingly weakening the binding strength of intermediates on the surface, which is consistent with the reported bimetallic Au-Pt alloys [31]. Fig. 5. Electronic properties of Au-Pt bimetallic ﬁlms. (a) Comparison of surface valence band XPS spectra of Au-Pt bimetallic ﬁlms with diﬀerent composition (the binding energy is the value of \|E-EF\|). The color line in the center represents the position of d-band center of the corresponding Au-Pt ﬁlm. (b) Energy of d-band center as a function of Pt content. The inset shows the d-band center (Ed) position of Au and Au-Pt alloys with respect to the Fermi level. Based on the catalytic CO2 reduction performance of Au-Pt bime- tallic ﬁlms, we propose a mechanism to correlate the binding strength of COOH, CO and H intermediates on Au-Pt bimetallic ﬁlms with the d- band center position relative to the Fermi level, as displayed in Fig. 6. For Pt, the Ed is very close to EF (3.67 eV), which corresponds to the strongest binding strength of COOH and CO, indicating that the CO2 activation for stabilizing COOH intermediate and the formation of ad- sorbed CO are facile. However, the desorption of CO from the surface is limited due to the strongest binding strength of CO on Pt (Fig. 6), which corresponds to the very low catalytic activity for the reduction of CO2 to CO (Fig. 3a). This result is consistent with DFT simulation work for CO2 reduction on Pt [17]. With increasing Au composition of Au-Pt bime- tallic ﬁlms, the Ed moves away from EF by getting d-electrons from Au [31], which reduces the binding strength of COOH and CO with gra- dually increasing Au content, resulting in the gradually increased cat- alytic activity for the conversion of CO2 into CO. 2.2. Electrocatalytic CO2 reduction activity Faradaic eﬃciency for CO on Au-Pt bimetallic alloys with diﬀerent compositions at −0.65 V versus RHE in CO2-saturated 0.1 M KHCO3 electrolyte (pH = 6.8). 53 Nano Energy 42 (2017) 51–57 M. Ma et al. Fig. 3. Comparison of the electrocatalytic performance of Au-Pt bimetallic alloys with diﬀerent compositions. Faradaic eﬃciency for CO (a) and H2 (b) on Au-Pt bimetallic alloys with diﬀerent compositions at various potentials in CO2-saturated 0.1 M KHCO3 electrolyte (pH = 6.8). (c) Partial current density for CO at various potentials. Fig. 3. Comparison of the electrocatalytic performance of Au-Pt bimetallic alloys with diﬀerent compositions. Faradaic eﬃciency for CO (a) and H2 (b) on Au-Pt bimetallic alloys with diﬀerent compositions at various potentials in CO2-saturated 0.1 M KHCO3 electrolyte (pH = 6.8). (c) Partial current density for CO at various potentials. center (Ed = 3.67 eV) relative to the Fermi level (EF). Notably, the d- band center of Au-Pt bimetallic ﬁlms was shifted away from the Fermi level with gradually increasing Au content, correspondingly weakening the binding strength of intermediates on the surface, which is consistent with the reported bimetallic Au-Pt alloys [31]. Based on the catalytic CO2 reduction performance of Au-Pt bime- tallic ﬁlms, we propose a mechanism to correlate the binding strength of COOH, CO and H intermediates on Au-Pt bimetallic ﬁlms with the d- Fig. 4. Faradaic eﬃciency for HCOOH formation at various potentials in CO2-saturated 0.1 M KHCO3 electrolyte. Fig. 5. Electronic properties of Au-Pt bimetallic ﬁlms. (a) Comparison of surface valence band XPS spectra of Au-Pt bimetallic ﬁlms with diﬀerent composition (the binding energy is the value of \|E-EF\|). The color line in the center represents the position of d-band center of the corresponding Au-Pt ﬁlm. (b) Energy of d-band center as a function of Pt content. The inset shows the d-band center (Ed) position of Au and Au-Pt alloys with respect to the Fermi level. Fig. 4. Faradaic eﬃciency for HCOOH formation at various potentials in CO2-saturated 0.1 M KHCO3 electrolyte. Fig. 4. Faradaic eﬃciency for HCOOH formation at various potentials in CO2-saturated 0.1 M KHCO3 electrolyte. Fig. 4. Faradaic eﬃciency for HCOOH formation at various potentials in CO2-saturated 0.1 M KHCO3 electrolyte. Fig. 5. Electronic properties of Au-Pt bimetallic ﬁlms. (a) Comparison of surface valence band XPS spectra of Au-Pt bimetallic ﬁlms with diﬀerent composition (the binding energy is the value of \|E-EF\|). 2.2. Electrocatalytic CO2 reduction activity The highest value of Ed (5.03 eV) was detected on pure Au, which oﬀers weaker binding strength for COOH and CO, which is the optimum binding strength among transition metals for the reduction of CO2 to CO [17], corresponding to the highest catalytic activity for CO2 reduction to CO on Au among the catalysts studied here. The above discussion indicates that the binding strength at a mixed site is the average of the properties of the constituents, which likely implies that the binding strength for intermediates on the Au-Pt bi- metallic ﬁlms are correlated linearly with each other with tuning the binary composition, corresponding to the gradually enhanced catalytic activity for the reduction of CO2 to CO with increasing Au content. The binding energies of the reaction intermediates on Au-Pt bime- tallic surface were further investigated by DFT simulations. The binding energies of COOH and CO intermediates on the Au-Pt alloys are shown in Fig. 7a together with the rate of CO2 reduction to CO based on a previously published micro-kinetic model [17]. According to this model, optimum activity for CO production is obtained for appropriate binding energies of COOH and CO, which simultaneously allow facile 54 Nano Energy 42 (2017) 51–57 M. Ma et al. Fig. 6. Schematic illustration of binding strength of COOH, CO and H intermediates on Au-Pt bimetallic catalysts as a function of the d-band center position. et al. Nano Energy 42 (201 Fig. 6. Schematic illustration of binding strength of COOH, CO and H intermediates on Au-Pt bimetallic catalysts as a function of the d-band center position. ustration of binding strength of COOH, CO and H intermediates on Au-Pt bimetallic catalysts as a function of the d-band center position. Fig. 6. Schematic illustration of binding strength of COOH, CO and H intermediates on Au-Pt bimetallic catalysts as a function of the d-b alloy ﬁlms lie in between pure Pt and pure Au. This result is consistent with our experimental work. In addition, the shift of the d-band center away from the Fermi level with increasing Au composition is also found from DFT calculation (Fig. S8). CO2 activation and CO release. The catalytic activity on late transition metal and coinage metal surfaces is limited by a correlation between COOH and CO binding energies. The Au-Pt alloys studied here follow the same correlation between COOH and CO binding energies as dis- covered previously [17]. 3. Conclusion 9] D. Kim, J. Resasco, Y. Yu, A.M. Asiri, P. Yang, Nat. Commun. 5 (2 f d d ll k b [19] D. Kim, J. Resasco, Y. Yu, A.M. Asiri, P. Yang, Nat. Commun. 5 (2014) 4948. [20] S. Sarfraz, A.T. Garcia-Esparza, A. Jedidi, L. Cavallo, K. Takanabe, ACS Catal. 6 (2016) 2842–2851. In summary, Au-Pt bimetallic thin ﬁlms with tunable compositions were prepared with a uniform morphology, oﬀering a platform for understanding the electronic eﬀect on the catalytic activity for CO2 reduction in a bimetallic system. The Au-Pt binary ﬁlms exhibited a gradually improved catalytic activity for the reduction of CO2 to CO with increasing the Au content, which is attributed to the variation of electronic properties caused by changing binary composition. We show that the d-band center was gradually shifted away from the Fermi level with increasing Au content, which weakens the binding strength of COOH and CO, resulting in the corresponding variation in catalytic activity for CO2 reduction. In addition, with increasing Au composition, the binding strength of intermediates on the Au-Pt bimetallic ﬁlms may still follow the scaling relation, which reveals that electronic eﬀect alone in bimetallic catalysts is likely unable to break the scaling relation to freely tune the binding strength of a certain intermediate without aﬀecting others for achieving a high CO2 reduction performance. This study shows that a single electronic eﬀect in bimetallic system could not reduce the required overpotential for the dramatically improved cata- lytic activity for CO2 reduction to CO, and the atomic arrangement also needs to be taken into account to design a bimetallic catalyst for driving highly selective and eﬃcient CO2 reduction to CO at the reduced overpotential. In addition, the formation of formic acid in the bimetallic systems at reduced overpotentials indicates that synergistic eﬀects can facilitate reaction pathways for products that are not accessible with the individual components. ( ) [21] Y. Zhao, C. Wang, G.G. Wallace, J. Mater. Chem. A 4 (2016) 10710–10718. [21] Y. Zhao, C. Wang, G.G. Wallace, J. Mater. Chem. A 4 (2016) 10 [ ] i i h i i h [22] M. Li, J. Wang, P. Li, K. Chang, C. Li, T. Wang, B. Jiang, H. Zhang, H. Liu, Y. Yamauchi, N. Umezawa, J. Ye, J. Mater. Chem. A 4 (2016) 4776–4782. [23] Z. Yin, D. Gao, S. Yao, B. Zhao, F. Cai, L. Lin, P. Tang, P. Zhai, G. (2017) 505–509. signiﬁcantly improved catalytic activity for CO2 reduction only ob- served on the nanostructured Cu-In and Cu-Sn bimetallic surfaces is mainly ascribed to the synergistic geometric and electronic eﬀects. It has been demonstrated that the geometric eﬀect which could lead catalysts to deviate from the scaling relation has a signiﬁcant eﬀect on the adsorption of intermediates [19,25]. Thereby, it seems that a solely electronic eﬀect of bimetallic alloys may not be enough to signiﬁcantly break the scaling relation for dramatically improving the electro- catalytic reduction of CO2. The atomic arrangement (nanostructure) should be also considered to design a bimetallic catalyst for the elec- troreduction of CO2, which may be attributed to the synergistic geo- metric and electronic eﬀects that contributes to the high-performance of bimetallic catalysts. [7] M. Mikkelsen, M. Jørgensen, F.C. Krebs, Energy Environ. Sci. 3 (2010) 43–81. [8] M. Gattrell, N. Gupta, A. Co, J. Electroanal. Chem. 594 (2006) 1–19. [7] M. Mikkelsen, M. Jørgensen, F.C. Krebs, Energy Environ. Sci. 3 (2010) 43–81. [8] M. Gattrell, N. Gupta, A. Co, J. Electroanal. Chem. 594 (2006 [9] K.P. Kuhl, E.R. Cave, D.N. Abram, T.F. Jaramillo, Energy Environ. Sci. 5 (2012) 7050–7059. [10] Y. Hori, R. Takahashi, Y. Yoshinami, A. Murata, J. Phys. Chem. B 101 (1997) 7075–7081. [11] Y. Hori, Modern Aspects of Electrochemistry, Vol. 42 Springer, New York, 2008, p. 89. . Chen, C.W. Li, M.W. Kanan, J. Am. Chem. Soc. 134 (2012) 19969–1 [13] C.W. Li, M.W. Kanan, J. Am. Chem. Soc. 134 (2012) 7231–7234. [14] A.A. Peterson, J.K. Nørskov, J. Phys. Chem. Lett. 3 (2012) 251–258. 5] P. Hirunsit, W. Soodsawang, J. Limtrakul, J. Phys. Chem. C 119 (20 [15] P. Hirunsit, W. Soodsawang, J. Limtrakul, J. Phys. Chem. C 119 (2015) 8238–8249. [16] H.A. Hansen, C. Shi, A.C. Lausche, A.A. Peterson, J.K. Nørskov, Phys. Chem. Chem. Phys. 18 (2016) 9194–9201. [16] H.A. Hansen, C. Shi, A.C. Lausche, A.A. Peterson, J.K. Nørskov, Phys. Chem. Chem. Phys. 18 (2016) 9194–9201. y [17] H.A. Hansen, J.B. Varley, A.A. Peterson, J.K. Nørskov, J. Phys. Chem. Lett. 4 (2013) 388–392. [18] S. Rasul, D.H. Anjum, A. Jedidi, Y. Minenkov, L. Cavallo, K. Takanabe, Angew. Chem. Int. Ed. 54 (2015) 2146–2150. 3. Conclusion His research interest mainly focuses on het- erogeneous nanocatalysts for the electrochemical reduction of CO2, including the fabrication, characterization, catalytic performance and reaction mechanism investigation of na- nostructured metal catalysts. Acknowledgements This work is supported by an NWO VIDI grant awarded to Wilson A. Smith. Funding from the Lundbeck foundation (R141-2013-A13204) is also gratefully acknowledged. The Niﬂheim supercomputer at DTU was used for the computations performed in this work. The authors would like to thank Prof. Dr. Bernard Dam, Dr. Recep Kas and Divya Bohra for helpful discussions. We also would like to thank Dr. Kristina Djanashvili for assistance in the 400 MHz NMR experiments. Heine Anton Hansen obtained his Ph.D. at the Department of Physics at the Technical University of Denmark (DTU) in 2009, and he was a postdoc at Northwestern University and Stanford University before becoming a researcher at the Department of Energy Conversion and Storage at DTU in 2013. His research is focused on the modeling of electro- chemical interfaces and reactions of small molecules based on density functional theory calculations and micro-kinetic modeling. He is particular interested in the electrochemical reduction of CO2 and CO to fuels, the oxygen reduction reaction, and water electrolysis. [1] D.T. Whipple, P.J. A. Kenis, J. Phys. Chem. Lett. 1 (2010) 3451–3458. [2] K.P. Kuhl, T. Hatsukade, E.R. Cave, D.N. Abram, J. Kibsgaard, T.F. Jaramillo, J. Am. Chem. Soc. 136 (2014) 14107–14113. [3] J. Qiao, Y. Liu, F. Hong, J. Zhang, Chem. Soc. Rev. 43 (2014) 631–675. [4] N. Kornienko, Y. Zhao, C.S. Kley, C. Zhu, D. Kim, S. Lin, C.J. Chang, O.M. Yaghi, P. Yang, J. Am. Chem. Soc. 137 (2015) 14129–14135. [5] M. Ma, K. Djanashvili, W.A. Smith, Angew. Chem. Int. Ed. 55 (2016) 6680–6684. [6] F. Li, L. Chen, G.P. Knowles, D.R. MacFarlane, J. Zhang, Angew. Chem. Int. Ed. 56 Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.nanoen.2017.09.043. , j , , g [6] F. Li, L. Chen, G.P. Knowles, D.R. MacFarlane, J. Zhang, Angew. Chem. Int. Ed. 56 3. Conclusion Wang, D. Ma, X. Bao, Nano Energy 27 (2016) 35–43. [24] D.A. Torelli, S.A. Francis, J.C. Crompton, A. Javier, J.R. Thompson, B.S. Brunschwig, M.P. Soriaga, N.S. Lewis, ACS Catal. 6 (2016) 2100–2104. 5] P. Liu, J.K. Nørskov, Phys. Chem. Chem. Phys. 3 (2001) 3814–381 [26] J. Ko, B.-K. Kim, J.W. Han, J. Phys. Chem. C 120 (2016) 3438–3447. [27] R. Kortlever, I. Peters, S. Koper, M.T.M. Koper, ACS Catal. 5 (2015) 3916–3923. [28] M. Li, J. Wang, P. Li, K. Chang, C. Li, T. Wang, B. Jiang, H. Zhang, H. Liu, Y. Yamauchi, N. Umezawa, J. Ye, J. Mater. Chem. A 4 (2016) 4776–4782. [29] C. Hahn, D.N. Abram, H.A. Hansen, T. Hatsukade, A. Jackson, N.C. Johnson, T.R. Hellstern, K.P. Kuhl, E.R. Cave, J.T. Feaster, T.F. Jaramillo, J. Mater. Chem. A 3 (2015) 20185–20194. [30] E. Irissou, F. Laplante, S. Garbarino, M. Chaker, D. Guay, J. Phys. Chem. C 114 (2010) 2192–2199. [31] F. Chang, S. Shan, V. Petkov, Z. Skeete, A. Lu, J. Ravid, J. Wu, J. Luo, G. Yu, Y. Ren, C.-J. Zhong, J. Am. Chem. Soc. 138 (2016) 12166–12175. [32] M. Ma, K. Djanashvili, W.A. Smith, Phys. Chem. Chem. Phys. 17 (2015) 20861–20867. [33] J.K. Nørskov, T. Bligaard, J. Rossmeisl, C.H. Christensen, Nat. Chem. 1 (2009) 37–46. [34] J.K. Nørskov, T. Bligaard, A. Logadottir, J.R. Kitchin, J.G. Chen, S. Pandelov, U. Stimming, J. Electrochem. Soc. 152 (2005) 23–26. [35] J.R. Kitchin, J.K. Nørskov, M.A. Barteau, J.G. Chen, Phys. Rev. Lett. 93 (2004) 156801. Dr. Ming Ma ﬁnished his Ph.D. research on the topic of selective electrocatalytic CO2 conversion over metal sur- faces, supervised by Dr. Wilson A. Smith at Department of Chemical Engineering, Delft University of Technology. Currently, he is a full-time researcher temporarily em- ployed at Faculty of Applied Sciences, Delft University of Technology. His research interest mainly focuses on het- erogeneous nanocatalysts for the electrochemical reduction of CO2, including the fabrication, characterization, catalytic performance and reaction mechanism investigation of na- nostructured metal catalysts. Dr. Ming Ma ﬁnished his Ph.D. research on the topic of selective electrocatalytic CO2 conversion over metal sur- faces, supervised by Dr. Wilson A. Smith at Department of Chemical Engineering, Delft University of Technology. Currently, he is a full-time researcher temporarily em- ployed at Faculty of Applied Sciences, Delft University of Technology. [4] N. Kornienko, Y. Zhao, C.S. Kley, C. Zhu, D. Kim, S. Lin, C.J. Chang, O.M. Yaghi, P. Yang, J. Am. Chem. Soc. 137 (2015) 14129–14135. Heine Anton Hansen obtained his Ph.D. at the Department of Physics at the Technical University of Denmark (DTU) in 2009, and he was a postdoc at Northwestern University and Stanford University before becoming a researcher at the Department of Energy Conversion and Storage at DTU in 2013. His research is focused on the modeling of electro- chemical interfaces and reactions of small molecules based on density functional theory calculations and micro-kinetic modeling. He is particular interested in the electrochemical reduction of CO2 and CO to fuels, the oxygen reduction reaction, and water electrolysis. [3] J. Qiao, Y. Liu, F. Hong, J. Zhang, Chem. Soc. Rev. 43 (2014) 631–675. [2] K.P. Kuhl, T. Hatsukade, E.R. Cave, D.N. Abram, J. Kibsgaard, T.F. J Chem. Soc. 136 (2014) 14107–14113. [1] D.T. Whipple, P.J. A. Kenis, J. Phys. Chem. Lett. 1 (2010) 3451–3458. [ ] hl k d b ib d ill [5] M. Ma, K. Djanashvili, W.A. Smith, Angew. Chem. Int. Ed. 55 (2016) 6680–6684. [6] F. Li, L. Chen, G.P. Knowles, D.R. MacFarlane, J. Zhang, Angew. Chem. Int. Ed. 56 2.2. Electrocatalytic CO2 reduction activity In addition, the variation in catalyst activity for CO production is linked to the change of hydrogen binding energy that is responsible for H2 production [34], due to a correlation between CO and H binding energies as presented in Fig. 7b. Furthermore, the variation of electronic properties on bimetallic surfaces are induced by strain and ligand eﬀects [35]. We ﬁnd no correlation between alloy lattice constants and binding energies of COOH, CO, H on Au-Pt alloy (Fig. S7). However, the electronic ligand eﬀect plays a signiﬁcant role, which may lead to Au atoms in Au-Pt alloys binding intermediates stronger than pure Au and Pt binding intermediates weaker than pure Pt, implying that the binding energies of intermediates on the Au-Pt This exploration of the electronic eﬀect on the catalytic activity for CO2 reduction in the bimetallic system suggests that the binding strength for intermediates on Au-Pt bimetallic ﬁlms likely follows the scaling relation, which is in agreement with the previous DFT simula- tion work for Au-Pd alloy surfaces (a linear relationship is obtained between the adsorption strength of intermediates and the composition of alloy) [25]. In addition, it was reported that although the dramati- cally improved FE for the reduction of CO2 to CO could be achieved on nanostructured Cu-In and Cu-Sn bimetallic catalysts at the reduced overpotentials, In and Sn deposited on ﬂat Cu catalysts experienced a predominant H2 evolution under similar conditions [18,20]. The Fig. 7. (a) COOH and CO binding energies of Au-Pt (211) steps calculated from DFT shown over an activity map for CO production. (b) Correlation of CO and H binding energies on Au-Pt (211) steps. Fig. 7. (a) COOH and CO binding energies of Au-Pt (211) steps calculated from DFT shown over an activity map for CO production. (b) Correlation of CO and H binding energies on Au-Pt (211) steps. of Au-Pt (211) steps calculated from DFT shown over an activity map for CO production. (b) Correlation of CO and H binding energies on Au-Pt 55 M. Ma et al. Nano Energy 42 (2017) 51–57 References properties of water splitting semiconductor photoelectrodes by plasmonic nanoparticle functionalization, and (ii) studying the activity/selectivity in the electrochemical CO2 reduction over noble metal alloy nanoparticles. Zegao Wang obtained his Ph.D. in Microelectronics and Solid-State Electronics from University of Electronic Science and Technology of China in 2014. Since 2014, he is a postdoctoral fellow in the Bio-SPM group, Aarhus University. His current research interest focuses on two- dimensional nanomaterials and their applications in elec- tronic devices, biosensors and energy devices. Anping Cao conducted her Ph.D. research on the topic of surface modiﬁed SiNWs for chemical sensing at the Department of Chemical Engineering, Delft University of Technology. Currently she is working as a post-doc in Eindhoven University of Technology, her research is focus on the liquid crystal based dynamic membrane for gas se- paration. Dr. Wilson A. Smith is an Associate P Department of Chemical Engineering at De Technology. He earned his Ph.D. in Ph University of Georgia in 2010. After his search associate at the Universite Pierre Sorbonne, he began his current position i group focuses on fundamental processes re electrochemical water splitting, electrocata dation, and CO2 reduction catalysis usin nostructured materials. Zegao Wang obtained his Ph.D. in Microelectronics and Solid-State Electronics from University of Electronic Science and Technology of China in 2014. Since 2014, he is a postdoctoral fellow in the Bio-SPM group, Aarhus University. His current research interest focuses on two- dimensional nanomaterials and their applications in elec- tronic devices, biosensors and energy devices. Dr. Wilson A. Smith is an Associate Professor in the Department of Chemical Engineering at Delft University of Technology. He earned his Ph.D. in Physics from the University of Georgia in 2010. After his postdoctoral re- search associate at the Universite Pierre et Marie Curie/ Sorbonne, he began his current position in 2012 and his group focuses on fundamental processes related to photo- electrochemical water splitting, electrocatalytic water oxi- dation, and CO2 reduction catalysis using inorganic na- nostructured materials. Dr. Wilson A. Smith is an Associate Professor in the Department of Chemical Engineering at Delft University of Technology. He earned his Ph.D. in Physics from the University of Georgia in 2010. Dr. Wilson A. Smith is an Associate Professor in the Department of Chemical Engineering at Delft University of Technology. He earned his Ph.D. in Physics from the University of Georgia in 2010. After his postdoctoral re- search associate at the Universite Pierre et Marie Curie/ Sorbonne, he began his current position in 2012 and his group focuses on fundamental processes related to photo- electrochemical water splitting, electrocatalytic water oxi- dation, and CO2 reduction catalysis using inorganic na- nostructured materials. References 56 Nano Energy 42 (2017) 51–57 M. Ma et al. Ph.D. student supervised by Prof. Dr. at Delft University of Technology, The earch is focused on developing eﬃcient o-fuel energy conversion. His main in- : (i) improving the opto-electronic plitting semiconductor photoelectrodes oparticle functionalization, and (ii) /selectivity in the electrochemical CO2 e metal alloy nanoparticles. ned his Ph.D. in Microelectronics and nics from University of Electronic ogy of China in 2014. Since 2014, he is ow in the Bio-SPM group, Aarhus ent research interest focuses on two- aterials and their applications in elec- nsors and energy devices. Mingdong Dong obtained his Ph.D. in Applied Physics from Aarhus University, Denmark in 2006. After post- doctoral research in Harvard University, he started his in- dependent academic career as Assistant Professor and Associate Professor in Interdisciplinary Nanoscience Center (iNANO), Aarhus University. Currently, he is the group leader of Bio-SPM group. His research focuses on both the implementation and further development of a novel scan- ning probe microscope technique for biomedical applica- tions and new functional materials. Dr. Wilson A. Smith is an Associate Professor in the Department of Chemical Engineering at Delft University of Technology. He earned his Ph.D. in Physics from the University of Georgia in 2010. After his postdoctoral re- search associate at the Universite Pierre et Marie Curie/ Sorbonne, he began his current position in 2012 and his group focuses on fundamental processes related to photo- electrochemical water splitting, electrocatalytic water oxi- dation, and CO2 reduction catalysis using inorganic na- nostructured materials. Marco Valenti is a Ph.D. student supervised by Prof. Dr. Andreas Schmidt-Ott at Delft University of Technology, The Netherlands. His research is focused on developing eﬃcient materials for solar-to-fuel energy conversion. His main in- terests are twofold: (i) improving the opto-electronic properties of water splitting semiconductor photoelectrodes by plasmonic nanoparticle functionalization, and (ii) studying the activity/selectivity in the electrochemical CO2 reduction over noble metal alloy nanoparticles. Mingdong Dong obtained his Ph.D. in Applied Physics from Aarhus University, Denmark in 2006. After post- doctoral research in Harvard University, he started his in- dependent academic career as Assistant Professor and Associate Professor in Interdisciplinary Nanoscience Center (iNANO), Aarhus University. Currently, he is the group leader of Bio-SPM group. His research focuses on both the implementation and further development of a novel scan- ning probe microscope technique for biomedical applica- tions and new functional materials. References After his postdoctoral re- search associate at the Universite Pierre et Marie Curie/ Sorbonne, he began his current position in 2012 and his group focuses on fundamental processes related to photo- electrochemical water splitting, electrocatalytic water oxi- dation, and CO2 reduction catalysis using inorganic na- nostructured materials. Dr. Wilson A. Smith is an Associate Professor in the Department of Chemical Engineering at Delft University of Technology. He earned his Ph.D. in Physics from the University of Georgia in 2010. After his postdoctoral re- search associate at the Universite Pierre et Marie Curie/ Sorbonne, he began his current position in 2012 and his group focuses on fundamental processes related to photo- electrochemical water splitting, electrocatalytic water oxi- dation, and CO2 reduction catalysis using inorganic na- nostructured materials. Anping Cao conducted her Ph.D. research on the topic of surface modiﬁed SiNWs for chemical sensing at the Department of Chemical Engineering, Delft University of Technology. Currently she is working as a post-doc in Eindhoven University of Technology, her research is focus on the liquid crystal based dynamic membrane for gas se- paration. 57 57
https://openalex.org/W4389494016	https://www.bio-conferences.org/articles/bioconf/pdf/2023/24/bioconf_icome2023_12001.pdf	English	null	Designing a Green Chemistry Integrated Separation and Purification Textbook Using the Four Steps Teaching Material Development (4STMD) Method: Selecting and Structuring Steps	Bio web of conferences/BIO web of conferences	2,023	cc-by	3,866	Designing a Green Chemistry Integrated Separation and Purification Textbook Using the Four Steps Teaching Material Development (4STMD) Method: Selecting and Structuring Steps Fitriah Khoirunnisa1,2, Sjaeful Anwar1, Asep Kadarohman1, and Hendrawan Hendrawan1 1Universitas Pendidikan Indonesia 2Universitas Maritim Raja Ali Haji Abstract. This research aims to develop a university chemistry textbook in the Separation and Purification course by integrating the principles of Green Chemistry through the Four Steps Teaching Material Development (4STMD) method. Several separation and purification techniques are designed in this textbook, including sublimation, recrystallisation, chromatography, and distillation techniques, which are packaged in a presentation that focuses on the principles of Green Chemistry. The textbook development uses the 4STMD method which includes the steps of selecting, structuring, characterisation, and didactic reduction. The 4STMD is a teaching material development method characterised by a well-organized and straightforward sequence of steps. The research is limited to the selecting and structuring steps so that the research results are more focused and in-depth. The instrument used in this study was a review sheet of the suitability of concepts, indicators, and core competencies; as well as the suitability of concept maps, macro structures, and textbook systematics. The instrument used in this study was a review sheet of the suitability of concepts, indicators, and core competencies; as well as the suitability of concept maps, macro structures, and textbook systematics. This research was motivated by the limited number of college textbooks with a Green Chemistry perspective, especially for prospective chemistry teachers in coastal areas. Separation and purification are basic skills that must be mastered before synthesising and isolating a compound. However, the synthesis and isolation process usually involves the use of relatively hazardous solvents. Therefore, a separation and purification textbook that integrates Green Chemistry is needed so that students can master specification and concentration materials that are oriented towards environmental sustainability. BIO Web of Conferences 79, 12001 (2023) ICOME 2023 BIO Web of Conferences 79, 12001 (2023) ICOME 2023 https://doi.org/10.1051/bioconf/20237912001 1 Introduction Separation and purification are basic skills that must be mastered by chemistry or chemistry education students before synthesising and isolating a compound, either organic or inorganic. In universities, the separation and purification of compounds are included in advanced chemistry courses, one of which is the Separation Chemistry course. Along with the development of technology, the techniques employed for the separation and purification of organic molecules are subject to ongoing development and refinement over time [1-3]. The purification technique also follows the concept of sustainable development, one of which begins with the reduction of solvents [2-4]. The use of solvents plays an important role in the purification process, where it is known that solvents commonly used in organic chemistry are relatively hazardous solvents, hence the need to develop purification techniques that are sustainable, especially in the field of education [5]. Sustainable development in education, especially in the Separation Chemistry course can be obtained through experimental activities in the laboratory. Laboratory experiments have long had a special and central role in the science curriculum and science educators have stated that many advantages are gained by involving students in laboratory activities [6]. Chemistry education students are required to be able to master skills in the laboratory as a provision when teaching at school. Green Chemistry refers to the deliberate and systematic approach employed in the development of chemical goods and processes with the aim of minimising or completely eradicating the utilisation and production of compounds that pose a threat to human health and the environment [7]. The essential aspect of Green Chemistry is the design concept consisting of twelve Green Chemistry principles which are design criteria or guidelines that provide a framework for sustainable design [7-9]. Green Chemistry embodies two main components, which concern the efficient utilisation of raw materials and relate to the issue of chemical waste removal [10]. The integration of Green Chemistry into the undergraduate curriculum is a necessity to prepare students for a sustainable future [11]. Green Chemistry can be realised through teaching materials that are in accordance with curriculum criteria and can be a learning resource for students in the classroom [12]. Teaching materials encompass a wide range of materials that are structured and systematically in accordance with the demands of the curriculum and become learning resources for students, as well as materials or materials for teachers in carrying out teaching and learning activities [12]. Corresponding author: saefulanwar@upi.edu © 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/20237912001 BIO Web of Conferences 79, 12001 (2023) ICOME 2023 1 Introduction Teaching materials have an essential part in facilitating effective learning experiences. Teaching materials can be a guide for educators in carrying out the learning process. One of the teaching materials that are commonly used in learning is a textbook. The textbook can be interpreted as a composition of teaching materials that can make it easier for students and students to learn well. The textbook is one of the effective curricular material dissemination strategies [13]. Acquisition of high-quality teaching materials necessitates a systematic approach that takes into consideration the requirements of the relevant curriculum [14]. The lack of theory about development that specialises in teaching materials makes a difficult to develop science teaching materials [15], in this research, the teaching materials developed are in the form of textbooks on the topic of separation and purification. The Four Steps Teaching Material Development (4STMD) is a method for developing teaching materials that offer distinct advantages when compared to other methodologies or models used in product development. The 4-step method for text mining and document analysis (4STMD) comprises four distinct stages, which are selecting, structuring, characterization, and didactic reduction. This method offers several advantages. Firstly, it enables the selection of subject matter from diverse sources. Additionally, it facilitates the exploration of the values acquired by students throughout the study of the chosen subject matter. Furthermore, the processes outlined in the 2 BIO Web of Conferences 79, 12001 (2023) ICOME 2023 https://doi.org/10.1051/bioconf/20237912001 Four-Step Teaching Model for Developing Material (4STMD) have the capacity to enhance cognitive abilities, construct conceptual frameworks, and classify material concepts according to their level of complexity, hence facilitating students' comprehension of the supplied content [16]. The problems obtained by researchers when conducting observations in the field are that there are no teaching materials, both in the form of textbooks and practicum guidelines that support sustainability during the Separation Chemistry lecture process. Consequently, students have encountered challenges in applying the concepts and theories they have acquired in the classroom, resulting in suboptimal development of essential abilities and values that are expected to be cultivated among students. Hence, it is imperative to create educational resources, specifically textbooks, that can effectively address the requirements of a sustainable future. This can be achieved by developing textbooks that adopt the principles of Green Chemistry and focus on the subject of Separation and Purification. The incorporation of the 4STMD method in the textbook development process is recommended. 2 Research Methods This research uses a research and development design that focuses on the development of teaching materials processes and products. The objectives of this study were to develop a Green Chemistry integrated Separation and Purification textbook for the university level; identify the suitability to the demands of the curriculum, ensure the correctness of the concept, and develop the context of the developed material; and identify the suitability of the concept map, macro structure, and three levels of representation. 2.1 Data collection techniques The development of textbooks in this study used the Four Steps Teaching Material Development (4STMD) method [12] because it has several advantages over other teaching material development models or methods. The 4STMD method offers the development of teaching materials with detailed stages and steps accompanied by clear criteria according to the demands of the applicable curriculum [12, 14]. In addition, the stages and steps used are work that can be followed easily by teaching material developers by following the established criteria [12]. Textbook development using the Four Steps Teaching Material Development (4STMD) method consists of the steps of selecting, structuring, characterisation, and didactic reduction [12]. However, this research is restricted to the selecting and structuring steps, and the other two steps will be continued in the subsequent research. The steps of 4STMD are illustrated in Figure 1. 3 https://doi.org/10.1051/bioconf/20237912001 BIO Web of Conferences 79, 12001 (2023) BIO Web of Conferences 79, 12001 (2023) ICOME 2023 Fig.1. Four Steps Teaching Material Development (4STMD) The data collection techniques used in this research are literature studies of the syllabus Fig.1. Four Steps Teaching Material Development (4STMD) The data collection techniques used in this research are literature studies of the syllabus and curriculum applicable to the Separation Chemistry course; and expert judgement on the suitability of predetermined criteria. The research instrument for the selecting step consists of instruments: 1) suitability of indicators or sub-course learning outcome with course learning outcomes; 2) suitability of concept labels with indicators or sub-course learning outcome; 3) suitability of values with material concepts. In the structuring step, the draft of teaching materials that have been prepared need to be reviewed independently by the developer of teaching materials by developing, inserting, refining, and adjusting [12]. 3.1 Selecting Step The development of the Separation and Purification textbook has been conducted using the 4STMD method. This research is limited to the selecting and structuring steps to obtain in- depth results according to the criteria. There are three stages in the selecting step, namely 1) development of indicators and concept labels; 2) development of material from concept labels; and 3) development of context (substantial context and pedagogical context). ) p ( p g g ) The selecting step was carried out by analysing the syllabus and curriculum for the Separation and Purification course. After the syllabus and curriculum analysis process were carried out, four course learning outcomes were obtained that were suitable for the development of separation and purification textbooks. Furthermore, developing indicators or sub-course learning outcome derived from the Course Learning Outcome (CLO) of the course that is the target of development. The selection of CLO is based on its suitability for the textbook to be developed. The CLO that has been selected, is then developed into indicators or sub-course learning outcome. After that, based on the indicators, the concept labels were developed. There were 17 indicators or sub-course learning outcome and 11 concept labels that were considered in accordance with the learning outcomes that had been previously determined. The next step is to collect information related to the concept or material according to the concept label that has been agreed upon previously. Concept/material descriptions are sourced from textbooks that have been used by many scientists around the world to ensure the correctness of the concepts used [12] and can be added with other relevant secondary sources. Table 1 shows the relationship between indicators and concept labels that form the basis for textbook development in the next stage. Table 1. The relationship between indicators and concept labels. No. Indicators/Sub-Course Learning Outcomes Concept Labels 1 Analyse the principles of separation and purification of organic compounds. Principles of Separation and Purification of Organic Compounds 2 Identify the types of laboratory equipment related to the separation of organic compounds. Introduction to Equipment, Safety, and Security in the Organic Chemistry Laboratory 3 Describe the properties and characteristics of solvents in organic chemistry laboratories. 4 Identify the maintenance and handling of glassware in the organic chemistry laboratory. 5 Identify specialised handling of hazardous materials in organic laboratories 6 Analyse the basic techniques of separation and purification of organic compounds. 2.2 Data analysis techniques Analyse the data in the selecting step using Content Validity Index (CVI). In this step, three experts (raters) were involved. The categorisation of the results of the CVI calculation is adjusted to the number of validators used based on certain criteria [17]. The rating scale for content validation in this step uses a Guttman scale with two alternative answers "Yes" and "No" [18] to get a firm and clear answer to a questioned problem. With 3 experts, a category or statement will be declared valid or accepted if V = 1 [19]. V = Σs n (c −1) V = agreement index s = r - lo r = the score given by the validator/rater lo = lowest number of judgements n = number of raters c = the number of categories that the rater can choose 4 https://doi.org/10.1051/bioconf/20237912001 BIO Web of Conferences 79, 12001 (2023) ICOME 2023 3.1 Selecting Step Basic Techniques of Separation and Purification of Organic Compounds 7 Analyse various techniques for testing the purity of an organic compound. Purity Testing of Organic Compounds 8 Analyse the principles of Green Chemistry in the practicum of separation and purification of organic compounds. Principles of Green Chemistry and Microscale in Separation and Purification of Organic Compounds Practicum 9 Analyse the Microscale criteria in the practicum of separation and purification of organic compounds. 10 Design practical procedures for the separation and purification of compounds based on aspects of Green Chemistry-Microscale. Practicum Project Proposal 11 Implement the principles of purification and separation through practicum by sublimation techniques. Principles of Separation and Purification by Sublimation Technique 12 Implement the principles of purification and separation through practicum by recrystallisation techniques. Principles of Separation and Purification by Recrystallisation Technique Table 1. The relationship between indicators and concept labels. 5 BIO Web of Conferences 79, 12001 (2023) ICOME 2023 https://doi.org/10.1051/bioconf/20237912001 No. Indicators/Sub-Course Learning Outcomes Concept Labels 13 Implement the principles of purification and separation through practicum by paper chromatography techniques. Principles of Separation and Purification by Chromatographic Techniques 14 Implement the principles of purification and separation through practicum by thin-layer chromatography technique. 15 Implement the principles of purification and separation through practicum by simple distillation techniques. Principles of Separation and Purification by Simple Distillation Techniques 16 Identify information and data from the results of the separation and purification practicum project. Scientific Articles based on Research Results 17 Synthesise information and data to describe the results of the separation and purification practicum project. The last stage in the selecting step is to develop values that are referred to as substantial and pedagogical contexts. Substantial context contains the context of the textbook that is connected to phenomena, symptoms, facts, data, and benefits in various fields of life [12]. The substantial context integrated into the textbook is related to the principles of Green Chemistry and Microscale criteria in practicum. Next is to develop a pedagogical context in the textbook, namely exploring the values, attitudes, and skills related to the material [12- 18]. The pedagogical context inserted in this textbook is related to science process skills and sustainability awareness. The collection of information in this selecting step is referred to as Draft 1 of the textbook. 3.1 Selecting Step As a guarantee that indicators of competency achievement, concept labels, concept/material descriptions, and contexts represent the learning outcomes demanded by the curriculum, a review was conducted by three experts in chemistry and chemistry education. This review process aims to determine 1) the suitability of indicators or sub-course learning outcome with learning outcomes; 2) the suitability of indicators or sub-course learning outcome with concept descriptions; and 3) the suitability of concept descriptions with related substantial and pedagogical contexts. Table 2 shows the agreement index (V) on the aspects reviewed based on the calculation using the Conten Validity Index [21]. Table 2. Results of the selecting step review. Raters Agreement Index (V) Indicator/Sub-Course Learning Outcome with Learning Outcome Concept Label with Indicator Value with Concept R1 1,00 1,00 1,00 R2 1,00 1,00 1,00 R4 1,00 1,00 1,00 Category Valid Valid Valid Table 2. Results of the selecting step review. 3.2 Structuring Step The structuring step aims to make the book develop to have an orderly arrangement of concepts according to the cognitive structure of students [22]. There are three important parts to this structuralisation step: the concept map, the macro structure, and the three levels of representation [12]. First, the concept map aims to provide a relationship between concepts in the textbook so that readers are able to relate new knowledge to their cognitive structure [12, 21]. In addition, concept maps can also give learners the opportunity to think deeply about science by helping them to better understand and organise what they are learning, and to store and retrieve information more efficiently [23]. Furthermore, the macro structure form describes the systematic presentation in the textbook by considering didactic elements in teaching materials so that readers can more 6 6 6 BIO Web of Conferences 79, 12001 (2023) ICOME 2023 https://doi.org/10.1051/bioconf/20237912001 easily understand the textbook developed [12]. In other words, the macro structure in the development of this textbook serves to show the order and arrangement of the material to be studied in the textbook. An example of a macro structure in one of the chapters is shown by Figure 2. Fig. 2. Macro structure of chapter 5 of green chemistry-integrated separation and purification textbook. Macro structure of chapter 5 of green chemistry-integrated separation and purification textbook. Subsequently, three levels of representation consisting of macro, sub-micro, and symbolic levels were determined [24]. With these three representation models, teaching material will help students create the mental models that they should have correctly [12]. However, at this stage, it is not a must to use, so it needs to be considered and adjusted to the characteristics of the concept or material [12]. Table 3 shows an example of the three levels of representation of one of the concepts in the developed textbook. The final product of this structuring step is the development of a draft textbook according to the macro structure and three levels of representation. The systematic presentation of textbooks is adjusted to the macro structure, while the presentation of concepts in the textbooks follows the three levels of representation. The concept map can be included at the beginning of each teaching material, the chapter, if the purpose of using the concept map is as a guideline to find out the scope of discussion in each chapter. 3.2 Structuring Step However, if the concept map aims as a summary of the discussion in the textbook, it should be included at the end of each chapter. 4 Conclusion The development of the separation and purification textbook was carried out using the 4STMD (Four Steps Teaching Material Development) method which consists of selecting, structuring, characterisation, and didactic reduction steps. In this research, it is limited to the selecting and structuring steps to get in-depth research results. As many as 4 course learning outcomes, 17 indicators of sub-course learning outcomes, and 11 concept labels were obtained so that it became a systematic material which consists of 10 chapters and 30 sub- chapters in the developed textbook. Green Chemistry and microscale principles are integrated as a substance context, while science process skills and sustainable awareness are integrated as pedagogical contexts in the development of separation and purification textbook. 5 Acknowledgment The author expresses gratitude towards the Indonesian Ministry of Education, Culture, Research, and Technology for facilitating the research by providing opportunities and grants in the form of Indonesian Education Scholarships (BPI). This collaboration involves the Higher Education Financing Agency (BPPT) Education Financing Service Centre (PUSLAPDIK) and the Indonesia Endowment Funds for Education (LPDP). 7 7 https://doi.org/10.1051/bioconf/20237912001 BIO Web of Conferences 79, 12001 (2023) ICOME 2023 Table 3. An example of three levels of representation of one of the textbook concepts developed. Material Concept Macroscopic (Sub) Microscopic Symbolic Sublimation is the direct change of a solid phase substance into the vapour phase of the substance without going through the liquid phase. It has been proven that the vapour pressure of a liquid increases as the temperature increases. Since the boiling point of a liquid occurs when its vapour pressure is equal to the applied pressure (usually atmospheric pressure), the vapour pressure of a liquid is equal to 760 mmHg at its boiling point. The vapour pressure of a solid also varies with temperature. Because of this behaviour, some solids can go directly into the vapour phase without going through the liquid phase. One example of separation that is often encountered in everyday life is the process of sublimation of camphor from its impurities, such as car fragrances that emit fragrance even though it is invisible to the naked eye. A cupboard with mothballs in it will emit a strong fragrance when opened. Naphthalene has the chemical formula C10H8 References 1. M. Pena-Abaurrea, V. S. García de la Torre, and L. Ramos, J. Chromatogr. A 1317, 223 (2013) 2. V. M. Vorotyntsev, V. M. Malyshev, and A. N. Petukhov, Theor. Found. Chem. Eng. 52, 819 (2018) 3. J. Zeng, W. Wang, J. Lin, Y. Zhang, H. Li, J. Liu, C. Yan, Y. Gu, and Y. Wei, J. Chromatogr. A 1674, 1 (2022) 4. D. A. Vosburg, ACS Symp. Ser. 1345, 63 (2020) 5. M. Chen, E. Jeronen, and A. Wang, Int. J. Environ. Res. Public Health 17, (2020) 6. K. Tobin, Sch. Sci. Math. 90, 403 (1990) 7. P. Anastas and N. Eghbali, Chem. Soc. Rev. 301 (2010) 8. E. S. Beach, Z. Cui, and P. T. Anastas, Energy Environ. Sci. 2, 1038 (2009) 9. R. Boethling and A. Voutchkova, Handbook of Green Chemistry Green Processes M. Chen, E. Jeronen, and A. Wang, Int. J. Environ. Res. Public Health 17, (2020) ( ) 7. P. Anastas and N. Eghbali, Chem. Soc. Rev. 301 (2010) P. Anastas and N. Eghbali, Chem. Soc. Rev. 301 (2010) 8. E. S. Beach, Z. Cui, and P. T. Anastas, Energy Environ. Sci. 2, 1038 (2009) R. Boethling and A. Voutchkova, Handbook of Green Chemistry Green Processes 9. R. Boethling and A. Voutchkova, Handbook of Green Chemistry Green Processe 8 BIO Web of Conferences 79, 12001 (2023) ICOME 2023 https://doi.org/10.1051/bioconf/20237912001 Designing Safer Chemicals (2012) 10. S. Ravichandran, Int. J. ChemTech Res. 3, 1046 (2011) 11. F. A. Etzkorn and J. L. Ferguson, Angew. Chemie - Int. Ed. 62, (2023) 12. S. Anwar, Metode Pengembangan Bahan Ajar: Four Steps Teaching Material Development (4STMD), I (Indonesia Emas Group, Bandung, 2023) 13. J. A. Haack and J. E. Hutchison, ACS Sustain. Chem. Eng. 4, 5889 (2016) 14. Arifin and S. Anwar, J. Pendidik. Fis. Indones. 12, 8 (2016) 15. D. Lotaningrat, Didakt. J. Pendidik. V, 80 (2019) 16. S. Suryaningsih, B. Muslim, and N. A. Wati, TARBIYA J. Educ. Muslim Soc. 7, 78=87 (2020) 17. F. R. Wilson, W. Pan, and D. A. Schumsky, Meas. Eval. Couns. Dev. 45, 197 (2012) ( ) 18. A. Saifuddin, Penyusunan Skala Psikologi (Kencana, Jakarta, 2020) 19. L. R. Aiken, J. Artic. Reports - Res. Numer. Data 45, 131 (1985) 20. A. Hasyim, Pros. Simp. Nas. Inov. Dan Pembelajaran Sains 2015 (SNIPS 2015) 2015, 605 (2015) ( ) 21. C. H. Lawshe, Pers. Psychol. 28, 563 (1975) 22. N. I. Syar and N. J. K. Gilbert and D. Treagust, Models and Modeling in Science Education: Multiple Representations in Chemical Education (Springer, Netherlands, 2009) References Meriza, Kwangsan J. Teknol. Pendidik. 08, 190 (2020) 23. J. Vanides, Y. Yin, M. Tomita, and M. A. Ruiz-Primo, Teach. Strateg. 28, 27 (2005) 24. J. K. Gilbert and D. Treagust, Models and Modeling in Science Education: Multiple Representations in Chemical Education (Springer, Netherlands, 2009) 9
https://openalex.org/W2167082355	https://academicjournals.org/journal/AJAR/article-full-text-pdf/EBC69FC37553.pdf	English	null	Antioxidants and chlorophyll in cassava leaves at three plant ages	African journal of agricultural research	2,013	cc-by	6,166	Full Length Research Paper Antioxidants and chlorophyll in cassava leaves at three plant ages Anderson Assaid Simão* , Mírian Aparecida Isidro Santos, Rodrigo Martins Fraguas, Mariana Aparecida Braga, Tamara Rezende Marques, Mariene Helena Duarte, Claudia Mendes dos Santos, Juliana Mesquita Freire and Angelita Duarte Corrêa Chemistry Department, Biochemistry Laboratory, Federal University of Lavras – UFLA, PO Box 3037, Zip Code 37200.000, Lavras, MG, Brazil. Chemistry Department, Biochemistry Laboratory, Federal University of Lavras – UFLA, PO Box 3037, Zip Code 37200.000, Lavras, MG, Brazil. Accepted 15 May, 2013 The aim of this study was to quantify antioxidant substances and chlorophyll content, as well as to measure the antioxidant activity in cassava leaf flour (CLF) of different cultivars at several plant ages, in order to lead to a higher utilization of these leaves, and consequently to an enhancement of this agricultural by-product. The contents of antioxidant substances (vitamin C, polyphenols and β- carotene) were regarded as high and increased as the plants matured. The chlorophyll content decreased with plant maturity and presented a negative correlation with antioxidant substances, which indicates that the highest antioxidant levels are found when the plant presents low chlorophyll levels. CLF showed a high antioxidant activity when the lipid oxidation inhibition method (β-carotene/linoleic acid) was used, and moderate when the free radical capture method (ABTS) was used. The main contribution to the CLF antioxidant activity seems to be provided by vitamin C, which presented the best correlation with the ABTS test. Out of the ages studied, that of 14 months presented the highest antioxidant levels; Mocotó and Pão da China cultivars stood out the most. Key words: Cassava leaf flour, antioxidant activity, antioxidant substances. Vol. 8(28), pp. 3724-3730, 26 July, 2013 DOI: 10.5897/AJAR2013.6746 ISSN 1991-637X ©2013 Academic Journals http://www.academicjournals.org/AJAR African Journal of Agricultural Research Vol. 8(28), pp. 3724-3730, 26 July, 2013 DOI: 10.5897/AJAR2013.6746 ISSN 1991-637X ©2013 Academic Journals http://www.academicjournals.org/AJAR African Journal of Agricultural Research African Journal of Agricultural Research INTRODUCTION Cassava (Manihot esculenta Crantz) is a perennial, bushy plant, of the Euphorbiaceae family. Originating on the American continent, probably in Central Brazil, it is cultivated all over the world, mainly in poor areas, where its cultivation constitutes one of the main agricultural activities, having a high social importance as a main carbohydrate source for more than 500 million people, essentially in developing countries (Brazilian Company of Agricultural Research-EMBRAPA, 2010). In recent years, the aerial part of the plant, which had been treated as an agricultural by-product, but that nutritionally, presents great potential for human and animal consumption, has been gaining prominence. Those leaves are rich in proteins and vitamins A and C (Corrêa et al., 2004; Wobeto et al., 2006), and minerals, especially Mg, Fe, Zn and Mn (Wobeto et al., 2006), obtained at a low cost, when compared to conventional leafy vegetables. Furthermore, their use can provide an extra income to various producers that live on the cassava culture. In the search for natural substances that can bring benefits, mainly to health, aggregating value to this agricultural by-product can contribute to a greater use of the cassava leaves in feeding and in many other applications. pp Experimental and epidemiological studies have been demonstrating that people who consume foods rich in Corresponding author. E-mail: andersonbsbufla@yahoo.com.br. Tel: +55-35-3829-1272. Fax: +55-35-3829- 1271. Corresponding author. E-mail: andersonbsbufla@yahoo.com.br. Tel: +55-35-3829-1272. Fax: +55-35-3829- Corresponding author. E-mail: andersonbsbufla@yahoo.com.br. Tel: +55-35-382 1271 E-mail: andersonbsbufla@yahoo.com.br. Tel: +55-35-3829-1272. Fax: +55-35-3829 ponding author. E-mail: andersonbsbufla@yahoo.com.br. Tel: +55-35-3829-1272. 3725 Simao et al. Phenolic compounds: The extraction of phenolic compounds was carried out with 1 g of sample in 50 ml of 50% methanol, under reflux for three consecutive times, at 80°C, and the extracts were collected, evaporated up to 25 ml and submitted to phenolic compound measurement, using Folin-Denis reagent, and tannic acid as a standard (Association of Official Analytical Chemists - AOAC, 2011). antioxidants could have a reduced risk of many diseases, such as cancer, cardiovascular diseases, chronic diseases and aging, among others. Antioxidants are substances that combat free radicals, which are extremely reactive species that cause oxidation of various biomolecules present in the organism. Moreover, besides the problems found with the synthetic antioxidants used in food conservation and their high production costs, it has also been demonstrated, by toxicological studies, that they can provoke undesirable effects in human and animal organism. Antioxidant activity Extract preparation: For the obtention of the extracts, the CLFs were maintained under maceration using 50% ethanol, 1:40 (w/v), for 30 min and soon afterwards centrifuged at 2,500 g, for 15 min. The supernatant was collected and the precipitate was again submitted to the previously described extraction process, substituting 50% ethanol for 70% acetone; the supernatants were collected and then subjected to the detection of antioxidant activity by the methods described subsequently. Therefore, this work was conducted with the objective of quantifying antioxidant substances, chlorophyll and to measure the antioxidant activity in cassava leaf flour of different cultivars, at several plant ages. INTRODUCTION Therefore, the search for natural substances that is efficient antioxidant sources, and less expensive than synthetic antioxidants, has been the objective of various studies (Gan et al., 2010; Surveswaran et al., 2007; Wojdylo et al., 2007). β-carotene: The extraction of β-carotene was carried out with 0.5 g of sample in a 40 ml extraction solution of isopropyl alcohol:hexane 3:1. The content was transferred to a 125 ml separation funnel wrapped in aluminum, where the volume was completed with distilled water. It was left at rest for 30 min, followed by washing of the material and discard of the aqueous phase. This operation was repeated three more times. The content was filtered with cotton sprayed with anhydrous sodium sulphate 99% to a 25 ml volumetric flask wrapped with aluminum, where 5 ml of acetone were added and the volume completed with hexane. Then, absorbance readings were taken in a spectrophotometer at four wavelengths (453, 505, 645 and 663 nm) (Nagata and Yamashita, 1992) and the results expressed in mg 100 g-1, calculated by the formula: In plant leaves, chlorophyll, the photosynthetic pigment, is directly related to their nutritional state. The amount of that pigment has been used as an evaluation index of the nutritional state for several types of cultures (Argenta et al., 2001). Variables, such chlorophyll and antioxidant levels, can be directly correlated, demanding studies to confirm that correlation and, after being confirmed, can serve as a parameter for obtaining antioxidant substances in cassava leaves. β-carotene (mg 100 g-1) = 0.216 A663 - 1.22 A645 - 0.304 A505 + 0.452 A453. β-carotene (mg 100 g-1) = 0.216 A663 - 1.22 A645 - 0.304 A505 + 0.452 A453. MATERIALS AND METHODS ABTS method: The methodology used was developed by Rufino et al. (2007). Four different dilutions were made from the obtained extracts for the assays and subsequent construction of analytical curves. Six point analytical curves were made with trolox (6- hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) (100 to 2,000 µmol L-1) and with ascorbic acid (10 to 200 mg mL-1), in addition to tests for comparison with the patterns BHT (butylhydroxytoluene–synthetic antioxidant) and with rutin and quercetin, that are flavonoids with proven antioxidant activity; these standards were prepared at a concentration of 200 mg L-1. Samples Mature leaves of four cassava cultivars, ‘Mocotó ', ‘Ufla ', ‘Pão da China ' and ‘Ouro do Vale’, obtained from the Agriculture Department of Federal University of Lavras, were picked in the morning, from plants at three different ages (TPA), in December (10 months), February (12 months) and April (14 months), in three repetitions. Intact leaves, free from diseases and pests, were selected, washed in running water and distilled water and, soon afterwards, dried in a forced-air oven, for 48 h, at temperatures ranging from 30 to 35°C. After drying, they were ground (without petioles) in a Willy type mill and the cassava leaf flours (CLF) were stored in hermetically sealed flasks under refrigeration, until the analyses were performed. β-carotene/linoleic acid method: The methodology used was developed by Rufino et al. (2006). The test was applied to the extracts, at a concentration of 10,000 mg L-1. g For the preparation of the β-carotene/linoleic acid solution system, 50 µl of β-carotene diluted in chloroform (20 g L-1) were used, to which 40 µl of linoleic acid were added, as well as 530 µl of tween 20 (emulsifier) and, for solubilization, 1 ml of chloroform. In a flask covered with aluminum for protection against light, chloroform was evaporated in a rotary-evaporator and 100 ml of oxygen saturated water (distilled water treated with oxygen for 30 min) were added, and the combination was agitated until the solution system presented a yellow-orange coloration. In test tubes, 2.5 ml of that solution system were added to 0.2 ml of each sample dilution used for the test. Control tubes were made containing 2.5 ml of the solution system with 0.2 ml of 2,6-di-tert-butyl-p-cresol (BHT), quercetin and rutin, all at the concentration of 200 mg L-1. In laboratory tests, it was found that the concentration of 200 mg L-1 BHT is the one that provides the greatest protection for the system, when compared to others; therefore, its use is suggested. After Statistical analysis The experiment was conducted in a completely randomized design, in a 4 × 3 factorial outline, with four cassava cultivars and three plant ages, and three repetitions. The statistical analysis was conducted using R (version 2.15.2) statistical software (R Core Team, 2012). Averages were compared by the Scott-Knott test, at 5% probability. Pearson correlation analysis was conducted among the antioxidant substances, antioxidant tests and chlorophyll, using the Statistical Analysis System program (1999). When compared with other unconventional foods, CLF at 14 months presented average levels of vitamin C superior to those found in 100 g, of carrot leaves (203.70 mg) (Pereira et al., 2003). However, in relation to the fruits, those levels are also superior to those found in 100 g of fresh matter (FM) for orange (66 mg) and papaya (149 mg) (Hernández et al, 2006). Nevertheless, they are inferior to those of fruits considered rich in vitamin C, such as acerola, that contains 1,500 mg in 100 g-1 FM. With the discovery of the antioxidant action of this vitamin, ingestion of substances with high vitamin C content has been recommended. As such, those plants are shown as good vitamin C sources with potential for use as antioxidants. Determination of antioxidant substances (1989) found an increase in vitamin C levels for cassava leaves dried at 45°C, up to 14 months of age. It was also certified by Wobeto et al. (2006) who, analyzing five cassava cultivars, in TIP, observed an increase in vitamin C with plant maturity. These results are consistent with the observations made in the present study. homogenization, their readings were taken in a spectrophotometer at 470 nm, using water to calibrate the spectrophotometer; this was considered to be the reading at time zero (initial). The tubes were placed in a water bath, at 40°C and readings were taken after 2 h. Chlorophyll determination The chlorophyll determination was conducted with Minolta SPAD- 502 equipment, directly on the leaf still attached to the plant, moments before the collection, according to the methodology described by Argenta et al. (2001). Each SPAD reading is equivalent to the result obtained by the measurement performed on six leaves, with five measurements taken on each leaf, which were performed at points situated in half to two thirds of the sampled leaf length. The results were expressed in SPAD units, which are equal to the average of thirty readings. y Higher vitamin C levels were found in this study, when compared to Wobeto et al. (2006) who, analyzing cultivars Mocotó and Ouro do Vale at 12 months of age, found levels of 55.72 and 64.12 mg 100 g-1 of dry matter (DM) for Mocotó and Ouro do Vale, respectively. In the present study, it was found, at the same age, levels of 294.77 and 310.88 mg 100 g-1 DM for Mocotó and Ouro do Vale, respectively. These differences may have occurred as a result of the analysis method used, which, in this study, was the chromatographic method, while Wobeto et al. (2006) used the colorimetric method, besides other factors, such as manuring, climatic conditions, among others. Determination of antioxidant substances Vitamin C: The extraction of vitamin C for chromatographic analyses was conducted in oxalic acid, according to Strohecker and Henning (1967). A Shimadzu LC 200A liquid chromatography with UV-VIS detector, wavelength detection at 254 nm; quaternary pump, degasser, and automatic injection was used. A C18 Nucleosil (250 × 4.6 mm × 5 µm) column and a C18 (15 × 3.2 mm × 7.5 µm) pre-column were used. As movable phase a pH 6.7 buffer was used, containing sodium acetate 0.04 mol L-1, EDTA 0.05 mol L-1, tributyl ammonium phosphate isocratic mode, flow rate 0.6 ml min-1, 15 min running time for each sample. 3726 Afr. J. Agric. Res. Table 1. Antioxidant levels in cassava leaf flour of four cultivars, at three plant ages. Table 1. Antioxidant levels in cassava leaf flour of four cultivars, at three plant ages. Table 1. Antioxidant levels in cassava leaf flour of four cultivars, at three plant ages. Antioxidant substances Cultivar 10 months 12 months 14 months Vitamin C (mg 100 g-1) Mocotó 281.53±14.84aB 294.77±5.47aB 521.19±31.60bA Ufla 149.29±3.84dC 249.68±1.32cB 474.05±30.07cA Pão da China 244.74±8.58bC 276.97±11.50bB 568.64±7.71aA Ouro do Vale 171.44±2.58cC 310.88±26.16aB 463.69±2.40cA Polyphenols (mg g-1) Mocotó 29.48±0.56aC 52.12±1.13bB 55.06±0.30aA Ufla 29.14±0.19aC 54.53±1.58aA 50.14±0.66bB Pão da China 29.18±0.30aB 55.57±1.54aA 56.21±1.39aA Ouro do Vale 16.46±0.16bC 31.73±0.86cB 36.26±0.67cA β-carotene (mg 100g-1) Mocotó 50.20±1.60bB 70.31±0.24aA 65.92±2.08aA Ufla 53.08±3.21bB 70.07±3.59aA 65.60±1.39aA Pão da China 65.90±0.83aB 72.72±1.09aA 61.63±1.38aB Ouro do Vale 51.82±4.59bB 68.79±4.74aA 51.44±6.67bB Data are the mean of three replicates ± standard deviation. Lowercase letters in columns compare among cultivars and uppercase on the lines compare among ages. Same letters do not differ among themselves by the Scott-Knott test at 5% probability. Data are the mean of three replicates ± standard deviation. Lowercase letters in columns compare among cultivars and uppercase on the lines compare among ages. Same letters do not differ among themselves by the Scott-Knott test at 5% probability China’ (14 months) presented the highest vitamin C level. Carvalho et al. (1989) found an increase in vitamin C levels for cassava leaves dried at 45°C, up to 14 months of age. It was also certified by Wobeto et al. (2006) who, analyzing five cassava cultivars, in TIP, observed an increase in vitamin C with plant maturity. These results are consistent with the observations made in the present study. China’ (14 months) presented the highest vitamin C level. Carvalho et al. Antioxidant substances Table 1 shows vitamin C, polyphenol and β-carotene levels of CLF in the four cultivars, at TPA. It was observed that vitamin C levels increased with plant maturity, for all cultivars. Among the cultivars, ‘Pão da Simao et al. Simao et al. 3727 significant differences in β-carotene levels for CLF from Baiana cultivar. The highest levels are found for the oven-drying process at 30°C (84.83 mg 100 g-1 DM) and the lowest ones for the shade-dried leaves (64.88 mg 100 g-1 DM). Consequently, the different levels of this substance can be probably attributed to plant age, cultivars and also to the forms of drying. It was possible to verify that, in general, polyphenol levels increased with plant maturity. Cultivars Pão da China and Mocotó, at 14 months of age, did not differ significantly amongst themselves and they presented the highest polyphenol levels. Ouro do Vale, at the three analyzed ages, was that which presented significantly lower polyphenol levels. y g The average β-carotene levels in the CLF studied, independent of cultivar and plant age, ranged from 50.20 to 72.72 mg 100 g-1 DM and, when compared to those found in 100 g DM of other green leaves, unconventionally used, are comparable to those of sweet potato (40 to 120 mg) (Almazan and Begun, 1996), superior to those of carrot (8.70 mg) (Pereira et al., 2003) and lower than those of peanut leaves (100 to 140 mg) (Almazan and Begun, 1996). However, in relation to the levels in 100 g FM of green vegetables, these are higher than those of lettuce (1.37 mg), watercress (5.26 mg), green onion (2.31 mg) and parsley (3.82 mg) (Campos et al., 2003). Wobeto et al. (2007), studying CLF from five cultivars, in TIP, also observed an increase in polyphenol levels with plant maturity. In that study, the authors also observed higher polyphenol levels for cultivar Ouro do Vale (61.49 mg 100 g-1 DM) and lower for Mocotó (44.13 mg 100 g-1 DM) at 12 months of age, when compared to those recorded in this study at the same age, for Ouro do Vale (31.73 mg 100 g-1 DM) and Mocotó (52.12 mg 100 g-1 DM). These different results are probably due to factors related to manuring, climatic conditions, among others. Antioxidant activity ( g ) g g Phenolic compounds act as antioxidants, due to their redox properties that allow them to act as reducing agents, hydrogen donors and metal chelators. Besides their role as antioxidants, these compounds present a wide spectrum of medicinal properties, such as antiallergic, anti-inflammatory, anti-bacterial and anti- thrombotic, plus they present cardioprotective and vasodilator effects (Balasundram et al., 2006), showing a broad field of application for the phenolics in these plants. It can be noticed that there is an increase in β-carotene levels until the age of 12 months and that those levels did not differ significantly from those at 14 months, except for Ouro do Vale and Pão da China cultivars, that presented a reduction in β-carotene levels between 12 and 14 months of age. The CLF antioxidant activity (AA) determined by the ABTS method for four cultivars, at TPA, is shown in Table 2. It was possible to observe that AA by the ABTS method increased with plant maturity for all cultivars, in equivalent of trolox, as well as ascorbic acid, and it can be explained by an increase in vitamin C and polyphenol levels with cultivar maturity, which is substances that have a strong action in capturing ABTS. The highest AA was observed at 14 months for Pão da China cultivar, in equivalent of trolox (680.62 µmol L-1 g-1), as well as ascorbic acid (102.42 mg g-1), probably due to higher levels of vitamin C and phenolic compounds in their leaves at this age. However, Ouro do Vale presented the lowest AA, in equivalent of trolox, as well as ascorbic acid, at TPA, which can be explained by lower levels of antioxidants found in the flour of that cultivar. Wobeto et al. (2006), analyzing five cassava cultivars, in TIP, also found, at 12 months of age, higher β- carotene levels. However, these authors observed a decline in these levels with plant maturity. It is observed that the CLF antioxidant potential by the ABTS method, when compared to BHT and rutin standards, in equivalent of trolox, as well as ascorbic acid, is considered of moderate potential, because at 14 months it reached on average, almost 50% of the potential of those standards, except for Ouro do Vale cultivar. In relation to quercetin, cultivar potential at any age was very inferior to the antioxidant potential of this standard. Antioxidant substances Polyphenol levels found in this study, independent of age and cultivar, are higher than those found in mg 100 g-1 FM for some vegetables, such as broccoli (0.68), onion (1.13) and cabbage (0.67) and for some fruits, such as pineapple (0.85), banana (2.16), orange (1.14), papaya (0.15) and mango (1.10) according to Faller and Fialho (2009). These are within the range related by Asolini et al. (2006) who, analyzing phenolic compounds in plants used as tea, found levels between 15 (mate) to 56 (lemongrass) mg g-1 DM. The consumption of natural antioxidants, such as vitamin C, polyphenols and β-carotene present in most plants, has been associated to a lower incidence of diseases caused by free radicals. Consequently, the levels of these substances found in these CLFs can contribute to their antioxidant capacity. Antioxidant activity It is observed that the CLF antioxidant potential by the ABTS method, when compared to BHT and rutin standards, in equivalent of trolox, as well as ascorbic acid, is considered of moderate potential, because at 14 months it reached on average, almost Lower β-carotene levels were found in this study, when compared to Wobeto et al. (2006) who, analyzing Mocotó and Ouro do Vale cultivars at 12 months of age, found levels of 126.57 and 124.24 mg 100 g-1 DM for Mocotó and Ouro do Vale, respectively. In the present study, we found, at the same age, levels of 70.31 and 68.79 mg 100 g-1 DM for Mocotó and Ouro do Vale, respectively. Corrêa (2004), analyzing different forms of cassava leaf drying (sun-dried, shade-dried and oven-dried at 30°C and 40°C), observed that these forms of drying cause 50% of the potential of those standards, except for Ouro do Vale cultivar. In relation to quercetin, cultivar potential at any age was very inferior to the antioxidant potential of this standard. The good antioxidant potential shown by CLF is Afr. J. Agric. Res. 3728 2. Antioxidant activity of cassava leaf flour of four cultivars, by the ABTS method, at three plant ages. ble 2. Antioxidant activity of cassava leaf flour of four cultivars, by the ABTS method, at three plant ages. Cultivar µmol trolox L-1 g-1 mg of vitamin C g-1 10 months 12 months 14 months 10 months 12 months 14 months Mocotó 238.62±1.05bC 320.25±1.02cB 636.12±1.41cA 38.54±0.04bC 49.11±0.10cB 100.83±0.25cA Ufla 359.85±0.86aC 444.65±0.59aB 658.71±1.84bA 50.11±0.12aC 52.07±0.20aB 101.20±0.03bA Pão da china 240.25±0.68bC 338.15±0.67bB 680.62±0.30aA 38.79±0.03bC 50.45±0.11bB 102.42±0.58aA Ouro do Vale 155.90±2.39cC 266.83±0.64dB 327.27±0.08dA 24.51±0.09cC 44.60±0.13dB 50.85±0.18dA BHT 1,418.81±97.98 259,55±3.27 Quercetin 7,491.59±119.04 942,96±1.89 Rutin 1,055.50±13.39 161,29±0.30 Data are the mean of three replicates ± standard deviation. Lowercase letters in columns compare among cultivars and uppercase on the lines compare among ages. Same letters do not differ among themselves by the Scott-Knott test at 5% probability. Data are the mean of three replicates ± standard deviation. Lowercase letters in columns compare among cultivars and uppercase on the lines compare among ages. Same letters do not differ among themselves by the Scott-Knott test at 5% probability. Table 3. Antioxidant activity of the cassava leaf flour of four cultivars, at three plant ages, in % inhibition by the β-carotene/linoleic acid method. Antioxidant activity Cultivar % inhibition 10 months 12 months 14 months Mocotó 93.04±3.14aA 94.05±0.20aA 89.81±0.19aB Ufla 85.17±0.96bA 86.47±0.33bA 84.61±0.66bA Pão da china 81.47±1.27cB 85.51±0.63bA 79.79±1.28cB Ouro do Vale 82.79±0.35cB 85.69±0.80bA 78.87±2.25cC BHT (200 mg L-1) 93.60±0.56 Quercetin (200 mg L-1) 76.20±1.15 Rutin (200 mg L-1) 19.21±0.88 Data are the mean of three replicates ± standard deviation. Lowercase letters in columns compare among cultivars and uppercase on the lines compare among ages. Same letters do not differ among themselves by the Scott-Knott test at 5% probability. The test was applied to the extracts at a concentration of 10,000 mg L -1. Table 3. Antioxidant activity of the cassava leaf flour of four cultivars, at three plant ages, in % inhibition by the β-carotene/linoleic acid method. Data are the mean of three replicates ± standard deviation. Lowercase letters in columns compare among cultivars and uppercase on the lines compare among ages. Same letters do not differ among themselves by the Scott-Knott test at 5% probability. The test was applied to the extracts at a concentration of 10,000 mg L -1. evidenced when compared to other studies, in which, independently of the extract, surpassed that AA found by Kuskoski et al. (2005), in frozen fruit pulps, in µmol trolox L-1 g-1 FM: acerola (66.5), mango (11.8), grape (8.5), guava (7.2) and passion fruit (1.02); that of two grape peels in DM: ‘Isabel' (89.22) and ‘Niágara' (157.31) according to Soares et al. (2008) and that of wine agroindustrial residues: (98.9), according to Cataneo et al. (2008). It also surpassed that detected by Bouayed et al. (2007) in several parts of medicinal plants, in mg g-1 vitamin C: 2.8 (Alcea kurdica flowers), 7.36 (Valerian officinalis root), 15.4 (Stachys lavandulifolium flowers), 19.2 (Lavandula officinalis leaves) and 19.3 (Melissa officinalis leaves), and the ones detected by Wojdylo et al. (2007) who, in 32 Polish herbs, verified potentials between 0.0045 (Archangelica officinalis) and 3.46 (Syzygium aromaticum) µmol trolox g-1. The lipidic oxidation inhibition results for CLF from four cultivars, by the β-carotene/linoleic acid method, at TPA after 2 h of reaction, are shown in Table 3. Plant maturity had little influence on the CLF AA, by the β- carotene/linoleic acid method. The greatest inhibition evidenced when compared to other studies, in which, independently of the extract, surpassed that AA found by Kuskoski et al. Chlorophyll The SPAD-502 equipment has been investigated as an instrument for fast diagnosis of the nutritional state of various cultures, adding advantages such as simplicity of use, besides enabling a non-destructive evaluation of the foliar tissue substituting, with good precision, the traditional chlorophyll level determination in plants (Argenta et al., 2001). Results of the correlations indicate that vitamin C and polyphenols are the antioxidant substances that most contributed to the increase in AA for CLF; vitamin C stood out, therefore it presented the highest correlation coefficients, 0.78 and 0.84, for equivalent of trolox and vitamin C, respectively. β-carotene levels did not contri- bute to the CLF AA increase. The negative correlation among the antioxidant substances levels and chlorophyll indicate that antioxidant substances are found in a higher amount when cassava leaves present low chlorophyll levels. ( g ) Chlorophyll reading results for CLF at TPA, using the SPAD-502 portable meter, are in Table 4. It was verified that the highest chlorophyll levels were found at 10 months of age and that there was a tendency of reduction of those levels with plant maturity, Ouro do Vale, at TPA, was the one which presented the highest chlorophyll levels. Mocotó cultivar, at TPA, was that which showed significantly lower chlorophyll levels. Cataneo et al. (2008) and Soares et al. (2008) also observed a positive correlation between total phenols and AA by the ABTS test, equivalent to trolox, in studies performed with wine agroindustrial residues and grape skins of two varieties, respectively, indicating that polyphenols are substances with a high antioxidant potential and are one of the main antioxidants present in medicinal plants and food. potential of CLF in relation to other plants. potential of CLF in relation to other plants. and with polyphenols, but there was no correlation with β- carotene. For the β-carotene/linoleic acid test, there was no correlation with antioxidant substances. The antioxidant substances presented a negative correlation with chlorophyll, that is, when antioxidant levels increased, chlorophyll contents decreased in the plant. This is easily explained for β-carotene, because, with the increase in maturity, chlorophyll begins to disappear and carotenoids protrude. Antioxidant activity Average levels of chlorophyll (SPAD units) for cassava leaf flour of four cultivars, at three plant ages. Table 4. Average levels of chlorophyll (SPAD units) for cassava leaf flour of four cultivars, at three plant ages. Table 4. Average levels of chlorophyll (SPAD units) for cassava leaf flour of four cultivars, at three plant ages. Cultivar Months 10 12 14 Mocotó 565.60±3.05dA 479.80±9.07dB 429.60±8.68dC Ufla 719.80±7.63bA 631.20±5.72bC 677.00±11.51bB Pão da China 589.40±10.43cA 491.60±1.52cB 468.40±13.67cC Ouro do Vale 771.00±5.20aA 743.20±4.38aC 758.40±0.63aB Data are the mean of three replicates ± standard deviation. Lowercase letters in columns compare among cultivars and uppercase on the lines compare among ages. Same letters do not differ among themselves by the Scott-Knott test at 5% probability. Data are the mean of three replicates ± standard deviation. Lowercase letters in columns compare among cultivars and uppercase on the lines compare among ages. Same letters do not differ among themselves by the Scott-Knott test at 5% probability. Table 5. Correlation among antioxidant substances with antioxidant activity and chlorophyll in cassava leaf flour at three plant ages. Table 5. Correlation among antioxidant substances with antioxidant activity and chlorophyll in cassava leaf flour at three plant ages. Antioxidant substances Vitamin C Polyphenols β-carotene ABTS (trolox) 0.78* 0.75* 0.28ns ABTS (vit C) 0.84* 0.70* 0.25ns β-carotene/linoleic acid -0.20ns 0.18ns 0.20ns Chlorophyll -0.40* -0.71* -0.45* Significant at 5% probability; ns = not significant. Significant at 5% probability; ns = not significant. Antioxidant activity (2005), in frozen fruit pulps, in µmol trolox L-1 g-1 FM: acerola (66.5), mango (11.8), grape (8.5), guava (7.2) and passion fruit (1.02); that of two grape peels in DM: ‘Isabel' (89.22) and ‘Niágara' (157.31) according to Soares et al. (2008) and that of wine agroindustrial residues: (98.9), according to Cataneo et al. (2008). It also surpassed that detected by Bouayed et al. (2007) in several parts of medicinal plants, in mg g-1 vitamin C: 2.8 (Alcea kurdica flowers), 7.36 (Valerian officinalis root), 15.4 (Stachys lavandulifolium flowers), 19.2 (Lavandula officinalis leaves) and 19.3 (Melissa officinalis leaves), and the ones detected by Wojdylo et al. (2007) who, in 32 Polish herbs, verified potentials between 0.0045 (Archangelica officinalis) and 3.46 (Syzygium aromaticum) µmol trolox g-1. potential of lipid oxidation occurred at 12 months of age for all cultivars, which can be explained by higher levels of β-carotene (nonpolar antioxidant) in CLF at this age, since the β-carotene/linoleic acid method shows a better response to antioxidants with apolar character. Among them, ‘Mocotó ' was that which presented the highest oxidation inhibition potential, at the TPA. The inhibition potential of lipid oxidation presented by CLF was considered high, because, compared to the BHT standard, it was verified that Mocotó cultivar, at TPA, showed AA similar to that standard. However, other cultivars presented lower AA in relation to BHT. In relation to rutin and quercetin standards, the cultivars, at TPA, presented higher AA than those standards. Many authors relate antioxidant potential above 70% as optimum for lipid oxidation inhibition. All cultivars, at TPA, reached AA above 70% and, as such, present high lipid oxidation inhibition potential. ( y yg ) µ g The lipidic oxidation inhibition results for CLF from four cultivars, by the β-carotene/linoleic acid method, at TPA after 2 h of reaction, are shown in Table 3. Plant maturity had little influence on the CLF AA, by the β- carotene/linoleic acid method. The greatest inhibition Melo et al. (2006) analyzing lipid oxidation inhibition potential of some plants, found an inhibition potential of 70% for broccoli in relation to BHT, but in other plants the potential was below 60%, emphasizing the high antioxidant 3729 Simao et al. Table 4. Average levels of chlorophyll (SPAD units) for cassava leaf flour of four cultivars, at three plant ages. Table 4. Conclusions ( ) Hernández Y, Lobo MG, González M (2006). Determination of vitamin C in tropical fruits: a comparative evaluation of methods. Food Chem. 96(4):654-664. CLFs are rich in antioxidant substances and show high antioxidant capacity in the protection of lipid peroxidation; however, they present moderate capacity in the capture of free radicals. Cassava leaves at 14 months of age present the highest antioxidant levels, and Mocotó and Pão da China cultivars stood out. The highest antioxidant levels (Vitamin C, polyphenols and β-carotene) are found when the cassava leaf presents low chlorophyll levels. ( ) Kubola J, Siriamornpum S (2008). Phenolic contents and antioxidant activities of bitter gourd (Momordica Charantia L.) leafs stem and fruit fraction extracts in vitro. Food Chem. 1110(4):881-890. Kuskoski EM, Asuero AG, Troncoso NA, Filho JM, Fett R (2005). Aplicación de diversos métodos químicos para determinar a actividad antioxidant en pulpa de frutos. Ciênc. Tecnol. Aliment 25(4):726-732 p p ( ) Melo EA, Maciel MIS, Lima VLAG, Araujo CR (2008). Total phenolic content and antioxidant capacity of frozen fruit pulps. Alimentos e Nutrição 19(1):67-72. Melo EA, Maciel MIS, Lima VLAG, leal FLL, Caetano ACS, Nascimento RJ (2006). Antioxidant capacity of vegetables commonly consumed. Ciênc. Technol. Aliment 26(3):639-644. Correlation among antioxidant substances with AA and chlorophyll Results of the correlations among antioxidant substances (Table 1) with AA (Tables 2 and 3) and with chlorophyll (Table 4) in CLF, at TPA, are shown in Table 5. The ABTS antioxidant tests (equivalent to trolox and vitamin C) presented a high positive correlation with vitamin C Kubola and Siriamornpun (2008) and Melo et al. (2008) found no correlation between the β-carotene/linoleic acid Afr. J. Agric. Res. Afr. J. Agric. Res. 3730 Carvalho VD, Chagas SJR, Morais AR, Paula MB (1989). Efeito da época de colheita na produtividade e teores de vitamina C e β- caroteno da parte aérea de cultivares de mandioca (Manihot esculenta Crantz). Revista Brasileira de Mandioca 8(1):25-35. test and polyphenol contents in fruits. Duarte-Almeida et al. (2006) found that acerola, rich in vitamin C, showed a low AA by the the β-carotene/linoleic acid test, while the DPPH test (method of scavenging free radicals, which has the same principle of the ABTS test), showed a high AA, which demonstrates that vitamin C little contributes to AA in the β-carotene/linoleic acid test, confirming the results of the present study, in which a negative correlation between antioxidants (polyphenols and vitamin C) was also observed, as the β-carotene/linoleic acid test, which can be explained by the fact that the β- carotene/linoleic acid test shows a better response to nonpolar antioxidants, thus polar antioxidants (vitamin C and polyphenols), and has little contribution in the inhibition of lipid oxidation. Cataneo CB, Caliari V, Gonzaga L, Kuskoski LM, Fett R (2008). Antioxidant activity and phenolic content of agricultural by-products from wine production. Semina Ciênc. Agrár. 29(1):93-102. Core Team R (2012). R: A language and environment for statistical computing. Viena: R Foundation for Statistical Computing; 2012. ISBN 3-900051-07-0. Available: http://www.R-project.org/. Corrêa AD, Santos SR, Abreu CMP, Jokl L, Santos CD (2004). Removal of polyphenols of the flour cassava leaves. Ciênc. Tecnol. Aliment 24(2):159-164. Duarte-almeida JM, Santos RJ, Genovese MI, Lajolo FM (2006). Evaluation of antioxidant activity using system b-carotene/linoleic acid and sequestration method DPPH radical. Ciênc. Tecnol. Aliment 26(2):446-452. ( ) Faller ALK, Fialho E (2009). Polyphenol availability in fruits and vegetables consumed in Brazil. Rev. Saúde Públ. 43(2):211-218. Gan R, Xu XR, Song FL, Kuang L, Li HB (2010). Antioxidant activity and total phenolic content of medicinal plants associated with prevention and treatment of cardiovascular and cerebrovascular diseases. J. Med. Plant. Res. 4(22):2438-2444. ACKNOWLEDGMENTS Nagata M, Yamashita I (1992). Simple method for simultaneous determination of chlorophyll and carotenoids in tomatoes fruit. J. Japan. Soc. Food Sci. Technol. 39(10):925-928. The expresses their thanks to CAPES, for the doctoral scholarship, and FAPEMIG, for financial support and scientific initiation scholarship. p Pereira GIS, Pereira RGFA, Barcelos MFP, Morais AR (2003). Carrot leaf chemical evaluation aiming its use in human feeding. Ciênc. Agrotec. 27(4):852-857. g ( ) Rufino MSM, Alves RS, Brito ES, Filho JM, Moreira AVB (2006). Determination of total antioxidant activity in fruit by the method β- caroteno/ácido linoleic. Fortaleza: Embrapa Agroindústria tropical. REFERENCES Almazan AM, Begum F (1996). Nutrients and antinutrient in peanut greens. J. Food Compos. Anal. 9(43):375-383. Rufino MSM, Alves RS, Brito ES, Morais SM (2007). Determination of total antioxidant activity in fruits by capturing the free radical ABTS +. Fortaleza: EMBRAPA Agroindústria tropical. Argenta G, Silva PRF, Forsthofer EL, Strieder ML (2001). Relationship of reading of portable chlorophyll meter with contents of extractable chlorophyll and leaf nitrogen in maize. Rev. Bras. Fisiol. Veg. 13(2):158-167. Soares M, Welter L, Kuskoski EM, Gonzaga L, Fett R (2008). Phenolic compounds and antioxidant activity in skin of Niagara and Isabel grapes. Rev. Bras. Frutic. 30(1):59-64. g p ( ) Statistical Analysis System (SAS) (1999). Users quide: statistcs : versão 6. 4. ed. Cary. 2:1686. Asolini FC, Tedesco AM, Carpes ST (2006). Antioxidant and Antibacterial Activities of Phenolic Compounds from Extracts of Plants Used as Tea. Bra. J. Food Technol. 9(3):209-215. Strohecker R, Henning HM (1967). Analisis de vitaminas: metodos comprobados. Madrid: Paz Montalvo. P. 428. Association of Oficcial Anlytical Chemists (AOAC) (2011). Official methods of analysis of the association of the analytical chemists (18 ed). Washington, DC. USA. Surveswaran S, Cai Y, Corke H, Sun M (2007). Systematic evaluation of natural phenolic antioxidants from 133 Indian medicinal plants. Food Chem. 102(3):938-953. ) g Balasundram N, Sundram K, Sammar S (2006). Phenolic compounds in plants and agri-industrial by-products: Antioxidant activity, occurrence, and potential uses. Food Chem. 99(1):191-203. Wobeto C, Corrêa AD, Abreu CMP, Santos CD, Abreu JR (2006). Nutrients in the cassava (Manihot esculenta Crantz) leaf powder at three ages of the plant. Ciência e Tecnologia de Alimentos. 26(4):865-869. p ( ) Bouayed J, Piri K, Rammal H, Dicko A, Desor F, Younos C, Soulimani S (2007). Comparative evaluation of the antioxidant potential of some Iranian medicinal plants. Food Chem. 104(1):364-368. Wobeto C, Corrêa AD, Abreu CMP, Santos CD, Abreu JR (2007). Antinutrients in the cassava (Manihot esculenta Crantz) leaf powder at three ages of the plant. Ciênc. Tecnol. Aliment 27(1):108-112. Brazilian Company of Agricultural Research (EMBRAPA). Disponível em: <http://sistemasdeprodução.cnptia.embrapa.br/FONTESHTML/Mandi oca/Mandioca centrosul/importância.htm>. Acesso em: 15 mai, 2010. Wojdylo A, Osmianski J, Czermerys R (2007). Antioxidant activity and phenolic compounds in 32 selected herbs. Food Chem. 105(3):940- 949. p Campos FM, Santana HM, Stringheta PC, Chaves JBP (2003). Levels of Beta Carotene in Leafy Vegetables Prepared in Commercial Restaurants in Viçosa, Brazil. Bra. J. Food Technol. 6(2):163-169.
https://openalex.org/W2826187237	https://europepmc.org/articles/pmc6041276?pdf=render	English	null	Direct electric field control of the skyrmion phase in a magnetoelectric insulator	Scientific reports	2,018	cc-by	7,601	Direct electric field control of the skyrmion phase in a magnetoelectric insulator Received: 9 March 2018 Accepted: 25 May 2018 Published: xx xx xxxx Received: 9 March 2018 Accepted: 25 May 2018 Published: xx xx xxxx A. J. Kruchkov1,2, J. S. White 3, M. Bartkowiak4, I. Živković 2, A. Magrez5 & H. M. Rønnow 2 Magnetic skyrmions are topologically protected spin-whirls currently considered as promising for use in ultra-dense memory devices. Towards achieving this goal, exploration of the skyrmion phase response and under external stimuli is urgently required. Here we show experimentally, and explain theoretically, that in the magnetoelectric insulator Cu2OSeO3 the skyrmion phase can expand and shrink significantly depending on the polarity of a moderate applied electric field (few V/μm). The theory we develop incorporates fluctuations around the mean-field that clarifies precisely how the electric field provides direct control over the free energy difference between the skyrmion and the surrounding conical phase. The quantitative agreement between theory and experiment provides a solid foundation for the development of skyrmionic applications based on magnetoelectric coupling. To realise skyrmion-based applications (skyrmionics)1–5, research into the creation, control and stabilisation of skyrmions is in an active phase1,6–13. In this context, it could seem problematic that in bulk materials the skyrmion phase is stable only for a narrow interval at finite temperature (T) just below the magnetic ordering temperature TC, and under an applied magnetic field (H)6,14–17. In Cu2OSeO3 for example, the skyrmion phase spreads down in T by just 3.5% of TC, occupying no more than 1% of the total ordered phase space that is otherwise dominated by topologically trivial helical or conical phases7,17,18. This limited skyrmion phase space is observed also in other known bulk hosts of skyrmions14–16,19. On the other hand, the finite extent of the skyrmion phase pocket can be considered to present an interesting advantage, since relatively small perturbations can dramatically influence the skyrmion phase stability. It follows that the ability to enhance or suppress the skyrmion phase space in a sample can provide a flexible platform for the respective creation or destruction of skyrmions, a process that can be tech- nologically useful. Here we describe a simple and reliable mechanism for the stabilisation and destabilisation of the skyrmion lattice (SkL) phase by exploiting electric (E) fields applied to the insulating magnetoelectric material Cu2OSeO3. To date, several approaches for skyrmion manipulation have been demonstrated that make use of either mod- erate electric currents, electric fields, or thermal gradients6,8–13,20–23. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 9 March 2018 Accepted: 25 May 2018 Published: xx xx xxxx Direct electric field control of the skyrmion phase in a magnetoelectric insulator In addition, progress towards tuning the bulk skyrmion phase stability was also demonstrated using both applied uniaxial24,25 and hydrostatic pressure18. For possible applications however, the use of E fields to manipulate skyrmions and skyrmion phase stability in insulators offers several potential advantages for applications, yet this approach remains still little-explored exper- imentally6,8,23,26. Moreover, the E field control of the SkL phase stability remains an outstanding theoretical issue. yi y g Here we report a combined experimental and theoretical study of skyrmion phase stability under moderate E-fields (V/μm) in the model insulating skyrmion host Cu2OSeO3. We use the microscopic probe of small-angle neutron scattering (SANS) to show that in Cu2OSeO3, the extent of the skyrmion phase stability either expands or shrinks in both T and H, dependent on the E field polarity. Theoretically, we address the role of the E field using first order perturbation theory to evaluate the free energy of the underlying phases. We show how an applied E field causes a relatively small shift of the SkL free energy that is nonetheless commensurate with the mean-field free energy difference between the competing skyrmion and conical phases, and thus dramatically controls the SkL phase stability. For the quantitative description of the experimental phase diagram, we develop 1Department of Physics, Harvard University, Cambridge, MA, 02138, USA. 2Laboratory for Quantum Magnetism (LQM), Insititute of Physics, École Polytechnique Fédérale de Lausanne (EPFL), CH-1015, Lausanne, Switzerland. 3Laboratory for Neutron Scattering and Imaging (LNS), Paul Scherrer Institut (PSI), CH-5232, Villigen, Switzerland. 4Laboratory for Scientific Developments and Novel Materials (LDM), Paul Scherrer Institut (PSI), CH-5232, Villigen, Switzerland. 5Crystal Growth Facility, Insititute of Physics, École Polytechnique Fédérale de Lausanne (EPFL), CH- 1015, Lausanne, Switzerland. Correspondence and requests for materials should be addressed to A.J.K. (email: akruchkov@g.harvard.edu) Scientific REPOrts \| (2018) 8:10466 \| DOI:10.1038/s41598-018-27882-4 1 www.nature.com/scientificreports/ Figure 1. Skyrmion phase tuning by electric fields. (a–d) Representative SANS diffraction patterns obtained from the SkL phase at a temperature of 56.8 K, and various (H, E) conditions for E H [111]. Here the [111] direction is into the page. Starting with only a weak intensity from the SkL at T = 56.8 K and H = 17 mT (a nearly conical state) (a), applying a positive E field enhances the SkL stability as evidenced by a significantly enhanced SkL intensity (b). Results C t ll Controlling skyrmion phase stability using electric fields. From recent bulk susceptibility χ(H) measurements of Cu2OSeO3 23, it was suggested that skyrmions may be “created” or “annihilated” by applying a dc E-field in suitable parts of the temperature–magnetic field (T,H) phase diagram. In that study23 the skyrmion phase is identified by a small drop in χ(H), which serves as an indirect indication for the existence of the skyr- mion phase. We have used the tool of SANS to observe directly the microscopic skyrmionic magnetism in Cu2OSeO3 and its response to an applied dc E-field. In SANS the SkL phase is typically evidenced by a sixfold symmetric dif- fraction pattern, consistent with the so-called multi-q (triple-q) magnetic structure ansatz for the SkL described by three propagation q-vectors rotated by 120° with respect to each other (note that both ±q each give a SANS diffraction spot)6,14,27. In our SANS experiments we oriented the sample so that E\|\|H\|\| [111], since according to previous bulk measurements7, the effect of the E field is expected to be maximal in this geometry. By measuring with the neutron beam also along [111], scattered intensity is only observed due to the SkL phase; for both the conical phase with q\|\|H and the zero field helical phase with q\|\|{100} type directions6, the expected scattering lies well out of the detector plane.i Figure 1 summarizes the direct E-field control of the SkL phase stability in Cu2OSeO3. Representative SANS data collected at constant T = 56.8 K, and at (H,E) coordinates selected to emphasise the E field effect, are shown in Fig. 1a–d. The initial states at T = 56.8 K were always prepared after zero-field cooling. By applying H = 17 mT at E = 0, the system the system is located on the border of the SkL phase and mostly in the conical phase, as evi- denced by a weak SkL signal on the SANS detector (Fig. 1a). Without changing T or H, applying a positive E field of +5.0 V/μm leads to the appearance of the characteristic 6-fold diffraction pattern in the SANS image (Fig. 1b), demonstrating the creation of a well-developed SkL. Conversely, starting from the SkL state at H = 0 38 mT and E = 0 (Fig. 1c), application of a negative electric field E = −2.5 V/μm erases the SkL as evidenced by the disap- pearance of the SANS diffraction peaks (Fig. 1d). Direct electric field control of the skyrmion phase in a magnetoelectric insulator Conversely, starting from a well-developed SkL state at T = 56.8 K and H = 38 mT (c), applying a negative E field erases the SkL (d). For the systematic analysis of the SANS data shown later in Figs 1(e) and 2(a–c), we evaluate the entire SANS intensity due to all SkL domains within the annular integration windows denoted by white rings in (a–d). This allows us to cater for scattering due to the major SkL domain (six strongest spots), and also any minority domains present that are signified by weaker diffraction spots in-between the strong ones. (e) shows the extent of the skyrmion phase pockets in the phase diagram for various E fields as determined by SANS. The skyrmion pocket doubles in temperature extent under a positive E field of +5.0 V/μm, while shrinks to half its size at E = 0 under a negative E field of −2.5 V/μm. The white and yellow stars on figure (e) mark experimental conditions of SANS patterns (a–d) on the phase diagram. Figure 1. Skyrmion phase tuning by electric fields. (a–d) Representative SANS diffraction patterns obtained from the SkL phase at a temperature of 56.8 K, and various (H, E) conditions for E H [111]. Here the [111] direction is into the page. Starting with only a weak intensity from the SkL at T = 56.8 K and H = 17 mT (a nearly conical state) (a), applying a positive E field enhances the SkL stability as evidenced by a significantly enhanced SkL intensity (b). Conversely, starting from a well-developed SkL state at T = 56.8 K and H = 38 mT (c), applying a negative E field erases the SkL (d). For the systematic analysis of the SANS data shown later in Figs 1(e) and 2(a–c), we evaluate the entire SANS intensity due to all SkL domains within the annular integration windows denoted by white rings in (a–d). This allows us to cater for scattering due to the major SkL domain (six strongest spots), and also any minority domains present that are signified by weaker diffraction spots in-between the strong ones. (e) shows the extent of the skyrmion phase pockets in the phase diagram for various E fields as determined by SANS. Direct electric field control of the skyrmion phase in a magnetoelectric insulator The skyrmion pocket doubles in temperature extent under a positive E field of +5.0 V/μm, while shrinks to half its size at E = 0 under a negative E field of −2.5 V/μm. The white and yellow stars on figure (e) mark experimental conditions of SANS patterns (a–d) on the phase diagram. a new approach for treating the fluctuative part of the free energy contributed by quasiparticle modes around TC. The inclusion of these modes proves pivotal for the correct evaluation of the free energy difference between the competing skyrmion and conical phases, both with and without E field, and thus represents an improved approach more generally for the calculation of the skyrmion phase diagram. Scientific REPOrts \| (2018) 8:10466 \| DOI:10.1038/s41598-018-27882-4 Results C t ll The dotted lines in (c) are guides for the eye. (d,e) The H-dependence of the calculated free energy difference between skyrmion and conical phases at (d) T = 55.8 K and (e) T = 56.8 K, for various E-fields. (f) The T- dependence of the minima in the calculated free energy differences like those shown in (d,e). In (d–f) calculations for E = +5 kV/mm, E = 0 kV/mm, E = −5 kV/mm are shown in red, green, blue, respectively (asymmetric æ2 effects neglected). The skyrmion phase stability is preferred over the conical phase when the calculated free energy differences are negative. (blue) E fields. We find that a positive E field of +5.0 V/μm expands the skyrmion pocket so that it becomes twice larger (in temperature), while a negative E field of just E = −2.5 V/μm shrinks the pocket by a factor of two. These SANS results provide microscopic experimental evidence for the electric field control of the skyrmion phase. Moreover, the results are quantitatively consistent with those of the indirect measurements reported in ref.23 after rescaling the data to take into account the difference between the ranges of applied electric field explored in the two studies. Optimum conditions for stabilising and destabilising the skyrmion phase. Examining our SANS data more closely reveals the systematic manner by which applied E fields modify the SkL stability in the phase diagram. Figure 2a,b, show H-scans of the total scattered SANS intensity from the SkL for various E fields, and at T = 55.8 K and 56.8 K, respectively. At each T, the extent in H over which the SkL intensity is observed at E = 0 becomes enhanced under positive E fields, and suppressed under negative E fields. This demonstrates clearly the importance of the E field polarity on either enhancing or suppressing SkL stability. The effect is very pronounced as seen in Fig. 2a; at T = 55.8 K a negative E field can completely destabilise the SkL that is otherwise stable in the unperturbed state (E = 0). p Since the total scattered SANS intensity is indicative of both the population (relative to the conical phase) and quality of SkLs in the sample, the maximum intensity in the H scans like those shown in Fig. 2a,b is identified to represent the optimal SkL stability at each E and T. Results C t ll f Since in our experimental geometry it is only scattering from the SkL that contributes to the observed SANS intensity, we determined the extent of the SkL phase by evaluating the total intensity within the region of interest on the detector (the annular range within the white rings shown in Fig. 1a–d) as a function of T, H, and E. The resulting stability range of the skyrmion phase is shown in Fig. 1e for zero (green), positive (red) and negative Scientific REPOrts \| (2018) 8:10466 \| DOI:10.1038/s41598-018-27882-4 2 www.nature.com/scientificreports/ Figure 2. Skyrmion lattice stability in electric field: experiment and theory (a,b) E- and H-field dependence of the total scattered SANS intensity from SkLs in the sample at (a) T = 55.8 K, and (b) T = 56.8 K. The intensity evaluated is that observed within the annular sectors defined by white rings in Fig. 1a–d. (c) The E- and T- dependence of the peak SANS intensity in the H-scans like those shown in (a,b). In (a–c), symbols and lines in red, green and blue denote data obtained under E = +5kV/mm, E = 0 and E = −2.5 kV/mm, respectively. The dotted lines in (c) are guides for the eye. (d,e) The H-dependence of the calculated free energy difference between skyrmion and conical phases at (d) T = 55.8 K and (e) T = 56.8 K, for various E-fields. (f) The T- dependence of the minima in the calculated free energy differences like those shown in (d,e). In (d–f) calculations for E = +5 kV/mm, E = 0 kV/mm, E = −5 kV/mm are shown in red, green, blue, respectively (asymmetric æ2 effects neglected). The skyrmion phase stability is preferred over the conical phase when the calculated free energy differences are negative. Figure 2. Skyrmion lattice stability in electric field: experiment and theory (a,b) E- and H-field dependence of the total scattered SANS intensity from SkLs in the sample at (a) T = 55.8 K, and (b) T = 56.8 K. The intensity evaluated is that observed within the annular sectors defined by white rings in Fig. 1a–d. (c) The E- and T- dependence of the peak SANS intensity in the H-scans like those shown in (a,b). In (a–c), symbols and lines in red, green and blue denote data obtained under E = +5kV/mm, E = 0 and E = −2.5 kV/mm, respectively. Results C t ll We present the T-dependence of this intensity peak at each E field in Fig. 2c, along with T windows identified as optimal for either enhancing or suppressing the SkL stability using the E field. Within the T window of 53–55.5 K, positive E fields enhance the SkL phase stability relative to the case at E = 0, while in the T-window of 55–56.5 K the SkL phase stability is readily suppressed by the negative E field relative to the E = 0 case. Positioning a sample at 55.5 K allows the demonstration of either a significant enhancement or suppression of the skyrmion phase at E = 0 by using E-fields of opposite polarity. In what follows, we develop a theory capable of explaining these observations quantitatively. Scientific REPOrts \| (2018) 8:10466 \| DOI:10.1038/s41598-018-27882-4 3 www.nature.com/scientificreports/ Free energy in electric fields. The underlying mechanism for the E field driven enhancement or suppres- sion of the skyrmion phase stability relies upon the magnetoelectric (ME) coupling in insulating Cu2OSeO3, which microscopically originates from the d-p hybridisation mechanism27–31. The emergent electric dipole moment P = λ(SySz, SzSx, SxSy) is generated by the underlying spin structure S(r) = (Sx, Sy, Sz), with the coupling between the magnetic and electric degrees of freedom described by a ME coupling parameter λ of relativistically small magnitude. Crucially, because the skyrmion phase now carries a non-vanishing electric-dipole moment, the ME coupling results in a P·E shift of the skyrmion free energy in E-field. This perturbation renormalises the elementary helices upon which the skyrmion phase is built, and slightly distorts the SkL6,32.f y p y p g y In this work, we apply the ME perturbation to the free energy described by an effective Ginsburg-Landau functional with Dzyaloshinski-Moriya interaction (DMI), and consider carefully the critical fluctuations that favour the skyrmion phase with respect to the competing conical phase (see Methods). Due to the relativistically small size of λ, the dimensionless E-field itself is rather small so that,  E Dk æ /4 1 0 λ = , and we can build a perturbation theory in æ; for the modified free energy, neglecting all the terms of order æ2 and higher. Our finding is that perturbations of the fluctuative terms are important only at second order, while the mean-field energy already shifts in the first order due to direct ME and nonlinear contributions (see Methods). Results C t ll The corresponding shift in free energy of the skyrmion phase depends on the direction of E-field (see Fig. 2d–f), thus either enhanc- ing (E > 0) or suppressing (E < 0) the skyrmion phase stability. While at first sight it can be surprising that the perturbation due to only a moderate applied E-field can play such a crucial role here, this is facilitated by the very close competition between the skyrmion and conical phases already in the mean-field. Calculation of the phase diagram. To calculate the response of the phase diagram to applied E-field, we use a new approach developed on the basis of effective models presented in refs6,14,33 (see Methods). Compared with these earlier studies, the new approach is self-consistent in the way that it reproduces the phase diagram, pro- vides a deeper understanding of the role of quasiparticle modes near TC, and includes the path-integral approach presented previously for calculating the fluctuative free energy14 as a limiting case. We thus treat the first-order perturbation in E field on top of the mean-field solution, and add the fluctuative contributions that stabilise the SkL in the bulk. The main contribution of the E field here is captured by the shift of the mean-field free energy dif- ference between the SkL and conical phases, while the fluctuative shift under voltage remains quadratically small.if plt g q y Figure 2d,e each show calculations of the H and E field-dependent free energy difference between the skyr- mion and conical phases, at T = 55.8 K and T = 56.8 K, respectively. In general, the calculations show the minima of the free energy difference curves deepen with increasingly positive E field. At T = 55.8 K changing between sufficiently negative and positive E fields can shift the curve so that the conical phase favoured for all H in a negative E field becomes unstable towards the skyrmion phase formation in the positive E field. This result is con- sistent with the experimental data shown in Fig. 2a. We also identify semi-quantitative agreement between these calculations and our experiments; the H location of the minima in the calculated free-energy difference curves [Fig. 2d,e] correspond well to the values of H where the peaks of SANS intensity are observed in the scans shown in Fig. 2a,b. Results C t ll In addition, we can link the calculated T and E field dependence of the free energy difference min- ima shown in Fig. 2f to our experimental data. The T s at which the free energy difference between the SkL and conical phases vanish for the theoretical E fields correspond to the T windows identified from the experimental data as optimal for either enhancing or suppressing SkL stability with E [Fig. 2c]. This correspondence between experiment and theory provides firm support for the validity of our theoretical treatment of the E-field effect on the SkL phase stability.i p y Independent of any applied E field, our theory provides a more general understanding of SkL stability on an intuitive, pictorial level: the critical fluctuations (waves) are superposed on top of the variationally minimised free energies. There are three critical modes k (0,1,2) ω around the mean-field (see Methods), with k (0) ω soft on the sphere = k k 0, which means that it costs very little energy to add many such fluctuations if they are coherent with the helix k0. Thus ωk (0) 0 is the so-called “dangerous” mode since it results in a Van-Hove-like singularity at TC and eventually breaks down the ordered phases into the disordered (paramagnetic) phase33. Below TC the symmetry-breaking can be observed using SANS by either a six-fold pattern (SkL phase) or a two-fold pattern (helical or conical phases), both circumscribed on a sphere of radius = k k 0 in reciprocal space. Our calculation shows that the skyrmion phase is favoured because adding fluctuations generates more entropy in the skyrmion phase. This analysis also leads to a qualitative criterion for capturing the magnetic-field-independent breakdown of the ordered phases at TC (see Methods). Asymptotically, the main contribution of the fluctuative free energy is given in the short-scale physics, where Cu2OSeO3 is “almost” a ferromagnet, thus reproducing the result of the path-integral approach14 as a limiting case. The model described here captures the qualitative physics of the sys- tem, as exemplified by the theoretical phase diagram shown in Fig. 3. To date, a quantitative theory for the skyr- mion phase diagram under electric fields has been missing. Discussion I In some respects, the effect of E field observed here resembles that achieved due to applied uniaxial pressure24,25 since, the SkL phase stability can be either enhanced or suppressed by appropriate selection of the uniaxial stress direction relative to the direction of H. However, integrating the pressure effect on skyrmion stability into a technological setting is very challenging. In contrast, the E field is a versatile and reliable external parameter; providing an efficient control of both the skyrmion position6,8,26 and, as we show here the stability of the phase as a whole. Since the E field controlled expansion or contraction of the skyrmion phase can occur in general for an insulating ME skyrmion host with any TC, our findings are very attractive for applications at room T; for a device layer of thickness 100 nm the skyrmion phase in a sample can be entirely destabilised (erased) or restabilised (written) with less than 1 V, a voltage compatible with modern microelectronics. Scientific REPOrts \| (2018) 8:10466 \| DOI:10.1038/s41598-018-27882-4 4 www.nature.com/scientificreports/ t c epo ts/ Figure 3. Calculation of the skyrmion phase in electric fields Calculated H and T extent of the skyrmion phase for E > 0 (red shading), E = 0 (green shading), E < 0 (blue shading) for the E H [111] geometry, and for the symmetric-response approach (æ ) 1 . The extent of each phase corresponds to the range over which the free energy of the skyrmion phase is less than that of the surrounding conical phase. Dashed lines denote phase boundaries, including the vertical breakdown regime at TC. Figure 3. Calculation of the skyrmion phase in electric fields Calculated H and T extent of the skyrmion phase for E > 0 (red shading), E = 0 (green shading), E < 0 (blue shading) for the E H [111] geometry, and for the symmetric-response approach (æ ) 1 . The extent of each phase corresponds to the range over which the free energy of the skyrmion phase is less than that of the surrounding conical phase. Dashed lines denote phase boundaries, including the vertical breakdown regime at TC. The present study further lays both theoretical and experimental foundations for fully exploring alternative H and E field configurations, not only in reciprocal-space measurements like SANS, but also by real-space imag- ing techniques such as cryo-Lorentz transmission electron microscopy (LTEM) on technologically-relevant, nanometrically-thin specimens. Methods Small-angle neutron scattering (SANS). For the SANS experiment, we used a single crystal crystal grown using chemical vapour transport34. The crystal with a TC = 58 K was of mass 6 mg and volume 3.0 × 2.0 × 0.50 mm3 with the thinnest axis \|\|[111], and [112] vertical. The sample was mounted onto a bespoke sample stick designed for applying dc E-fields35, and oriented with the orthogonal [112] and [110] directions lying in the SANS scattering plane. For the SANS images shown in Fig. 1, the [112] direction is aligned with the vertical axis, and the [110] direction aligned with the horizontal axis. In our experiments we achieved E-fields ranging from +5.0 kV/mm to −2.5 kV/mm. Evidence of electrical breakdown was detected for E-fields outside this range.hih i g The sample was loaded into a horizontal field cryomagnet at the SANS-II beamline, SINQ, PSI. The magnetic field (μ0H) was applied parallel to both the [111] direction of the sample and the incident neutron beam to give the experimental geometry E\|\|μ0H\|\|[111]. In this geometry, the SANS signal is only detected from the skyrmion phase, which typically presents as a hexagonal scattering pattern with propagation vectors q ⊥μ0H. In this geom- etry, we avoid detecting any SANS signal due to either of the neighbouring helical (q \|\|{100}) or conical phases (q \|\|μ0H), since the propagation vectors of these phases lie well out of the SANS detector plane. Åh \|\|μ0 p p g p p We used incident neutrons with a wavelength of 10.8 Å (Δλ/λ = 10%). The scattered neutrons were detected using a position-sensitive multidetector. The SANS measurements were done by rotating (‘rocking’) the sample and cryomagnet ensemble over angles that brought the various SkL diffraction spots onto the Bragg condition at the detector. Data taken at 70 K in the paramagnetic state were used for background subtraction. Before start- ing each μ0H-scan, the sample was initially zero field-cooled from 70 K to a target temperature, with the E-field applied when thermal equilibrium was achieved. The E-field was maintained during the μ0H-scan. At each T we define the μ0H extent of the SkL phase as that over which SANS intensity is detected. We use this criterion to extract the parametric extent of the SkL phase for (μ0H,T,E) as shown in Figs 1 and 2. See Supplemental Material for more details. Mean-field free energy. Discussion I Crucial next steps experimentally concerning the skyrmion writing and erasing includes exploring directly the expected E field-driven switching hysteresis between the competing conical and skyrmion states, and the associated volatility of remnant states in zero biasing E field. In addition, learning how E field influences out-of-equilibrium and metastable skyrmion configurations in confined geometries can provide progressive insights for assessing the merits of insulating skyrmions for practical uses. g g g g y In conclusion, we have demonstrated both theoretically and experimentally the mechanism by which a mod- erate electric field can either enhance or suppress skyrmion phase stability in the magnetoelectric chiral magnet Cu2OSeO3. In addition, we have provided the parameters by which our theoretical approach achieves quantitative agreement with experiment, and which can be extended towards describing the effect of E field on both stable and metastable skyrmion states, these being of paramount technological importance. Scientific REPOrts \| (2018) 8:10466 \| DOI:10.1038/s41598-018-27882-4 Methods The effective mean-field theory is based on the coarse-grained magnetisa approach = M M r S r ( ) ( ) s as described in14. One starts with the mean-field approach with free energy = Θ + ∇ + ⋅∇× + − ⋅ F J D U M M M M M M H M [ ] ( ) ( ) (1) T 2 2 4 (1) with spatial average ∫ 〈...〉= ... dV V , and α Θ ∝ − T T ( ) T C near TC, J is the Heisenberg stiffness and D is DMI, H is the magnetic field, and the higher-order term U grants the formation of the crystalline phase14. In the mean-field, with spatial average ∫ 〈...〉= ... dV V , and α Θ ∝ − T T ( ) T C near TC, J is the Heisenberg stiffness and D is DMI, H is he magnetic field, and the higher-order term U grants the formation of the crystalline phase14. In the mean-field Scientific REPOrts \| (2018) 8:10466 \| DOI:10.1038/s41598-018-27882-4 5 www.nature.com/scientificreports/ the interplay between Heisenberg and DMI energies determines the helical vector as k0 = D/2J. The long- range-ordered hexagonal skyrmion lattice is approximated as  μ + ∑ + . . φ + e S r m S ( ) c c i i q q q r n n n n , where the summation runs over the magnetocrystalline wave vectors q1 + q2 + q3 = 0. In the mean-field, the skyrmion phase is slightly gapped with respect to the conical phase, however the two are closely competing. Further details of the mean-field theory described in ref.14. the interplay between Heisenberg and DMI energies determines the helical vector as k0 = D/2J. The long- range-ordered hexagonal skyrmion lattice is approximated as  μ + ∑ + . . φ + e S r m S ( ) c c i i q q q r n n n n , where the summation runs over the magnetocrystalline wave vectors q1 + q2 + q3 = 0. In the mean-field, the skyrmion phase is slightly gapped with respect to the conical phase, however the two are closely competing. Further details of the mean-field theory described in ref.14. Perturbation theory in electric fields. Methods The magneto-electric coupling in Cu2OSeO3 is relativistically small, so the perturbation parameter is E Dk æ /4 1 0  λ = . It is sufficient to use the first order perturbation the- ory on top of the non-perturbed free energy. We go to the rotated frame defined by the magnetic field direction along [111], and re-write the free energy. The first order perturbation theory gives eigenvectors: ˆH O S S S S S (æ ), (2) n n n n k k k k k k k (æ) (0) 0 ( ) ( ) (æ) (0) (0) ( ) 2 ∑ ε ε = + 〈 \| − + ≠ (2) which are now the basis for constructing the distorted skyrmion lattice. For other (H, E)-field configurations, we re-do the calculations in the new rotated frames. See Supplemental Materials for further details. Fluctuation-induced phase stabilisation. We use a new approach, which captures as a limiting case the fluctuation free energy from14. The essential physics is captured already in Gaussian (noninteracting) fluctuations with free energy density ∑∑ω = − \| \|<Λ F f T S , (3) i i i k k k k fluct ( ) ( ) fluct (3) where Λ = 2π/a is the natural cut-off, f i k ( ) is the critical modes distribution, and the entropy of Gaussian fluctua- tions is where Λ = 2π/a is the natural cut-off, f i k ( ) is the critical modes distribution, and the entropy of Gaussian fluctua- tions is ∑∑ = + + − \| \|<Λ f f f f S {(1 )ln(1 ) ln } (4) i i i i i k k k k k k fluct ( ) ( ) ( ) ( ) (4) in the case of bosons. Fluctuations around mean-field are described by the generalised susceptibility χ ′ = δ δ δ − ′ r r ( , ) ij T F M M r r 1 1 ( ) ( ) i j 2 , giving rise to several collective modes (See Supplemental Material). On the local scale  k J D ( / ), the chiral magnet is reminiscent of a ferromagnet, so the modes behave asymptotically ωk ∝ k2 for large k, thus asymptotically F k log log k fluct 2  βω ∝ , which covers the model of ref.14. Methods The main contribution to (5) is given by the short length-scale (“ferromagnetic”) physics,  F U Da T S S 10 , (5) fluct SkL 2 con 2 π Δ − (5) The electric field also slightly affects the fluctuative energy, because it modifies the correlation length near TC and so renormalises Jeff, which is neglected here as a higher-order (æ ) 2 effect. See [Supplemental Material] for further details. Parameters of the effective model. For our numerical calculations we use TC = 58 K, which approximately sets the Heisenberg stiffness as J = 4.85 × 10−23 Jm/A2. From the SANS measurement we establish directly the modu- lation period of 60 nm, which estimatively differs by a few percents from the mean-field value 2π/k0, because the mean-field ordering vector k0 = D/2J is slightly renormalised by the fluctuations near TC. This sets the “bare” DM interaction entering (1) as D = −9.85 × 10−15 J/A2. The lattice parameter is a = 8.91 × 10−10 m, which gives the natural cutoff Λ = 2π/a ≈ 70k0. The saturation magnetization in Cu2OSeO3 is Ms = 1.11 × 105 A/m and scales with temperature as = − α α M T M T T ( ) (1 ( / ) ) s s C 1 2, with α1 = 1.95 and α2 = 0.39336. We choose the nonlinear coupling responsible for SkL formation U = 6.2 × 10−6 Jm−1 A−2 and Landau parameter Jk T T / ( ) 3 5 K T T C 0 2 m f 1 α θ = − = . . . −. For the qualita- tive phase diagram shown in Fig. 3, we use a symmetric-response model (\ae ) 1 , for which the best fit to SANS data is for = . \ae 0 02 1 , which corresponds here to E = ±5 × 106 V/m coupled with λ/Dk0 = 9.23 × 10−3 μm/V to the underly- ing spin structure through ME mechanism. Scientific REPOrts \| (2018) 8:10466 \| DOI:10.1038/s41598-018-27882-4 References 1. Fert, A., Cros, V. & Sampaio, J. Skyrmions on the track. Nat. Nanotech. 8(3), 152–156 (2013). 1. Fert, A., Cros, V. & Sampaio, J. Skyrmions on the track. Nat. Nanotech. 8(3), 152–156 (2013). 1. Fert, A., Cros, V. & Sampaio, J. Skyrmions on the track. Nat. Nanotech. 8(3), 152–156 (2013 2. Nagaosa, N. & Tokura, Y. Topological properties and dynamics of magnetic skyrmions. Nat. Nanotech. 8(12), 899–911 (2013). ll l f h d f k k h l 2. Nagaosa, N. & Tokura, Y. Topological properties and dynamics of magnetic skyrmions. Nat. Nanotech. 8(12), 899–911 (2013). 3. Tomasello, R. et al. A strategy for the design of skyrmion racetrack memories. Sci. Rep. 4, 6784, https://www.nature.com/articles/ 2. Nagaosa, N. & Tokura, Y. Topological properties and dynamics of magnetic skyrmions. Nat. Nanotech. 8(12), 899–911 (2013). 3. Tomasello, R. et al. A strategy for the design of skyrmion racetrack memories. Sci. Rep. 4, 6784, https://www.nature.com/articles srep0678410.1038/srep06784 (2014). 4. Zhang, X. et al. Skyrmion-skyrmion and skyrmion-edge repulsions in skyrmion-based racetrack memory. Sci. Rep. 5, 7643, https:// www.nature.com/articles/srep0764310.1038/srep07643 (2015). 4. Zhang, X. et al. Skyrmion-skyrmion and skyrmion-edge repulsions in skyrmion-based racetrack memory. Sci. Rep. 5, 7643, https:// www.nature.com/articles/srep0764310.1038/srep07643 (2015). 4. Zhang, X. et al. Skyrmion-skyrmion and skyrmion-edge repulsions in skyrmion-based racetrack memory. Sci. Rep. 5, 7643, https:/ www.nature.com/articles/srep0764310.1038/srep07643 (2015). p p 5. Zhang, X., Ezawa, M. & Zhou, Y. Magnetic skyrmion logic gates: conversion, duplication and merging of skyrmions. Sci. Rep. 5 9400, https://www.nature.com/articles/srep0940010.1038/srep09400 (2015).i p p 5. Zhang, X., Ezawa, M. & Zhou, Y. Magnetic skyrmion logic gates: conversion, duplication and merging of skyrmions. Sci. Rep. 5, 9400, https://www.nature.com/articles/srep0940010.1038/srep09400 (2015).i p p 5. Zhang, X., Ezawa, M. & Zhou, Y. Magnetic skyrmion logic gates: conversion, duplication and merging of skyrmions. Sci. Rep. 5, 9400, https://www.nature.com/articles/srep0940010.1038/srep09400 (2015).i p p p ( ) White, J. S. et al. Electric-field-induced skyrmion distortion and giant lattice rotation in the magnetoelectric insulator Cu2OSeO3. hys. Rev. Lett. 113(10), 107203 (2014). p p p 6. White, J. S. et al. Electric-field-induced skyrmion distortion and giant lattice rotation in the magnetoelectric insulator Cu2OS Phys. Rev. Lett. 113(10), 107203 (2014). y 7. Seki, S., Yu, X. Z., Ishiwata, S. & Tokura, Y. Observation of skyrmions in a multiferroic material. Science 336(6078), 198–201 (2012). 8 White J S et al Electric field control of the skyrmion lattice in Cu OSeO J Phys Condens Matter 24(43) 432201 (2012) y 7. Seki, S., Yu, X. References Microscopic theory of spin-polarization coupling in multiferroic transitio Phys. Rev. B 76(14), 144424 (2007). y ( ) ( ) 30. Arima, T. H. Ferroelectricity induced by proper-screw type magnetic order. J. Phys. Soc. Jpn. 76, 073702 (2007).f 31. Belesi, M. et al. Magnetoelectric effects in single crystals of the cubic ferrimagnetic helimagnet Cu2OSeO3. Phys. Rev. B 85(22), 224413 (2012). 31. Belesi, M. et al. Magnetoelectric effects in single crystals of the cubic ferrimagnetic he 224413 (2012). ( ) 32. Kruchkov, A. J. & Rønnow, H. M. Preprint at https://arxiv.org/abs/1702.08863 (2017).i 32. Kruchkov, A. J. & Rønnow, H. M. Preprint at https://arxiv.org/abs/1702.08863 (2017).i 32. Kruchkov, A. J. & Rønnow, H. M. Preprint at https://arxiv.org/a 33. Janoschek, M. et al. Fluctuation-induced first-order phase transition in Dzyaloshinskii-Moriya helimagnets. Phys. Rev. B 87(13), 134407 (2013). 34. Belesi, M. et al. Ferrimagnetism of the magnetoelectric compound Cu2OSeO3 probed by 77Se NMR. Phys. Rev. B 82(9), 09 (2010). 34. Belesi, M. et al. Ferrimagnetism of the magnetoelectric compound Cu2OSeO3 probed by 77Se NMR. Phys. Rev. B 82(9), 094422 (2010). 35 B tk i k M Whit J S R H M & P š K N t V til l ti k f t tt i i t i hi h (2010). 35. Bartkowiak, M., White, J. S., Rønnow, H. M. & Prša, K. Note: Versatile sample stick for neutron scattering experiments in high i ( ) 35. Bartkowiak, M., White, J. S., Rønnow, H. M. & Prša, K. Note: Versatile sample stick for neutron scattering experiments in electric fields. Rev. Sci. Instrum. 85, 026112 (2014). i ( ) 36. Živković, I. et al. Two-step transition in a magnetoelectric ferrimagnet Cu2OSeO3. Phys. Rev. B 85(22), 224402 (2012). References Precursor phenomena at the magnetic ordering of the cubic helimagnet FeGe. Phys. Rev. Lett. 107, 127203 (2011). 20. Mochizuki, M. & Watanabe, Y. Writing a skyrmion on multiferroic materials. Appl. Phys. Lett. 107(8), 082409 (2015). 19. Wilhelm, H. et al. Precursor phenomena at the magnetic ordering of the cubic helimagnet FeGe. Phys. Rev. Lett. 107, 127203 (2011). 20. Mochizuki, M. & Watanabe, Y. Writing a skyrmion on multiferroic materials. Appl. Phys. Lett. 107(8), 082409 (2015). 20. Mochizuki, M. & Watanabe, Y. Writing a skyrmion on multiferroic materials. Appl. Phys. Lett. 107(8), 082409 (2015). 20. Mochizuki, M. & Watanabe, Y. Writing a skyrmion on multiferroic materials. Appl. Phys. Lett. 107(8), 082409 (2015). 21. Mochizuki, M. Creation of Skyrmions by Electric Field on Chiral Lattice Magnetic Insulators. Adv. Electron. Mater. 2(1), https:/ org/10.1002/aelm.20150018010.1002/aelm.201500180 (2016).i g adhyaya, P., Yu, G., Amiri, P. K. & Wang, K. L. Electric-field guidin 22. Upadhyaya, P., Yu, G., Amiri, P. K. & Wang, K. L. Electric-field guiding of magnetic skyrmions. Phys. Rev. B 92(13), 134411 (2015). 23 Okamura, Y , Kagawa, F , Seki, S and Tokura, Y Transition to and from the skyrmion lattice phase by electric fields in a 22. Upadhyaya, P., Yu, G., Amiri, P. K. & Wang, K. L. Electric-field guiding of magnetic skyrmions. Phys. Rev. B 92(13), 134411 (2015). k k d k d f h k l h b l f ld 23. Okamura, Y., Kagawa, F., Seki, S. and Tokura, Y. Transition to and from the skyrmion lattice phase by electric fields in a magnetoelectric compound. Nat. Commun. 7, 12669, https://www.nature.com/articles/ncomms1266910.1038/ncomms12669 (2016). g y p y magnetoelectric compound. Nat. Commun. 7, 12669, https://www.nature.com/articles/ncomms1266910.1038/ncomms12669 (2016). ( ) 24. Chacon, A. et al. Uniaxial pressure dependence of magnetic order in MnSi. Phys. Rev. Lett. 115(26), 267202 (2015). 25. Nii, Y. et al. Uniaxial stress control of skyrmion phase. Nat. Commun. 6, 8539, https://www.nature.com/articles/ncomms95391 ncomms9539 (2015). 26. Omrani, A. A. et al. Exploration of the helimagnetic and skyrmion lattice phase diagram in Cu2OSeO3 using magnetoele susceptibility. Phys. Rev. B 89(6), 064406 (2014). 27. Seki, S., Ishiwata, S. & Tokura, Y. Magnetoelectric nature of skyrmions in a chiral magnetic insulator Cu2OSeO3. Phys. Rev. B 060403 (2012). ( ) 28. Liu, Y. H., Li, Y. Q. & Han, J. H. Skyrmion dynamics in multiferroic insulators. Phys. Rev. B 87(10), 100402 (2013). 29. Jia, C., Onoda, S., Nagaosa, N. & Han, J. H. Acknowledgements g We thank Achim Rosch, Naoto Nagaosa and Jiadong Zang for fruitful discussions. The work was supported by the Swiss National Science Foundation, its Sinergia networks “NanoSkyrmionics” and “Mott Physics Beyond the Heisenberg Model (MPBH)”, and projects 153451, 166298 and P2ELP2_175278. Neutron scattering experiments were carried out at the Swiss Spallation Neutron Source (SINQ), Paul Scherrer Institut, Switzerland. g We thank Achim Rosch, Naoto Nagaosa and Jiadong Zang for fruitful discussions. The work was supported by the Swiss National Science Foundation, its Sinergia networks “NanoSkyrmionics” and “Mott Physics Beyond the Heisenberg Model (MPBH)”, and projects 153451, 166298 and P2ELP2_175278. Neutron scattering experiments were carried out at the Swiss Spallation Neutron Source (SINQ), Paul Scherrer Institut, Switzerland. References Z., Ishiwata, S. & Tokura, Y. Observation of skyrmions in a multiferroic material. Science 336(6078), 198–201 (2012). 8. White, J. S. et al. Electric field control of the skyrmion lattice in Cu2OSeO3. J. Phys. Condens. Matter. 24(43), 432201 (2012). 9. Jonietz, F. et al. Spin transfer torques in MnSi at ultralow current densities. Science 330(6011), 1648–1651 (2010). y 7. Seki, S., Yu, X. Z., Ishiwata, S. & Tokura, Y. Observation of skyrmions in a multiferroic material. Science 336(6078), 198–201 (2012). 8. White, J. S. et al. Electric field control of the skyrmion lattice in Cu2OSeO3. J. Phys. Condens. Matter. 24(43), 432201 (2012). 9 Jonietz F et al Spin transfer torques in MnSi at ultralow current densities Science 330(6011) 1648 1651 (2010) Scientific REPOrts \| (2018) 8:10466 \| DOI:10.1038/s41598-018-27882-4 6 www.nature.com/scientificreports/ 10. Yu, X. Z. et al. Skyrmion flow near room temperature in an ultralow current density. Nat.Commun. 3, 988 (2012). yl y 11. Jiang, W. et al. Blowing magnetic skyrmion bubbles. Science 349(6245), 283–286 (2015).i 12. Everschor, K. et al. Rotating skyrmion lattices by spin torques and field or temperature gradients. Phys. Rev. B. 86(5), 054432 (2 3. Mochizuki, M. et al. Thermally driven ratchet motion of a skyrmion microcrystal and topological magnon Hall effect. Nat. Mater 13(3), 241 (2014). ( ) ( ) 14. Mühlbauer, S. et al. Skyrmion lattice in a chiral magnet. Science 323(5916), 915–919 (2009). 14. Mühlbauer, S. et al. Skyrmion lattice in a chiral magnet. Science 323(5916), 915–919 (2009). al. Skyrmion lattice in the doped semiconductor Fe1xCoxSi. Phys. R 15. Münzer, W. et al. Skyrmion lattice in the doped semiconductor 16. Tokunaga, Y. et al. A new class of chiral materials hosting magnetic skyrmions beyond room temperature. Nat. Commun. 6, 7 https://www.nature.com/articles/ncomms863810.1038/ncomms8638 (2015). p 7. Adams, T. et al. Long-wavelength helimagnetic order and skyrmion lattice phase in Cu2OSeO3. Phys. Rev. Lett. 108(23), 237204 (2012). 18. Levatić, I. et al. Dramatic pressure-driven enhancement of bulk skyrmion stability. Sci. Rep. 6, 21347, https://www.nature.com/ articles/srep2134710.1038/srep21347 (2016). p p lm, H. et al. Precursor phenomena at the magnetic ordering of the cubic helimagnet FeGe. Phys. Rev. Lett. 107, 127203 (2011). izuki M & Watanabe Y Writing a skyrmion on multiferroic materials Appl Phys Lett 107(8) 082409 (2015) p p 19. Wilhelm, H. et al. Precursor phenomena at the magnetic ordering of the cubic helimagnet FeGe. Phys. Rev. Lett. 107, 127203 (2 19. Wilhelm, H. et al. Author Contributions A.J.K. performed calculations, J.S.W. performed SANS measurements, H.M.R. planned the project. A.J.K., J.S.W., H.M.R. interpreted results. A.M. provided crystals for SANS, M.B. and I.Z. provided technical support. A.J.K., J.S.W., H.M.R. wrote the manuscript. A.J.K. performed calculations, J.S.W. performed SANS measurements, H.M.R. planned the project. A.J.K., J.S.W., H.M.R. interpreted results. A.M. provided crystals for SANS, M.B. and I.Z. provided technical support. A.J.K., J.S.W., H.M.R. wrote the manuscript. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-27882-4. Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-27882-4. Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-27882-4. Competing Interests: The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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 Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted 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 license, visit http://creativecommons.org/licenses/by/4.0/. 7 Scientific REPOrts \| (2018) 8:10466 \| DOI:10.1038/s41598-018-27882-4
https://openalex.org/W2477696513	http://eprints.whiterose.ac.uk/106552/1/radwapaper.pdf	English	null	The influence of intercalating perfluorohexane into lipid shells on nano and microbubble stability	Soft matter	2,016	cc-by	8,656	a Molecular and Nanoscale Physics Group, School of Physics and Astronomy, University of Leeds, LS2 9JT, UK. E-mail: s.d.evans@leeds.ac.uk b Biophysics Group, Department of Physics, Faculty of Science, Mansoura University, Egypt c Leeds Institute of Molecular Medicine, Wellcome Trust Brenner Building, St. James’s University Hospital, Leeds, LS9 7TF, UK d School of Chemistry, University of Leeds, LS2 9JT, UK † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c6sm00956e Open Access Article. Published on 28 July 2016. Downloaded on 24/10/2016 11:42:11. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Radwa H. Abou-Saleh,ab Sally A. Peyman,a Benjamin R. G. Johnson,a Gemma Marston,c Nicola Ingram,c Richard Bushby,d P. Louise Coletta,c Alexander F. Markhamc and Stephen D. Evans*a Microbubbles are potential diagnostic and therapeutic agents. In vivo stability is important as the bubbles are required to survive multiple passages through the heart and lungs to allow targeting and delivery. Here we have systematically varied key parameters aﬀecting microbubble lifetime to significantly increase in vivo stability. Whilst shell and core composition are found to have an important role in improving microbubble stability, we show that inclusion of small quantities of C6F14 in the microbubble bolus significantly improves microbubble lifetime. Our results indicate that C6F14 inserts into the lipid shell, decreasing surface tension to 19 mN m1, and increasing shell resistance, in addition to saturating the surrounding medium. Surface area isotherms suggest that C6F14 incorporates into the acyl chain region of the lipid at a high molar ratio, indicating B2 perfluorocarbon molecules per 5 lipid molecules. The resulting microbubble boluses exhibit a higher in vivo image intensity compared to commercial compositions, as well as longer lifetimes. Received 22nd April 2016, Accepted 28th July 2016 Received 22nd April 2016, Accepted 28th July 2016 Soft Matter, 2016, 12, 7223--7230 \| 7223 This journal is ©The Royal Society of Chemistry 2016 1 Introduction Furthermore, such MBs should not only be stable in vitro but also in vivo, where they are required to survive multiple pas- sages through the heart and lungs for successful diagnostic imaging, and possibly longer for therapeutic applications requiring the effective targeting and release of drug payloads. Control over the in vitro and in vivo stability (or lifetime) of MBs depends upon three main considerations; (i) the condi- tions of the surrounding medium, such as the temperature, pressure and concentration of the dissolved gas, (ii) the MB shell composition (which controls the surface tension and the resistance to permeation) and (iii) the solubility of the encapsulated gas core in the medium.23 In 1950 Epstein and Plesset introduced a model describing the rate of growth and/or dissolution of ‘‘shell-less’’ or ‘‘uncoated’’ bubbles in aqueous media and showed them to be critically dependent on the diﬀusion of gas away from the bubble surface, Dw, the degree of saturation of the solution, f (=C0/Cs), and the initial bubble radius, r (Fig. 1). C0 is the concentration of dissolved gas and Cs is the concentration at saturation.24 More recently Borden and Longo suggested a modified version of the Epstein–Plesset model, as shown in eqn (1), that included the effect of the MB coating as a barrier against gas diffusion, Rshell, and a modifier of the surface tension, s.25 dr dt ¼ H r Dw þ Rshell 1 þ 2s Par f 1 þ 4s 3Par ! (1) (1) (1) where H is the Ostwald coefficient for the gas (the ratio of the gas concentration in the aqueous phase to that in the gas phase in contact with the aqueous phase).26,27 The Laplace pressure, DP, places the gas core under increased pressure due to surface tension and curvature eﬀects and thus provides a strong driving force for MB dissolution.28 The inclusion of surfactants, in our case lipids, at the gas/liquid interface significantly reduces the surface tension and, con- sequently, the Laplace pressure.29,30 Such surfactants also provide a resistive barrier, Rshell, to gas leaving the MB and dissolving into the aqueous phase. 1 Introduction introduced a rapid pressure-drop on a chip technique, which led to the production of nano and micron sized bubbles that were typically less than 2 mm in diameter, at high concentrations (41010 and 108 MB mL1 respectively).22 Post production MBs are prone to fusion and Soft Matter, 2016, 12, 7223--7230 \| 7223 This journal is ©The Royal Society of Chemistry 2016 Soft Matter View Article Online View Article Online View Article Online Paper Soft Matter Soft Matter hardening, and resistance to gas transport.30–32 Borden and Longo25 showed that MB stability is strongly dependent on the lipid shell resistance and increases with the increasing acyl chain length of the lipids.33–36 They also showed that increasing the acyl chain length between DPPC (16) and DBPC (24) increases the lipid rigidity (Wrinkling threshold) and con- sequently the MB stability and circulation time. The use of higher molecular weight, less soluble gases such as SF6 and perfluoro- carbons (PFCs) also significantly enhances MB lifetime and permits diagnostic and therapeutic applications.5,23,27,37 Several groups have investigated the effect of different gases, or gas mixtures, on MB stability. Sarker et al.27,38 developed a modified EP model for gas diffusion from MBs filled with air and PFC gas and showed that the dependence of shell permeability to gas type predicts a 500 increase in the time taken for MB dissolution for PFC compared to air filled bubbles. Kraft et al. used acoustical methods, to investigate the effect of the gas composition on the size and stability of shelled MBs. Using bubbles filled with nitrogen and saturated with perfluorohexane, they reported that the PFC gas increased the compressibility of the lipid monolayer resulting in more flexible and stable MBs.39,40 Kabalnov et al.26,41 presented a detailed model and in vivo experimental studies on the efficacy of different PFC gases (as osmotic agents) mixed with oxygen or nitrogen on MB dissolution in the blood stream. They showed an increase in the bubble lifetime with the increasing molecular weight of the osmotic agent when mixed with oxygen. Increasing the PFC molecular weight beyond C6F14 does not have a positive effect. Finally, Schutt et al. encapsulated a mixture of different PFCs in their gaseous state, to show that MBs containing mixtures of C4F10 and C6F14 persist for 3 min, which was longer than with either gas on its own.28,42 dissolution unless they are stabilized with a suitable coating. This journal is ©The Royal Society of Chemistry 2016 1 Introduction sonoporation of cells in the immediate vicinity of the MBs, leading to an enhanced therapeutic uptake.9,10 Their changing role requires that MBs be re-engineered to improve their func- tional performance, with the key MB parameters being size and dispersity, biocompatibility, shell stiffness and MB lifetime.5,11 Microbubbles (MBs) used for contrast enhanced ultrasound (CEUS) imaging are micron sized gas encapsulated spheres, stabilized with a shell made of biocompatible materials such as proteins, surfactants, phospholipids or polymers. These MBs are capable of circulating in the vasculature and their high acoustic impedance mismatch with the surrounding tissue leads to strong ultrasound scattering resulting in enhanced contrast in US imaging. Recently there has been considerable interest in the development of MBs as vehicles for drug delivery1,2 by loading them with drug-filled liposomes, attaching genes to the shell,3–5 or filling the core with a therapeutic gas.6,7 The MB complex can be targeted to the required location using antibodies, or other ligands, to provide a route for targeted, triggered delivery.8 Furthermore, it has been demonstrated that bursting the MBs using an ultrasound pulse gives rise to For clinical and preclinical applications MBs are typically desired to be between 1–8 mm in diameter. Control over size can be achieved by selecting a suitable production technique. Sonication and mechanical agitation are the common methods for MB production.12,13 They produce broad poly-disperse size distributions with a polydispersity index of B150%, with the majority of MBs being in the range of o8 mm,14 and are currently used for diagnostic imaging.15,16 Coaxial electro- hydrodynamic atomisation14,17 produces bubbles which can be of a controllable size between 1 and 25 mm and with a moderate dispersity index of B30%. For the highest quality monodisperse MB production, the best control over the size and the dispersity index has come about through the use of flow-focus microfluidic (MF) technology.18–21 However, in this approach the MB concentration tends to be low (B106 MB mL1) compared to the 108 MB mL1 used in a single bolus for in vivo imaging experiments. Recently Peyman et al. 2.5 Langmuir trough A Langmuir trough (KSV Nima) was used to measure the diﬀerences in monolayer compressability and elasticity. The trough was equipped with two movable PTFE barriers to compress the monolayer symmetrically. A Wilhelmy plate tensiometer (paper method) was used to measure the surface pressure of the monolayer. This experiment was performed for the same lipid monolayer composition found to form the most stable MBs. 20 mL of a 1 mg mL1 solution of (DPPC + 5% DSPE-PEG2000), in chloroform, was spread on the surface of a subphase of water A typical production run produced 1 mL of MBs. For each MB population formed, a 10 mL sample (collected from the middle of the homogenous MB solution) was diluted 10-fold to facilitate counting and sizing of the bubbles. From this diluted sample 30 mL was introduced into a 50 mm chamber on a glass slide. The MBs were allowed to rise for B1 minute before acquiring images. An inverted microscope (Nikon, Japan) was used to image the MBs at 60 magnification. A CCD camera (DS-Fil 5 Mega pixel, Nikon, Japan) was used to capture 40 images of each sample, from which the concentration and size distribution were obtained using Image J freeware (http:// rsbweb.nih.gov/ij/) and statistically analysed using Origin Pro (Version 8.5 or later). Fig. 2 In vivo imaging of mouse aorta with high frequency ultrasound. (A) A region of interest (ROI) within the aorta was selected for post-processing analysis (blue region) to determine the contrast intensity within the aorta over time. (B) Example image showing the aorta post injection filled with MBs. Fig. 2 In vivo imaging of mouse aorta with high frequency ultrasound. (A) A region of interest (ROI) within the aorta was selected for post-processing analysis (blue region) to determine the contrast intensity within the aorta over time. (B) Example image showing the aorta post injection filled with MBs. 2.1 Materials The lipids used throughout this study were 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanol- amine-N-[methoxy(polyethylene-glycol)-2000] (DSPE-PEG2000), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy- (polyethyleneglycol)-5000] (DPPE-PEG5000) which were purchased from Avanti Polar Lipids (Alabaster, AL, USA) and used without further purification. All lipids were received in the powder form, and then dissolved in a 50/50 chloroform/methanol solution. Open Access Article. Published on 28 July 2016. Downloaded on 24/10/2016 11:42:11. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. For in vivo measurements 50 to 100 mL of the MB solution (in PBS) was injected via a syringe attached to a tail vein catheter. The injection of the MB bolus was controlled by a syringe driver at a rate of 0.6 mL min1. A typical bolus contained B108 MBs in 100 mL. 2 Materials and methods medium [RPMI from Invitrogen, Life Technologies, UK, with 10% (v/v) foetal calf serum (Sigma Aldrich, UK)] and incubated at 37 1C in a digital dryblock heater (Model D1100, Labnet Inter- national, USA). The vial containing the sample was left open exposed to air and 10 mL samples were collected every 15 min for sizing and counting. 2.2 Microbubble preparation and characterisation All animal work was performed under licence and in accordance with the UK Animals (Scientific Procedures) Act 1986 following local ethical review and procedures. Eighteen athymic CD1-nu/nu male mice (6–8 weeks old) were maintained in individual ventilated cages under specific pathogen-free conditions with free access to diet and water. The lipid composition as specified for each MB formulation was prepared as described previously.19,44 Briefly, the lipid mixture was dried under a steady stream of nitrogen gas on the vial walls. This dried film was then suspended in a solution containing 4 mg mL1 NaCl and 1% glycerine (499%, Sigma- Aldrich, St. Louis, MO, USA) to a final lipid concentration of 1 mg mL1 unless otherwise specified. This solution was vortexed for 1 minute before being placed in an ultrasonic bath for 1 hour. The lipid solutions were allowed to cool down in the fridge for 5 minutes prior to use in the MF-MB maker. The mouse aorta was identified and imaged by pulsed wave (PW) Doppler imaging using a Vevo770 (FUJIFILM VisualSonics, Inc.) using the 40 MHz transducer. For post-processing analysis, a region of interest (ROI) was drawn within the aorta such that the ROI was maintained within the aorta for the whole video loop, ensuring that with respiration motion, the ROI was not sampling tissue outside the aorta, as indicated by the blue circle in Fig. 2A. Fig. 2B shows a snapshot of the accumulation of MBs in the aorta post injection. Polymethylmethacrylate (PMMA) MF-chips, designed in Leeds and produced by Epigem plc (Redcar, UK), were used to prepare MBs according to our previously described protocols, in which gas flow is focussed through a nozzle before being rapidly expanded to create a microspray regime.19 Two gases were investigated of diﬀerent molecular weight: perfluoro- propane gas (C3F8), Mw = 188 g mol1; and perfluorobutane (C4F10), Mw = 238 g mol1. The gas pressure was controlled using a Kukuke microprecision regulator (RS supplies, Leeds, UK). The flow rate of the liquid phase, containing the lipid products, was controlled using an Aladdin AL 2000 syringe pump (World Precision Instruments, Stevenage, UK). The flow rate was fixed for all MB preparations at 80 mL min1. In some cases tetradecafluorohexane (C6F14, Sigma-Aldrich, St. Louis, MO, USA) was used to saturate the lipid solution (10 mL). 2 Materials and methods Soft Matter Soft Matter Paper This journal is ©The Royal Society of Chemistry 2016 1 Introduction Important shell properties that aﬀect the bubble stability are surface tension, surface The combined eﬀect of saturation of the surrounding medium, f, and diﬀerent MB shells, Rshell, was investigated by Kwan and Borden in 2010.43 Sulphur hexafluoride, SF6, was encapsulated in SDS or lipid shell MBs and used in a modified perfusion chamber to observe the MB dissolution behaviour in a SF6 or air-saturated medium. When suddenly placed in an air-saturated medium, MBs initially grow (air influx) and then decrease in size as a result of SF6 eﬄux. Lipid coated MBs deviated from the model, as when placed in an air-saturated environment, the initial growth regime was shorter and it was followed by rapid non-uniform dissolution to the original diameter, and then a steady dissolution with a constant wall velocity and final stability at around 10 mm in diameter. Fig. 1 Schematic representation of a lipid shelled MB illustrating the factors that aﬀect MB lifetime. The diﬀerent terms are defined in the text. For the studies reported here, MF-MBs were produced with an average diameter of 2 mm. The stability of the population was examined in vitro, at 37 1C, and in vivo using mouse models. The aim of the study was to optimize the in vivo lifetime of the MF-MBs. Factors investigated include: shell resistance, Rshell, through control over the lipid compositions, and gas dissolution, (Dw, f, H), through control over the molecular weight of the gas and saturation of the surrounding medium. By optimising these factors we achieved a prolonged lifetime of MBs in vivo. Our optimised MBs have lifetimes of 414 minutes in vivo and also retain excellent ultrasound contrast enhancement properties. Fig. 1 Schematic representation of a lipid shelled MB illustrating the factors that aﬀect MB lifetime. The diﬀerent terms are defined in the text. This journal is ©The Royal Society of Chemistry 2016 7224 \| Soft Matter, 2016, 12, 7223--7230 View Article Online 3.1 Microbubble production and characterisation MBs were prepared in the spray regime of a specially designed MF-chip19 shown schematically in Fig. 3A. The image (inset) in Fig. 3B shows a typical MB population, with an average MB diameter of 1.7 1.1 mm and concentrations of B2 109 MB mL1. No bubbles were produced with a diameter 47 mm. Of the factors identified above only two showed a significant eﬀect on MB stability. Summary data has been taken from a larger data set of the variables investigated, which is detailed in the ESI† (S2). The first significant eﬀect was that on changing from the C3F8 core to the higher molecular weight C4F10 gas, the MB population displayed a doubling in lifetime from the order of one to two hours (at 37 1C) Fig. 4A. This eﬀect is well understood and is due to the reduced solubility of the higher 3.2 MB lifetime In vitro MB lifetime was initially determined by measuring the MB concentration, using optical microscopy, every 30 min in a closed vial at room temperature and atmospheric pressure. MB concentration versus time (in vitro results) for MBs prepared with shells of DPPC (or DSPC) with 10% DSPE-PEG2000 encap- sulating C3F8 gas was tested (see S1, ESI†). The data showed that the MBs were relatively long lived in both cases, i.e. for both lipid types, with no significant diﬀerence in their stability over a period of B3 h following production. In contrast, the in vivo lifetime in the mouse aorta, as determined from time– intensity curves (TICs), showed that both MBs decayed within 2 minutes in vivo. A slightly higher acoustic intensity signal was observed for the DPPC MBs compared to the DSPC ones, which is consistent with previous observations for such MBs.46 These initial results showed that the in vitro study of MBs, at room temperature and atmospheric pressure, is not a good indicator of the in vivo behaviour and that physiological conditions (increased temperature, pressure and potential for gas exchange around the MBs) are likely to significantly affect the MB stability. Thus, in order to better test our MBs under more realistic conditions the in vitro experiment was modified to use a cell medium (RPMI with 10% (v/v) foetal calf serum) incubated at Fig. 4 (A) MB concentration versus time for DPPC + 5% PEG DSPE MBs with C3F8 and C4F10 gas cores. (B) The eﬀect of adding liquid C6F14 to the MB bolus, in increasing amounts from 0 to 15 mL mL1, on MB lifetime. (C) Calculated lifetime expected for the MB distribution profile shown in Fig. 3 for 3 cases, (i) with a C3F8 core, (ii) with a C4F10 core and (iii) C4F10 + C6F14 (open circles). The parameters for calculations were taken from Sarkar27 and given in detail in the ESI† (S4). All data was taken at 37 1C in cell media and at atmospheric pressure. Fig. 3 Bubble production and characterisation. (A) A schematic of the microfluidic chip design emphasizing the 3D expansion region for the production of MBs in the spray regime. (B) Graph displaying the size distribution for one of the bubble populations. The inset shows a bright field image representing the produced MBs (sample is 10-fold diluted). Fig. 2.3 In vitro lifetime The MB lifetime was measured in vitro as described previously.45 Briefly, 500 mL of MB solution was introduced into 500 mL of cell Soft Matter, 2016, 12, 7223--7230 \| 7225 This journal is ©The Royal Society of Chemistry 2016 View Article Online Paper Soft Matter 37 1C and the sample was left in an open tube exposed to air for gaseous exchange. Based on the in vivo data (S1, ESI†), and that DPPC MBs have a more homogenous lipid distribution on the bubble shell,47 the slightly shorter DPPC (16:0) lipid was chosen for subsequent MB studies over the DSPC (18:0). A series of factors were investigated to detect their effect on MB stability, firstly in vitro and then in vivo. These included: (i) the degree of pegylation in the MB shell, which affects the potential for phase separation;45,48 (ii) the gas core, by switching to a heavier gas (C4F10) to reduce solubility and diffusivity in the surrounding medium; and finally (iii) factors related to the surrounding solution, such as super saturating the lipid solution with C3F8 gas prior to bubble produc- tion, and also increasing the concentration of NaCl in the lipid solution to over 0.1 M to prevent bubble fusion.49 in the presence and absence of C6F14. Pressure–area isotherms were made with the compression rate of the barriers set to 5 mm min1 and the surface pressure was not allowed to rise beyond 30 mN m1; this ensured the reversibility of the isotherms and allowed us to observe multiple compression/ expansion cycles, which is important for the determination of whether any material is lost from the monolayer during compression. Open Access Article. Published on 28 July 2016. Downloaded on 24/10/2016 11:42:11. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 5A shows the in vivo TIC for four diﬀerent MB variants, comparing both the eﬀect of the presence of C6F14 in the bolus and the concentration of the lipid. Comparison of the area under the curves for the cases with and without C6F14 clearly indicates that the presence of C6F14 significantly enhances MB persistence in the bloodstream. Furthermore, by doubling the lipid concentration, from 1 mg mL1 to 2 mg mL1, during the production of the MBs, the concentration of the MBs was doubled from 1 109 to 2 109 MB mL1, and the lifetime was modestly enhanced, leading to a larger area beneath the TIC curves. Fig. 5B shows the TIC curves for our best in vivo MB in comparison to those obtained using commercially available MBs (Micromarker (MM), Definity). To control the saturation, f, or solubility of the gas in the solution, H, the medium containing the MBs was saturated with C6F14, which is a liquid at room temperature. The solubility of C6F14 in water, at 25 1C, is 2.7 104 mol L1, which is equivalent to B0.05 mL for 1 mL of the lipid solution.11,50 The volume of C6F14 per mL of MB was varied between 0 and 15 mL mL1 in the lipid solution (DPPC + 5% DSPE-PEG2000) to determine the optimum concentration at which the MBs have the longest life- time. Fig. 4B shows the fraction of bubbles remaining in the medium at 37 1C, as a function of time for diﬀerent volumes of C6F14 added to a 1 mL MB bolus. The data shows that MB lifetime was significantly increased upon the addition of C6F14 for con- centrations Z6 mL mL1, with the 15 mL mL1 sample producing the most stable MB population. However, the addition of such volumes of C6F14 adversely aﬀected the MB production leading to a 10-fold reduction in the concentration of MBs produced. Thus the addition of 10 mL of C6F14 was selected as being the optimal amount. At this concentration MBs were still produced at a high concentration of B1 109 MB mL1 whilst also increasing MBs stability. The MB decay rate was reduced to only 0.07% over the 2 h period. For each MB population the intensity versus time curves were collected from 5 mice, and from each curve 6 diﬀerent parameters were extracted, as shown in Fig. 6A. Soft Matter Soft Matter Paper Paper be 25 mN m1 and the saturation, f, was taken as unity, and all other parameters are given in the ESI.† We note that in this model MBs of a diameter o0.5 mm were considered to not be observable optically and so bubbles were removed from the MB population when their size decreased below 0.5 mm. Essentially we see the expected trend that C4F10 4 C3F8, however we note that the model predicts longer lifetimes than were experi- mentally observed, probably indicating that the MB surface tension, shell resistance and/or the degree of saturation are not accurately modelled in our system. Mw gas in solution (the Ostwald coeﬃcient reduces from 5.2 104 for C3F8 to 2.0 104 for C4F10), a slightly reduced diﬀusion rate in water (from 7.45 1010 for C3F8 to 6.9 1010 for C4F10) and an increase in the resistance of the shell to the increased Mw gas. These eﬀects have been modelled in S3 in the ESI†, following the work of Sarkar et al.27,38 for MBs with diﬀerent cores and assuming the surface tension of the shelled MBs to be 25 mN m1. Mw gas in solution (the Ostwald coeﬃcient reduces from 5.2 104 for C3F8 to 2.0 104 for C4F10), a slightly reduced diﬀusion rate in water (from 7.45 1010 for C3F8 to 6.9 1010 for C4F10) and an increase in the resistance of the shell to the increased Mw gas. These eﬀects have been modelled in S3 in the ESI†, following the work of Sarkar et al.27,38 for MBs with diﬀerent cores and assuming the surface tension of the shelled MBs to be 25 mN m1. Decreasing the PEG concentration from 10% to 5% led to a modest increase in the average lifetime (S4, ESI†). However, in spite of this significant improvement in the in vitro lifetime, at body temperature, these formulations showed negligible improvement in the in vivo MB lifetime, as indicated by the TIC curves (S4, ESI†). 3.2 MB lifetime 4 (A) MB concentration versus time for DPPC + 5% PEG DSPE MBs with C3F8 and C4F10 gas cores. (B) The eﬀect of adding liquid C6F14 to the MB bolus, in increasing amounts from 0 to 15 mL mL1, on MB lifetime. (C) Calculated lifetime expected for the MB distribution profile shown in Fig. 3 for 3 cases, (i) with a C3F8 core, (ii) with a C4F10 core and (iii) C4F10 + C6F14 (open circles). The parameters for calculations were taken from Sarkar27 and given in detail in the ESI† (S4). All data was taken at 37 1C in cell media and at atmospheric pressure. Fig. 4 (A) MB concentration versus time for DPPC + 5% PEG DSPE MBs with C3F8 and C4F10 gas cores. (B) The eﬀect of adding liquid C6F14 to the MB bolus, in increasing amounts from 0 to 15 mL mL1, on MB lifetime. (C) Calculated lifetime expected for the MB distribution profile shown in Fig. 3 for 3 cases, (i) with a C3F8 core, (ii) with a C4F10 core and (iii) C4F10 + C6F14 (open circles). The parameters for calculations were taken from Sarkar27 and given in detail in the ESI† (S4). All data was taken at 37 1C in cell media and at atmospheric pressure. Fig. 3 Bubble production and characterisation. (A) A schematic of the microfluidic chip design emphasizing the 3D expansion region for the production of MBs in the spray regime. (B) Graph displaying the size distribution for one of the bubble populations. The inset shows a bright field image representing the produced MBs (sample is 10-fold diluted). This journal is ©The Royal Society of Chemistry 2016 7226 \| Soft Matter, 2016, 12, 7223--7230 View Article Online This journal is ©The Royal Society of Chemistry 2016 Open Access Article. Published on 28 July 2016. Downloaded on 24/10/2016 11:42:11. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. These can facilitate the comparison of the diﬀerent MBs and aid the determination of the one with the best properties for in vivo imaging. These parameters are presented with the codes as follows: (1) rate of decay. This can be calculated from the slope of the curve after the peak point. The smaller the decay rate the longer the MB lifetime. (2) Peak enhancement, which is the point at the highest contrast intensity. This mainly depends on the MB shell properties and concentration. (3) Area under the curve, which is proportional to both the MB concentration and persistence time. A greater area indicates better quality and stability of MBs. (4) Time to peak, which is the time to reach the maximum intensity at the ROI. It is better if this is short. (5) Peak enhancement duration, which is the time at which there was the highest contrast intensity after injection. (6) Time to Fig. 4C shows the calculated MB population profile expected based on the calculation of a typical MB distribution, given in Fig. 3B, and that the MBs decay in accordance with the modified Epstein–Plesset model proposed by Borden25 and Sarkar.27 In these models the surface tension was assumed to Fig. 5 Ultrasound time intensity curves in mice aorta. (A) shows the eﬀect of increasing the final lipid concentration, and saturating the lipid solution with C6F14 on MB lifetime for MBs made with DPPC + 5% PEG2000 and encapsulating C4F10 gas. (B) compares TIC curves for our improved in-house MBs and commercial MBs. Fig. 5 Ultrasound time intensity curves in mice aorta. (A) shows the eﬀect of increasing the final lipid concentration, and saturating the lipid solution with C6F14 on MB lifetime for MBs made with DPPC + 5% PEG2000 and encapsulating C4F10 gas. (B) compares TIC curves for our improved in-house MBs and commercial MBs. Soft Matter, 2016, 12, 7223--7230 \| 7227 This journal is ©The Royal Society of Chemistry 2016 Fig. 7 Langmuir isotherms of DPPC + 5% DSPE-PEG2000 monolayers, on water sub-phase (black) and water with 10 mL mL1 C6F14 added to the sub-phase (red). Dashed lines show the extrapolated average molecular area for the DPPC/DSPE-PEG2000 phase. Soft Matter View Article Online Fig. 6 In vivo TIC analysis. Open Access Article. Published on 28 July 2016. Downloaded on 24/10/2016 11:42:11. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 6B shows the data analysis for the TIC curves comparing the six parameters between our in-house MF-MBs and the commercially available MBs. The data suggests that the MF-MBs produced in-house display an improved peak enhancement and area under the curve, and a significantly longer time to half peak. In order to understand the role of C6F14 in enhancing MB lifetime, a Langmuir trough was used to plot the relationship of the surface pressure, P (mN m1), versus the average molecular area, for a 1 mg mL1 (DPPC + 5% PEG2000) solution spread at the air/water interface in the presence and absence of C6F14 in the sub-phase. Fig. 7 shows the compression isotherms of the Open Access Article. Published on 28 July 2016. Downloaded on 24/10/2016 11:42:11. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. (A) shows an example time–intensity curve (TIC) for MB flow in a mouse aorta and defines six key parameters for defining MB lifetime: (1) is the decay rate calculated from the slope of the curve after the peak intensity, the slower the decay rate the greater the MB stability; (2) is the peak enhancement, which is the maximum intensity signal, and is due to the MB shell properties as well as the gas used; (3) is the area under the curve, which indicates the signal from the total MB population present and therefore the concentration of MBs; (4) is the time to peak, which is the time to reach the peak signal intensity, it should be short enough to enable imaging within a realistic time-frame; (5) is the peak enhancement duration, which is the length of time the MB signal persists, this needs to be of sufficient duration to allow a useful imaging session following MB injection; (6) is the time to half peak, which is the time it takes the signal to reduce to half the peak intensity, which is a further measure of the duration of the signal. (B) The six parameters for our in-house MBs and two commercial MB formulations. In-house MBs showed (1) slower decay rate, (2) improved peak enhancement signal and (5) duration, (3) larger area under the curve, (4) shorter time after injection to reach the maximum intensity, and (6) significantly longer time to half peak, indicating better in vivo stability, better contrast enhancement and longer imaging time. The isothermal compressibility, C, and the compression modulus (elasticity), K, of the lipid modified interface are calculated from the extrapolated lines shown on the isotherm (Fig. 7). These calculations showed that the existence of PFC in the surrounding solution increased the compressibility (C) from 1.0 102 to 1.2 102 m mN1, which corresponds to a decrease in the elasticity (K) from 97 mN m1 to 88 mN m1 in the presence of PFC molecules. These results are in good agreement with the previously published work by Kraﬀt39 on the eﬀect of saturating the surrounding air with diﬀerent PFC gases on the compressibility and elasticity of lipid monolayers. half peak contrast. The longer this parameter is, the more prolonged the lifetime of the MBs is at the ROI. Fig. Open Access Article. Published on 28 July 2016. Downloaded on 24/10/2016 11:42:11. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. (A) shows an example time–intensity curve (TIC) for MB flow in a mouse aorta and defines six key parameters for defining MB lifetime: (1) is the decay rate calculated from the slope of the curve after the peak intensity, the slower the decay rate the greater the MB stability; (2) is the peak enhancement, which is the maximum intensity signal, and is due to the MB shell properties as well as the gas used; (3) is the area under the curve, which indicates the signal from the total MB population present and therefore the concentration of MBs; (4) is the time to peak, which is the time to reach the peak signal intensity, it should be short enough to enable imaging within a realistic time-frame; (5) is the peak enhancement duration, which is the length of time the MB signal persists, this needs to be of sufficient duration to allow a useful imaging session following MB injection; (6) is the time to half peak, which is the time it takes the signal to reduce to half the peak intensity, which is a further measure of the duration of the signal. (B) The six parameters for our in-house MBs and two commercial MB formulations. In-house MBs showed (1) slower decay rate, (2) improved peak enhancement signal and (5) duration, (3) larger area under the curve, (4) shorter time after injection to reach the maximum intensity, and (6) significantly longer time to half peak, indicating better in vivo stability, better contrast enhancement and longer imaging time. Paper Open Access Article. Published on 28 July 2016. Downloaded on 24/10/2016 11:42:11. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 28 July 2016. Downloaded on 24/10/2016 11:42:11. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 7 Langmuir isotherms of DPPC + 5% DSPE-PEG2000 monolayers, on water sub-phase (black) and water with 10 mL mL1 C6F14 added to the sub-phase (red). Dashed lines show the extrapolated average molecular area for the DPPC/DSPE-PEG2000 phase. Fig. 7 Langmuir isotherms of DPPC + 5% DSPE-PEG2000 monolayers, on water sub-phase (black) and water with 10 mL mL1 C6F14 added to the sub-phase (red). Dashed lines show the extrapolated average molecular area for the DPPC/DSPE-PEG2000 phase. Fig. This journal is ©The Royal Society of Chemistry 2016 7228 \| Soft Matter, 2016, 12, 7223--7230 Open Access Article. Published on 28 July 2016. Downloaded on 24/10/2016 11:42:11. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 7 Langmuir isotherms of DPPC + 5% DSPE-PEG2000 monolayers, on water sub-phase (black) and water with 10 mL mL1 C6F14 added to the sub-phase (red). Dashed lines show the extrapolated average molecular area for the DPPC/DSPE-PEG2000 phase. monolayer. All conditions are fixed for both cases with the only diﬀerence being the sub-phase; either just with MilliQ water or MilliQ water plus C6F14 at the same concentration used during MB production (10 mL mL1). The mean molecular area was found to increase from 0.5 nm2 to 0.63 nm2 upon the inclusion of C6F14 in the sub- phase, which indicates the incorporation of the C6F14 molecules within the lipid layer. The fractional component of C6F14 in the MB shell was calculated to be 0.4, which means an estimation of 2 PFC molecules for every 5 lipid molecules. As a consequence of incorporating C6F14 (surface tension 11 mN m1 51) at this propor- tion in the lipid layer, the surface tension of the monolayer is reduced from 25 mN m127,38 to 19 mN m1. Consequently, this leads to a reduction in the Laplace pressure from 50 103 N m1 in the absence of C6F14, to 38 103 N m1 in the presence of C6F14, which leads to an improved estimate in the MB lifetime. The hollow circles in Fig. 4C indicate the predicted enhancement in MB lifetime due to the reduction in surface tension. This is shown more clearly in S3 (ESI†). Fig. 6 In vivo TIC analysis. Open Access Article. Published on 28 July 2016. Downloaded on 24/10/2016 11:42:11. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. (A) shows an example time–intensity curve (TIC) for MB flow in a mouse aorta and defines six key parameters for defining MB lifetime: (1) is the decay rate calculated from the slope of the curve after the peak intensity, the slower the decay rate the greater the MB stability; (2) is the peak enhancement, which is the maximum intensity signal, and is due to the MB shell properties as well as the gas used; (3) is the area under the curve, which indicates the signal from the total MB population present and therefore the concentration of MBs; (4) is the time to peak, which is the time to reach the peak signal intensity, it should be short enough to enable imaging within a realistic time-frame; (5) is the peak enhancement duration, which is the length of time the MB signal persists, this needs to be of sufficient duration to allow a useful imaging session following MB injection; (6) is the time to half peak, which is the time it takes the signal to reduce to half the peak intensity, which is a further measure of the duration of the signal. (B) The six parameters for our in-house MBs and two commercial MB formulations. In-house MBs showed (1) slower decay rate, (2) improved peak enhancement signal and (5) duration, (3) larger area under the curve, (4) shorter time after injection to reach the maximum intensity, and (6) significantly longer time to half peak, indicating better in vivo stability, better contrast enhancement and longer imaging time. Fig. 6 In vivo TIC analysis. Open Access Article. Published on 28 July 2016. Downloaded on 24/10/2016 11:42:11. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Recently, C6F14 has been encapsulated together with Dox or gold nano-rods in nanoparticles and locally activated at the site of a tumour to form MBs, and C6F14 had been shown to enhance the acoustic imaging, cavitation and therapeutic delivery eﬀects.53,54 In this work, saturating the medium sur- rounding the MBs with liquid C6F14 and using C4F10 in the gas core are considered key to the improvements in MB lifetime. The presence of C6F14 in the surrounding medium increased the stability and lifetime of the MBs in vitro and showed an improvement of up to 14 min in vivo (Fig. 5). We believe this improvement in stability arises for two reasons (Fig. 8); firstly, the addition of C6F14 eﬀectively results in the saturation of the surrounding medium, reducing the ability for C4F10 to partition into the aqueous phase. Secondly, the incorporation of C6F14 molecules into the lipid shell of the MB, as concluded from Fig. 7, leads to a 25% reduction in the surface tension of the membrane, and hence also a reduction in the Laplace driving force for dissolution. This also modifies the mechanical properties of the shell, where a 17% increase in the compressibility is observed accompanied by a 10% decrease in the shell elasticity. The eﬀect of saturating the gas medium with PFC gases on the lipid monolayers has been studied by Gerber et al.,55,56 who saturated the atmosphere above the Langmuir monolayer of DPPC with vapours of C6F14. They reported that saturating the gaseous medium with PFC gas has a fluidizing eﬀect on the DPPC monolayer adsorption at the gas/water interface, as it prevents the liquid condensed phase formation and hinders lipid crystal- lisation. Kraﬀt et al.39 also demonstrated that DMPC lipid shell In vivo MB lifetime improved from 2 min to 14 min through a combination of changes to the lipid shell, the gas core and the addition of C6F14 to the medium. Furthermore, comparing in-house MF-MBs and commercial MBs (Fig. 5) showed that the sat-C4F10 MBs provided enhanced lifetime and contrast properties compared to their commercial counterparts. In conclusion, control of MB properties including decay rate, intensity and enhancement duration, was achieved by changing the phospholipid shell composition, the encapsu- lated gas and, most significantly, the degree of saturation of the surrounding medium with C6F14. Open Access Article. Published on 28 July 2016. Downloaded on 24/10/2016 11:42:11. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Our data suggests the C6F14 molecules are incorporated in the lipid shell reducing the surface tension and also increasing the mechanical com- pressibility and collapse pressure. MBs for drug delivery appli- cations are likely to require longer lifetimes to allow for accumulation at the ROI for optimal dose delivery and will thus benefit greatly from such improvements. Acknowledgements The authors would like to acknowledge the EPSRC for funding (EP/I000623, EP/K023845) and thank the Microbubble Consortium (http://www.microbubbles.leeds.ac.uk/) at the University of Leeds for useful discussions. The data presented in this article are openly available from the University of Leeds Data Repository http://doi. org/10.5518/68. This journal is ©The Royal Society of Chemistry 2016 4 Discussion and conclusion MB lifetime stability has been investigated by changing diﬀerent factors that are known to have an eﬀect on MB lifetime. The MB architectures have a shell comprising a DPPC/DSPE-PEG2000 7228 \| Soft Matter, 2016, 12, 7223--7230 This journal is ©The Royal Society of Chemistry 2016 View Article Online Soft Matter Paper MBs showed a 20% increase in compressibility of the membrane and a 26% decrease in the surface tension upon saturating the air with C6F14 in the gas medium. Kraﬀt and Fainerman et al.57,58 theoretically modelled and practically showed that in case of DPPC monolayers, saturating the gas phase with C6F14 led to C6F14 molecules being adsorbed at the lipid surface, causing a reduction in the energy of attraction between the DPPC molecules, which leads to a fluidization of the monolayer. From the data shown here it appears that the surface tension alone is not a significant enough change to account for the increased MB stability and thus we also believe that the inclusion of the C6F14 molecules into the lipid chain must also increase the shell resistance. binary system, the components of which are completely miscible below 15% PEG2000 concentrations forming a single condensed phase with low permeability.52 We have previously shown that reducing the PEG2000 concentration from 10 to 5% helped increase the MB lifetime.45 MB lifetime was also improved by changing the encapsulated gas from C3F8 to one with a larger molecular weight, i.e. C4F10. This decreases the coeﬃcient of diﬀusivity from 7.45 1010 to 6.9 1010 m2 s1.27 Combining the changes outlined above has resulted in a considerable improvement in the in vitro lifetime of the MBs, as discussed in the Results section above. Open Access Article. Published on 28 July 2016. Downloaded on 24/10/2016 11:42:11. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 37 K. Ferrara, R. Pollard and M. Borden, Annu. Rev. Biomed. Eng., 2007, 9, 415–447. 10 Y. Luan, Z. Chen, G. Xie, J. Chen, A. Lu, C. Li, H. Fu, Z. Ma and J. Wang, J. Nanosci. Nanotechnol., 2015, 15, 1357–1361. 38 A. Katiyar and K. Sarkar, J. Colloid Interface Sci., 2010, 343, 42–47. 11 E. G. Schutt, D. H. Klein, R. M. Mattrey and J. G. Riess, Angew. Chem., Int. Ed., 2003, 42, 3218–3235. 39 C. Szijjarto, S. Rossi, G. Waton and M. P. Kraﬀt, Langmuir, 2012, 28, 1182–1189. 12 C. Christiansen, H. Kryvi, P. C. Sontum and T. Skotland, Biotechnol. Appl. Biochem., 1994, 19(pt 3), 307–320. 40 S. Rossi, G. Waton and M. P. Kraﬀt, Langmuir, 2010, 26, 1649–1655. 13 G. Pu, M. A. Borden and M. L. Longo, Langmuir, 2006, 22, 2993–2999. 41 A. Kabalnov, J. Bradley, S. Flaim, D. Klein, T. Pelura, B. Peters, S. Otto, J. Reynolds, E. Schutt and J. Weers, Ultrasound Med. Biol., 1998, 24, 751–760. 14 E. Stride and M. Edirisinghe, Med. Biol. Eng. Comput., 2009, 47, 883–892. 42 E. G. Schutt, C. D. Anderson and D. P. Evitts, US Pat., 5605673, 1997. 15 J. R. Lindner, M. P. Coggins, S. Kaul, A. L. Klibanov, G. H. Brandenburger and K. Ley, Circulation, 2000, 101, 668–675. 43 J. J. Kwan and M. A. Borden, Langmuir, 2010, 26, 6542–6548. 16 J. R. Lindner, P. A. Dayton, M. P. Coggins, K. Ley, J. Song, K. Ferrara and S. Kaul, Circulation, 2000, 102, 531–538. 44 R. H. Abou-Saleh, S. A. Peyman, K. Critchley, S. D. Evans and 44 R. H. Abou-Saleh, S. A. Peyman, K. Critchley, S. D. Evans and N H Thomson Langmuir 2013 29 4096 4103 44 R. H. Abou-Saleh, S. A. Peyman, K. Critchley, S. D. Evans an N. H. Thomson, Langmuir, 2013, 29, 4096–4103. 17 U. Farook, H. B. Zhang, M. J. Edirisinghe, E. Stride and N. Saﬀari, J. Biomed. Eng., 2007, 29, 749–754. N. H. Thomson, Langmuir, 2013, 29, 4096–4103. 45 R. H. Abou-Saleh, S. D. Evans and N. H. Thomson, Langmuir, 2014, 30, 5557–5563. 18 M. Hashimoto and G. M. Whitesides, Small, 2010, 6, 1051–1059. 46 T. V. Rooij, Y. Luan, G. Renaud, A. F. W. van der Steen, M. Versluis, N. De Jong and K. Kooiman, Ultrasound Med. Biol., 2015, 41, 1432–1445. 19 S. A. Peyman, R. H. Abou-Saleh, J. R. McLaughlan, N. Ingram, B. R. G. Johnson, K. Open Access Article. Published on 28 July 2016. Downloaded on 24/10/2016 11:42:11. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Critchley, S. Freear, J. A. Evans, A. F. Markham, P. L. Coletta and S. D. Evans, Lab Chip, 2012, 12, 4544–4552. 47 K. Kooiman, T. J. A. Kokhuis, T. V. Rooij, I. Skachkov, A. Nigg, J. G. Bosch, A. F. W. van der Steen, W. A. van Cappellen and N. De Jong, Eur. J. Lipid Sci. Technol., 2014, 116, 1217–1227. 20 E. Talu, K. Hettiarachchi, R. L. Powell, A. P. Lee, P. A. Dayton and M. L. Longo, Langmuir, 2008, 24, 1745–1749. 48 A. K. Kenworthy, S. A. Simon and T. J. McIntosh, Biophys. J., 1995, 68, 1903–1920. 21 E. Talu, K. Hettiarachchi, S. Zhao, R. L. Powell, A. P. Lee, M. L. Longo and P. A. Dayton, Mol. Imaging, 2007, 6, 384–392. 49 B. W. Ninham and P. Lo Nostro, Molecular forces and self assambly: in colloid, nano science and biology, Cambridge University Press, USA, 2010, ch. 8, pp. 232–249. 22 S. A. Peyman, J. R. McLaughlan, R. H. Abou-Saleh, G. Marston, B. R. Johnson, S. Freear, P. L. Coletta, A. F. Markham and S. D. Evans, Lab Chip, 2016, 16, 679–687. 50 A. S. Kabalnov, K. N. Makarov and O. V. Scherbakova, J. Fluorine Chem., 1990, 50, 271–284. 23 J. J. Kwan and M. A. Borden, Adv. Colloid Interface Sci., 2012, 183, 82–99. 51 Y. Luan, J. Xu, B. Huang, X. Liu, Y. Liu, L. Chen and X. Chu, Zhonghua Xueyexue Zazhi, 2015, 36, 286–290. 24 P. S. Epstein and M. S. Plesset, Adv. Colloid Interface Sci., 1950, 183, 82–99. 52 M. M. Lozano and M. L. Longo, Soft Matter, 2009, 5, 1822–1834. 25 M. A. Borden and M. L. Longo, Langmuir, 2002, 18, 9225–9233. 53 J. Zhong, S. Yang, L. Wen and D. Xing, J. Controlled Release, 2016, 226, 77–87. 26 A. Kabalnov, D. Klein, T. Pelura, E. Schutt and J. Weers, Ultrasound Med. Biol., 1998, 24, 739–749. 27 K. Sarkar, A. Katiyar and P. Jain, Ultrasound Med. Biol., 2009, 35, 1385–1396. 54 N. Zhang, X. Cai, W. Gao, R. Wang, C. Xu, Y. Yao, L. Hao, D. Sheng, H. Chen, Z. Wang and Y. Zheng, Theranostics, 2016, 6, 404–417. 28 E. G. Schutt, D. P. Evitts, C. D. Anderson and J. G. Weers, US Pat., US 6953569 B2, 2005. 55 F. Gerber, M. P. Kraﬀt, T. F. Vandamme, M. Goldmann and P. Fontaine, Angew. Chem., Int. Ed., 2005, 44, 2749–2752. 29 H. D. References Fig. 8 Schematic diagram showing (i) the incorporation of C6F14 into the MB shell thereby reducing surface tension and the Laplace driving force for dissolution and (ii) the saturation of the medium with C6F14 thereby reducing the solubility of C4F10 in the ambient phase. 1 S. B. Feinstein, Am. J. Physiol.: Heart Circ. Physiol., 2004, 287, H450–457. 2 J. R. Lindner, Nat. Rev. Drug Discovery, 2004, 3, 527–532. 3 B. Geers, I. Lentacker, N. N. Sanders, J. Demeester, S. Meairs and S. C. De Smedt, J. Controlled Release, 2011, 152, 249–256. 4 J. R. McLaughlan, N. Ingram, R. H. Abou-Saleh, S. Harput, T. Evans, S. D. Evans, L. Coletta and S. Freear, High-Frequency Subharmonic Imaging of Liposome-Loaded Microbubbles, Prague, 2013. 5 E. C. Unger, T. Porter, W. Culp, R. LaBell, T. Matsunaga and R. Zutshi, Adv. Drug Delivery Rev., 2004, 56, 1291–1314. Fig. 8 Schematic diagram showing (i) the incorporation of C6F14 into the MB shell thereby reducing surface tension and the Laplace driving force for dissolution and (ii) the saturation of the medium with C6F14 thereby reducing the solubility of C4F10 in the ambient phase. 6 J. Zhang, Y. Luan, Z. Lyu, L. Wang, L. Xu, K. Yuan, F. Pan, M. Lai, Z. Liu and W. Chen, Nanoscale, 2015, 7, 14881–14888. Soft Matter, 2016, 12, 7223--7230 \| 7229 This journal is ©The Royal Society of Chemistry 2016 View Article Online Paper 33 B. A. Lewis and D. M. Engelman, J. Mol. Biol., 1983, 166, 211–217. 7 F. Cavalieri, I. Finelli, M. Tortora, P. Mozetic, E. Chiessi, F. Polizio, T. B. Brismar and G. Paradossi, Chem. Mater., 2008, 20, 3254–3258. 34 K. H. Kim, S. Q. Choi, J. A. Zasadzinski and T. M. Squires, Soft Matter, 2011, 7, 7782–7789. 8 B. Geers, I. Lentacker, N. N. Sanders, J. Demeester, S. Meairs and S. C. De Smedt, J. Controlled Release, 2011, 152, 249–256. 35 D. H. Kim, M. J. Costello, P. B. Duncan and D. Needham, Langmuir, 2003, 19, 8455–8466. 9 R. Chen, Y. Luan, Z. Liu, W. Song, L. Wu, M. Li, J. Yang, X. Liu, T. Wang, J. Liu and Z. Ye, BioMed Res. Int., 2015, 2015, 514234. 36 J. J. Kwan and M. A. Borden, Soft Matter, 2012, 8, 4756–4766. Open Access Article. Published on 28 July 2016. Downloaded on 24/10/2016 11:42:11. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Van Liew and M. E. Burkard, Adv. Exp. Med. Biol., 1997, 411, 395–401. 56 F. Gerber, M. P. Kraﬀt, T. F. Vandamme, M. Goldmann and P. Fontaine, Biophys. J., 2006, 90, 3184–3192. 30 D. E. Yount, Aviat., Space Environ. Med., 1979, 50, 44–50. . Yount, Aviat., Space Environ. Med., 1979, 50, 44– 57 M. P. Kraﬀt, V. B. Fainerman and R. Miller, Colloid Polym. Sci., 2015, 293, 3091–3097. 31 H. D. Van Liew and S. Raychaudhuri, J. Appl. Physiol., 1997, 82, 2045–2053. 32 C. Li, J. Chen, J. Wang, Z. Ma, P. Han, Y. Luan and A. Lu, Sci. Total Environ., 2015, 521–522, 101–107. 32 C. Li, J. Chen, J. Wang, Z. Ma, P. Han, Y. Luan and A. Lu, Sci. Total Environ., 2015, 521–522, 101–107. 58 V. B. Fainerman, E. V. Aksenenko and R. Miller, Adv. Colloid Interface Sci., 2015, DOI: 10.1016/j.cis.2015.11.004. 58 V. B. Fainerman, E. V. Aksenenko and R. Miller, Adv. Colloid Interface Sci., 2015, DOI: 10.1016/j.cis.2015.11.004. 7230 7230 \| Soft Matter, 2016, 12, 7223--7230 This journal is ©The Royal Society of Chemistry 2016
https://openalex.org/W4289533379	https://www.scielo.br/j/rbla/a/XkbBgBdQpmmtgqC3d9FbsTr/?lang=en&format=pdf	English	null	In Service EAP Teachers’ Beliefs About Academic Writing and its Influence on Instruction	Revista Brasileira de Lingüística Aplicada	2,022	cc-by	11,652	http://dx.doi.org/10.1590/1984-6398202217330 Data de recebimento: 03/09/2020. Data de aprovação: 29/06/2021. http://dx.doi.org/10.1590/1984-6398202217330 Data de recebimento: 03/09/2020. Data de aprovação: 29/06/2021. http://dx.doi.org/10.1590/1984-6398202217330 Data de recebimento: 03/09/2020. Data de aprovação: 29/06/2021 In Service EAP Teachers’ Beliefs About Academic Writing and its Influence on Instruction Crenças sobre escrita acadêmica de professores de inglês para fins específicos em serviço e sua influência no ensino. Marilia Mendes Ferreira* Universidade de São Paulo (USP), São Paulo, São Paulo / Brasil mmferreira@usp.br http://orcid.org/0000-0001-5315-0377 Marilia Mendes Ferreira *Universidade de São Paulo (USP), São Paulo, São Paulo / Brasil mmferreira@usp.br http://orcid.org/0000-0001-5315-0377 Abstract: This paper aims to discuss how experienced English for Academic Purposes (EAP) teachers’ beliefs about academic writing have affected their instruction. The data comprised two semi-structured interviews conducted with three experienced Brazilian EAP teachers working at a large university language center. Their course programs were also collected. The analysis of the six interviews revealed that the instructors´ structuralist view of language affected the product-based teaching of writing. In order to develop themselves as EAP instructors, they have to face some challenges: this traditional view of language which hinders the work with the specificity of academic discourse and the structuration of sustainable EAP professional development. Keywords: beliefs about academic writing; in-service EAP teacher education; academic writing instruction Resumo: Este artigo objetivou discutir como crenças sobre escrita acadêmica de professores experientes de inglês para fins específicos afetou seu ensino. Os dados consistiram em duas entrevistas semiestruturadas realizadas com três professores brasileiros experientes do inglês para fins específicos que trabalham em um grande centro universitário de línguas. Seus programas de cursos também foram coletados. A análise das seis entrevistas revelou que a visão estruturalista de linguagem dos professores afetou o ensino da escrita baseada no produto. Para se desenvolverem como instrutores de inglês para fins específicos, eles precisam enfrentar alguns desafios: essa visão tradicional da linguagem que dificulta o trabalho com a especificidade do discurso acadêmico e a estruturação de um programa de formação continuada. Palavras-chave: crenças sobre escrita acadêmica; formação de professsores em serviço de inglês para fins específicos; ensino de escrita acadêmica 395 Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 1 University of São Paulo: Laboratory of Academic Literacy (http://letramentoacademico.fflch.usp.br) Federal University of Paraná: http://www.capa.ufpr.br/ Nowadays there are around 13 centers in Brazil according to ROCA´s (Rodas de Conversas Acadêmicas) survey. 1 University of São Paulo: Laboratory of Academic Literacy (http://letramentoacademico.fflch.usp.br) Federal University of Paraná: http://www.capa.ufpr.br/ Nowadays there are around 13 centers in Brazil according to ROCA´s (Rodas de Conversas Acadêmicas) survey. 2 An initiative is Language without Borders which will be explained later in this paper. 3 Science without Borders aimed to send Brazilian undergraduate and graduate students to well ranked universities abroad. 3 Science without Borders aimed to send Brazilian undergraduate and graduate students to well ranked universities abroad. 1 Introduction Internationalization is a worldwide phenomenon in higher education. Similarly in Brazil, this movement has increased recently across the country’s universities renewing the academic community’s need to learn English, be it to publish in highly ranked international journals, to teach disciplines in English or to communicate adequately in conferences or research meetings. Thus, more collaborative and internationalized knowledge production can be achieved. Unfortunately, the country’s material conditions to meet this goal have lagged this pressure on academics (ABREU-E-LIMA ET AL, 2016; CRISTÓVÃO; VIEIRA, 2016; FINARDI; PORCINO,2015; FERREIRA, 2016).f In a survey regarding the support offered by universities to their academics and students to publish in English, Ferreira (2016) identified three types of assistance: 1) workshops on English publication often sponsored by international publishers, 2) translation and editing services, 3) writing center services1. These initiatives are not part of a national or local policy to foster academic literacy learning in English or in any additional language. Instead, they are often isolated actions which aim to assist students with publication in English. Concerning writing centers, the great majority of Brazilian universities do not offer this kind of service as Brazilian universities are still struggling with the challenge of increasing their students’ level of proficiency in English2. Before this contradictory picture of great linguistic demands and pressure for academic performance in English on one hand and insufficient support for meeting them on the other, Brazilian universities need to foster more academic literacy development grounded on policies that guide this endeavor (ABREU-E-LIMA ET AL, 2016; CRISTÓVÃO; VIEIRA, 2016; FINARDI; PORCINO,2015; FERREIRA, 2016). One of the most relevant programs that addressed academic communities’ need for additional language proficiency was Languages without Borders. At the beginning, it was created to support the Science without Borders3 program by teaching English and other additional languages to the students who were applying Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 396 to study abroad. Later, the program, terminated in 20194, focused on improving pre-service teacher education in foreign language teaching for academic purposes and increasing the level of proficiency of Brazilian university students so that they could more effectively engage into internationalization (GIRI; MARTINS, 2017). Despite its novelty, the program did not have a policy for academic writing instruction at the universities either in English or in L1 yet and focused exclusively on pre-service teacher education. 1 Introduction Despite the fact teacher education has received considerable attention from the Brazilian government (with actions like PIBID5 ) and the fields of Education and Applied Linguistics, little has been written about EAP teacher education (see ABREU-E-LIMA et al, 2016 for a compilation of some studies involving EAP teacher education in the Language without Borders program). The situation is similar abroad (ALEXANDER, 2011; BARSTURKMEN, 2014; CAMPION, 2016; WORDEN 2018; WU; BADGER, 2009). ) The present paper aims at discussing three experienced EAP teachers’ beliefs about academic writing and their influence on their instruction. The instructors worked for a language center in a Brazilian public university and did not participate in any continuous EAP teacher education program at the university. This study despite its explorative nature, attempts to capture a situation that may be common at other universities: the increasing internationalization of universities and the pressure on academic communities to publish in English are not necessarily followed by proper EAP teacher education of experienced English teachers who are needed to better assist the community in this enterprise. 5 PIBID- Scholarship institutional program to foster initial teacher education. 4 The program now is run by Rede Andifes. 2 EAP teacher education EAP is one branch of English for Specific Purposes (ESP) (PALTRIDGE; STARFIELD, 2014). ESP has established itself as an important subfield of English Language Teaching with seminal publications (DUDLEY-EVANS; ST JOHNS, 1998; FLOWERDEW; PEACOCK, 2001; HUTCHINSON; WATERS, 1987). While ESP addresses specific needs in different contexts (vocational, occupational, technological, for example), EAP involves essentially one specific context, i.e., academic (higher education and previous levels) (CHARLES, 2014). EAP has continuously achieved greater relevance, mainly nowadays, due Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 397 to the internationalization of higher education (HYLAND; HAMP-LYONS, 2002). However, traditionally EAP has privileged the investigation of learners’ needs, materials and course development rather than teacher development (BASTURKMEN, 2014; CAMPION, 2016; WU; BADGER, 2009)6. As a result, EAP teacher development is often unstructured and nonconsensual (ALEXANDER, 2007; CAMPION, 2016)7, which can contribute to the low prestige these types of instructors have in the academic environment (HYLAND; HAMP-LYONS, 2002). In addition, there is a lack of EAP teacher development programs even in countries where the demand for such professionals is growing such as the UK (ALEXANDER, 2011). This shortage contrasts with the crucial supporting roles of these professionals in current internationalizing higher education.h pp g p g g The literature has pointed out some challenges faced by these EAP instructors: the understanding of EAP, the need to focus on different subjects and on the specificity of discourse communities’ linguistic and communicative preferences (ALEXANDER, 2007; HUTTNER ET AL, 2009; WU; BADGER, 2009). Another challenge is the various EAP instructors’ tasks. They have to research fields’ specificities and needs to be course and material developers (DUDLEY-EVANS; ST JOHN, 1998) and more recently they have to be consultants at universities which adopt EMI (English as a Medium of Instruction) (TAILLEFER, 2013). One of the crucial challenges for these instructors is understanding and dealing with a defining feature of the EAP approach: the specificity of the academic context composed of different fields which is manifested through their discourse communities’ communicative needs and epistemology. g Alexander (2011) distinguishes the communicative approach from EAP. While the first one focuses on linguistic items realized through communicative functions applicable to different contexts and on the speaking skill, EAP is ruled by discourse communities’ linguistic and discursive special needs. 7 As Campion (2016) points out the release of guidelines for EAP teacher development such as Baleap Competencies Framework and TEAP scheme has not altered much the situation. 6 ESP seems to face the same problem. For example, The Handbook of English for Specific Purposes does not have a chapter on ESP teacher development. 2 EAP teacher education The former also relies on a myriad of coursebooks whilst the latter counts on its own material development based on the understanding of these singularities.hi The specificity happens in three types of knowledge needed by the EAP teacher (FERGUNSON,1997): sociological, which refers to disciplinary cultures and values; epistemological, which relates to the way an area of study produces Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 398 knowledge; and linguistic (of genres and discourses). Also, there are different levels of EAP courses based on the degree of focus on specific needs of the fields: from zero in general English courses to some level in specialized courses which aims at a broader area (for example, medicine or engineering) to a maximum level in very specific ESP courses (for example, theoretical math) (HYLAND, 2002). i As can be seen, EAP teachers face many challenges in their work which could be facilitated by continuous teacher education programs. As part of these programs, an important element to study is instructors’ cognition. 8 This average was calculated based on just the years of experience in the university language center. It should also be considered that the ESP/EAP courses during these years have changed as the university needs modified as well: from focus on reading to academic writing and presentation courses 2.1 Beliefs as part of teachers’ cognition The term belief can be associated with words such as opinions, ideas, knowledge, assumptions, myths, and representations (BARCELOS, 2003) and can be seen in its cognitive and/or social aspects (BARCELOS, 2003). Teachers’ cognition has established itself as a solid area of research in teacher education that emerged out of concerns with educational reforms (SKOTT, 2015). Teachers were recognized as central actors in implementing reforms; for this reason, the study of their knowledge, thinking and beliefs has become relevant for teacher education (BORG, 2003). This cognition is related to any aspect of their work: learning, teaching, language, materials development, to name some. Thus, this article will consider beliefs as part of teachers’ cognition (BORG, 2003). h Similar to beliefs in general, teachers’ beliefs are considered complex, dynamic, contextual and multifaceted (BARCELOS; KALAJA, 2013). For example, these beliefs have been studied in relation to their process of change (RICHARDS; GALLO; RENANDYA, 2001), to learners’ beliefs (BARCELOS, 2003), to teachers’ pedagogical actions (WOODS, 1996), and to mediational means (NEGUERUELA-AZAROLA, 2011). The focus of research on beliefs has shifted from a mere description of beliefs to its process of change and its interaction with the many aspects of the teaching-learning activity. g g Teachers’ beliefs in general (BORG, 2003; ZHENG, 2009) and more specific about writing instruction have been extensively studied (TAGLE ET AL, 2017; TENG, 2016; TSUI, 1996). Moreover, these studies have shown how influential these constructs can be on teaching (FARRELL; BENNIS, 2013; FARRELL; IVES, 2015). However, studies on beliefs about English academic writing are nonexistent to the best of my knowledge. This study aims to fill this gap by investigating in service EAP teachers’ beliefs about academic writing and Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 399 their influence on instruction. Therefore, valuable information can be obtained to better contextualize EAP teacher education into the higher education context. From the several possible aspects to investigate teacher’ beliefs (BARCELOS; KALAJA, 2013), this exploratory study will focus on their interpretation of their past experiences as learners and teachers, on the relationship between their beliefs and one pedagogical action – feedback correction – and on the relationship between beliefs and new concepts that emerged out of their practice. Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 3.1 Participants The participants in this study are experienced EAP instructors who work in a language center from a large university located in the Southeast of Brazil. Three out of five were selected because they were teaching writing courses at the time of the study. Despite the small number of instructors, their profile given in table 1 might exemplify the history of other in-service EAP teachers who often come from general English courses (ALEXANDER, 2011). As can be seen, the three teachers have a graduate degree (either Master or a PhD) in languages or Applied Linguistics. Although their background as students covers different decades, they did not experience the recent internationalization movement of the Brazilian universities as students. Also, apparently, they did not have formal writing classes in English. Their socialization derived mostly from self-study, previous instructors’ feedback provision and an ESP course taken. h They are all female and have been teaching EAP for an average period of 11 years8. During most of this time they were not supervised by a pedagogical coordination. Further details on the participants’ background will be given in the data analysis section. Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 3.1 Participants 395-422, 2022 400 Table 1: Instructors´ profiles Table 1: Instructors´ profiles Tutor Degree Place Date Background on ESP/EAP F e e d b a c k experience as students A Master in Applied Linguistics PhD in Education p r i v a t e university and th en p u b l i c university (the same she taught a s a n E A P instructor) 1 9 8 0 ´ s (graduation) 1990´s (Master’s a n d P h D degrees) Master- courses on ESP- focused on reading had an English instructor who helped her a lot with feedback had an English i n s t r u c t o r who provided clear detailed feedback N Master in English Language and Literatures p u b l i c university (the same she taught a s a n E A P instructor) 1 9 9 0 ´ s (graduation) 2000´s (Master’s degree) took ESP course in USA- business writing and oral presentations self-learning a s a n undergraduate r e c e i v e d feedback from her instructors R M a s t e r a n d PhD in English Language and Literatures p u b l i c university (the same she taught a s a n E A P instructor) 1 9 7 0 ´ s (graduation) 2 0 0 0 ´ s (master´s and PhD degrees took Academic writing course in Portuguese did not take ESP/EAP courses in English a s a n undergraduate received general comment (good, very good) and a grade no feedback Source: own author Source: own author Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 3.2 Data collection The participants were interviewed twice, totalizing six interviews. Although the two semi-structured interviews focused on feedback provision, the present analysis investigates another relevant topic that emerged out of the data: teachers’ beliefs about academic writing and its influence on their practice. gl The interviews were conducted in English and transcribed for content analysis. The first aimed to gather background information on their studies and their experience with EAP learning and teaching. The second, which was a stimulated recall interview, aimed to obtain information on the teachers´ writing tasks and the feedback given to students. The writing assignments given and the course syllabus were collected from the instructors. The first interview occurred after one month of the beginning of the courses and the second, at the end of the course period. The assignments were collected during the course and provided by the teachers. Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 401 10 The questions were generated from the coding of the data rather than being elaborated beforehand. The initial goal of the data collection did not aim to investigate beliefs but instead the feedback practice of the teachers. 3.3 Data coding The interview data were analyzed using conventional content analysis procedures of codification (HSIEL; SHANNON, 2005) which could reveal the teachers’ beliefs about academic writing and how they influenced their practice as manifested in the assignments collected. The themes – academic writing beliefs, academic writing instruction, factors affecting academic writing instruction – emerged from the data after extensive reading of the transcripts and were divided into subthemes which were followed by excerpts of the interviews. The subthemes for theme 1 were language focused and product-focused approaches to writing, genre and skills; for theme 2, they were genre, skills and techniques; for theme 3, negative influences9. The subthemes and themes generated the research questions below. h l The syllabi and the written assignments were analyzed according to the themes derived from the interviews. They were used to verify to what extent the beliefs identified affected their pedagogical practice. 3.4 Research questions10 Based on the codification of the data explained above the following questions were asked: 1) what is the tutors’ background regarding academic writing?, 2) what are the tutors’ beliefs about academic writing?, 3) how do the tutors teach academic writing?, 4) what factors influence this instruction? Thus, question 2 focused on tutors’ beliefs; questions 3 and 4 approached their practice; the first and the fourth attempted to investigate the reasons for the beliefs and their practice. 9 Positive influences were not mentioned by the participants.h Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 9 Positive influences were not mentioned by the participants.h 4.1 Instructors´ background with academic writing Participant A studied at a private university which launched a pioneering ESP project in the 1980s in Brazil (HOLMES; CELANI, 2006). It was carried out in association with the British Council, which sent to the country several Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 402 English scholars for its implementation. The project developed reading skills among the Brazilian tertiary student population who had to learn how to read academic texts despite their little knowledge of the language. Writing in English or publishing in it was not an academic demand as it is nowadays. For this reason, her training was probably on ESP reading courses rather than on writing instruction. AI1: When I finished my MA, it was in (…) a long time ago. (…) And then we had material development of the steps of develop material and I had a course on English for specific purpose, methodology and then we learned everything concerning this kind of methodology how to build up materials also procedures in classroom to work with awareness the students this is to help students to become independent. AI1: I took many disciplines in EAP Material Development … although he was not my tutor he also helped me when I was writing my dissertation. Participants N and R took their undergraduate and graduate studies at the same university, which was not involved in this ESP project. Instructor A also took her PhD at this same public university. They all worked at this same public university language center. This lack of experience with ESP during the undergraduate studies might explain N’s seeking professional development in the USA and R’s not approaching genres more extensively in her course. NI1: As a student yeah I took EAP courses in the United States I took there writing business writing and how to give a presentation in English. RI1: As a student I don’t remember I don’t think we had much of this EAP orientation in class I didn’t do much of that through the teaching. NI1: As a student yeah I took EAP courses in the United States I took there writing business writing and how to give a presentation in English. RI1: As a student I don’t remember I don’t think we had much of this EAP orientation in class I didn’t do much of that through the teaching. 4.1 Instructors´ background with academic writing N does not say much about this EAP course maybe because it was easy: NI1: (…) grading the writing that we did and the teachers making some comments brief comments grading the paper because it was paper I think it was all writing not something big it was quite easy I thought but the teachers wrote something on the paper on the essay. Instructor R reports an influential experience with an academic writing course in Portuguese offered by her colleague from the language center. This person motivated her to follow the same approach to academic writing instruction in English: Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 403 RI1: I did a course here when I was doing my Master with the teacher of Portuguese and she did that in class academic writing in Portuguese (…) I thought it was very nice interesting so it was not a kind of course formal coursebook like content and grammar and style, (…) at the time I was teaching academic discourse of all areas, not only biological and she suggested: why don’t you do what I did in my course. And I started to I really love it it was something nice This colleague’s piece of advice reveals the lack of teacher education in this setting and, for this reason, R had to be proactive to seek for professional development. Her experience with a writing class, even in L1, provided her a way to teach her own academic writing courses in English. g g R also refers to the influence of thesis/dissertation writing into her socialization to academic writing in Portuguese as a parameter to understand it in English. Here, we can see the assumption that basic skills of writing such as clarity and coherence are transferable: RI1: I’d take my example as a Master ,PhD [student] when I did my thesis all this the demand of something correct clear with coherence that I learned when doing my academic paper here not before in the context The interviews clearly show that, as students, the instructors had their socialization into academic writing hampered as there was little support such as lack of clear feedback and of a writing process or the assessment based on one single text. AI1: Basically producing writing papers... 4.1 Instructors´ background with academic writing and also sometimes… we had the presentations… but the … we usually had to write a paper in the end of the course. NI1: (…) there were not a lot of assignments… they just had to ask one paper… and then you had to write one paper… and you should do it very well, because they’d score it 10 RI1: Assessment was really conventional information, twice a semester …. More or less AI1: Basically producing writing papers... and also sometimes… we had the presentations… but the … we usually had to write a paper in the end of the course. NI1: (…) there were not a lot of assignments… they just had to ask one paper… and then you had to write one paper… and you should do it very well, because they’d score it 10 RI1: Assessment was really conventional information, twice a semester …. More or less AI1: Basically producing writing papers... and also sometimes… we had the presentations… but the … we usually had to write a paper in the end of the course. NI1: (…) there were not a lot of assignments… they just had to ask one paper… and then you had to write one paper… and you should do it very well, because they’d score it 10 RI1: Assessment was really conventional information, twice a semester …. More or less AI1: Basically producing writing papers... and also sometimes… we had the presentations… but the … we usually had to write a paper in the end of the course. The feedback provision is problematic. For example, R is very emphatic about the lack of feedback. The sentence below summarizes her reaction to the description of feedback provision in her institution: general assessment of their performance without further comments to help students understand this assessment: Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 404 I: And could you figure it out? For example you got a good grade and they [other classmates] didn´t because there was just a good or a very good … could you together [because students studied in group to help each other] decide? RI1: Grammar ok ...it was really punctual... in terms of … you write about an author, describe … discuss something … but the others were more difficult to see why she [a student] got 0 and I got 5. This lack of feedback was compensated later by further professional experience:r RI1: I couldn’t have from the teacher [in undergraduate studies] any signalization of my language awareness. I discovered that later when I left the university and started working as an English teacher at British Council in [city] … and there were teachers there that I learned there teaching… at that time there g NI1: … I think the learning [in undergraduate studies] was a little bit faulty because of this, I learned a lot by myself later on g NI1: … I think the learning [in undergraduate studies] was a little bit faulty because of this, I learned a lot by myself later on On the other hand, A’s report reveals the positive experience with feedback due to the dedicated teacher whose practice she described in detail. AI1: Yeah depends on the subject the disciplines I took and usually we had both types … oral and written feedback but I had a very very very special professor for me he is an example.(…) I learned many things with him but he used to give us feedback he used to give us specially when we finished the paper he also asked us to submit the paper if we wanted and not to get a better score I remember specially a paper he gave me the feedback and it was about 5 pages of comments very detailed he just put the number and wrote the comments and then it was very easy to rewrite the paper because you had just to follow. AI1: Yeah depends on the subject the disciplines I took and usually we had both types … oral and written feedback but I had a very very very special professor for me he is an example.(…) I learned many things with him but he used to give us feedback he used to give us specially when we finished the paper he also asked us to submit the paper if we wanted and not to get a better score I remember specially a paper he gave me the feedback and it was about 5 pages of comments very detailed he just put the number and wrote the comments and then it was very easy to rewrite the paper because you had just to follow. In sum, based on the interviews, these instructors did not have any formal EAP instruction, writing classes or continuous EAP teacher development. 4.1 Instructors´ background with academic writing RI1: The feedback we got in English was none, was none, was none. RI1: I remember there was a teacher ... who gave us literature and she used to write a comment, she was one of the exceptions … write a comment on the margin … like good … really good and then the mark in numbers but the others would just put the marks… This lack of feedback was compensated later by further professional experience:r This lack of feedback was compensated later by further professional experience:r This lack of a rich background in academic writing likely influenced the beliefs about it and its instruction (CROSS, 2009; TORNER ET AL, 2010). Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 405 4.2 Instructors´ beliefs about academic writing in English Table 1 provides information on the academic writing courses the participants taught. This information was obtained from the first interview and the objectives, content, and pedagogical tools sections of the syllabi: Table 2: the academic writing courses Tutors Name of the course Objectives11 Content Pedagogical tools A Academic English for graduate students To develop students’ autonomy To help students develop strategies to use English in oral and written forms To integrate the fours skills in the academic context To make academic oral and written genres familiar to the students Emphasis, repetition, rhetorical questions in written texts12, symbols and abbreviations in note taking, Abstract writing, Citations Dictionaries group work (I) Moodle (I) explicit and implicit feedback on students’ production (I) interactions N English for academic writing To write paragraphs and abstracts To analyze the most common linguistic and organizational structure of academic genres To review grammar and vocabulary related to academic texts To use corpora To identify the most common mistakes to foster students’ autonomy (I) Organization, writing and revision of paragraphs and abstracts, sections of the research article and their linguistic structures, academic vocabulary, verb tense, passive voice, modals, hedging Discourse markers, corpora and dictionaries, word combinations genres (résumé, letter of recommendation, cover letter (I) and abstract) explicit feedback on students´ production (I) R English for academic writing in life sciences To know linguistic structures related to the academic text To review crucial grammatical structures to be used in academic writing To make students edit their own texts Paragraph structure, abstracts, verb tenses, hedging, passive voice, prepositions, adverbs, academic vocabulary, editing and paraphrasing skills plagiarism whole class edition of students’ texts led by the instructor , lectures Table 2: the academic writing courses 11 This table was created based on the syllabus and on the interviews. I stands for interview and the absence of a letter means syllabus. 12 Only the writing component of the course was analyzed and the content related to it is listed here. 12 Only the writing component of the course was analyzed and the content related to it is listed here. Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 406 Based on the data, the three participants seemed to believe academic writing in English is primarily focused on grammar and vocabulary and is product-based. Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 R I1: (…) mistakes fossilize so if you are not aware they will stay forever Despite the fact that A taught the genre abstract, she also focused on linguistic structures as a way to understand the moves of the genre. This will be addressed later. Their beliefs about academic writing are also revealed by the skills these instructors promoted in their classes. 4.2 Instructors´ beliefs about academic writing in English Her feedback and discussion of texts (as will be seen in excerpt on editing skills) centered around grammar. RI1: I look at the text and give them the feedback from the point of view of something academic with scientific style very well made (…) I really demand this scientific demand, let´s say rigor they have to have… without mistakes and being clear and with the grammar parts really well well done R I1: (…) mistakes fossilize so if you are not aware they will stay forever 4.2 Instructors´ beliefs about academic writing in English This approach to writing, based on behaviorism, claims that this skill is taught through models and extensive focus on grammar correction (ZAMEL, 1976). N’s and R’s syllabi contain a list of linguistic items as shown in table 1. This belief could possibly originate from their own past language learning experience at a time in which a structuralist view of language was prevailing. In the interviews, this perception becomes evident when they were asked about the focus of their feedback or when describing the course syllabus: A I1: Language mistakes, organizations, organizations of ideas, the structure of the paper of the composition (…), collocations, vocabulary, basically A I1: Language mistakes, organizations, organizations of ideas, the structure of the paper of the composition (…), collocations, vocabulary, basically N I1: (…) I write a comment, I write something more correct, I correct their English R I2: I have this [course syllabus] based on the content which is this part of the course we did grammar points like the academic vocabulary. I have exercise with academic vocabulary and then grammar grammar grammar and paraphrase and then I follow that in searching as I said a lot in the first 4 weeks, and then a little by little by the end of the semester with their own texts [each student´s text being discussed by the whole class] The types of exercises given also reveal the assumption that writing is taught by teaching and correcting grammar. For example, N provided structural exercises on verb tenses and quizzes on collocations, countable and uncountable nouns whereas R focused on proofreading skills. N I1: The organization, the structure, if written if they follow the correct structure that I taught …the choice of words, you know … I tell them that I am going to analyze … verb tenses … choice of words, vocabulary, technical words and then grammar I mean …collocations, linking words, collocations, the use of noun phrase As shown before, R taught her writing classes following the Portuguese teacher style. From the excerpt above it can be noticed that the first part of the course involved a grammar review, whereas the last part focused on students’ texts Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 407 discussion. Her feedback and discussion of texts (as will be seen in excerpt on editing skills) centered around grammar. discussion. Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 (a) Summarizing skills AI2: The moves … if they are correct and First of all … he had to write this abstract based on his reading so he was interested to understand how he could get the main ideas because in this article, the ideas were not very clear … the moves of the abstract so they had to read and try to put in a proper sequence and then to show to see if he was using the patterns of the abstract … and also his English usage , grammar and vocabulary and he had all kinds of RI2: (…) part of the lesson, I still give something like now between paraphrase and plagiarism RI2: (…) part of the lesson, I still give something like now between paraphrase and plagiarism R I2: Basically the same here it was a text I gave them, 3 texts one about pollution and animal agriculture and the other about in biological area for them to choose one and make a summary, make a paragraph (…) The only aspect of the first excerpt to be pointed out here refers to her focus on grammar and vocabulary in the summaries to be written in the form of abstracts. This particular use of the genre abstract will be discussed later. h p g The last two excerpts show R’s emphasis on important elements of a summary: paraphrases and plagiarism. Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 408 (b) Editing skills were taught through group work in the case of A, instructor’s guidance to the whole class in the case of R or lists of words or websites and translation tips in the case of N. These practices seem to be fostering students’ autonomy. For example, N clearly stated that it was one of her teaching goals (“the students should be a little more autonomous in the way they write in the way they search”) AI1: Through the process … as I said … I like talking to the students and making them work in groups and reflect upon what maybe the mistakes they made. Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 13 She refers to Lei de Diretrizes e Bases da Educação. In English, Law of Directives and Bases of National Education. FGTS stands for Fundo de Garantia por Tempo de Serviço. In English, Time of Service Guarantee Fund. (a) Summarizing skills RI2: (…)during the lesson we go to the board and everything I put here I ask and then we change then we change everything sometimes we change 2 lines or they change the lines, the rest of the group doesn’t have to change , because we are here to discuss unless they really want to of course the owner of the text wants to know the correct form and he’s going to write and erase and write again edit it the text . (…) So they come with I expect them to give the suggestions first did you see anything wrong, anything different or not clear in the first sentence? ) N I2: (…) they have learned about verbs, (…) search material on the internet, or dictionary they have used a variety of dictionaries, NI2: (…) they know I gave them ways and tools, and where to search. For example, this feedback from this website that teaches translation, words that we translate from Portuguese, names of laws, and things regarding our laws legal system The instructors’ concern for the development of students’ autonomy may reflect their own experience with EAP learning and teaching grasped by the interviews and characterized by their autonomy and self-study. Moreover, the editing skill reflects a structured focused belief about writing. It is possible that the instructors see their roles as language teachers rather than specialists of or knowledgeable about the specificity of EAP discourse; that would be left for the advisors. Moreover, the language is seen as a system of structures devoid of social contextualization. It is worth remarking the role of translation in N´s course. She provided lists of websites that could assist students with translating their texts, especially Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 409 very specific Brazilian terms. This might indicate that her approach to academic writing instruction is sensitive to cross-linguistic issues. N I2: (…) there is another name for lei de diretrizes the base13 …this is something we should discuss, I tell them there is a website you can find all these names FGTS, NI1: (…) a kind of genre... of course… it was a genre… I didn’t know it was a genre, but you had to follow a format you know there was not much move away from the format required by the teacher NI1: (…) a kind of genre... of course… it was a genre… I didn’t know it was a genre, but you had to follow a format you know there was not much move away from the format required by the teacher NI1: (…) a kind of genre... of course… it was a genre… I didn’t know it was a genre, but you had to follow a format you know there was not much move away from the format required by the teacher A’s excerpt above reveals that this genre is understood as linguistic structures composing the moves. Moves can be identified not only by these formulaic expressions but also through the meaning of the sentences. It seems then that A uses the linguistic expressions rather than meaning to identify the moves. Also, her understanding of the abstract genre does not encompass the macro social relations or functions it has in a particular social activity. Her correction of the students’ abstracts does not address these social issues either, being limited to grammar issues or to the formulaic expressions. For example, she provides the following comment on a piece: “may is a modal. We use the infinitive without to after it”. Or she underlines linguistic structures that signal a move of the abstract: “Prior studies have linked”/”The analysis included”. h Moreover, there seems to be some confusion about the social function of the genre abstract as she uses it to guide students to summarize a piece of news. AI2: then as homework they had to transform .. a text they read a text … it was not a journal… it was … I can’t remember where it was published … AI2: then as homework they had to transform .. a text they read a text … it was not a journal… it was … I can’t remember where it was published … 4.3 Instructors’ approach to teaching academic writing in English Overall, their approach to writing reflects the belief discussed above: writing as a language accuracy exercise detached from social contextualization. N and R list grammar and vocabulary items in the goal and content sections of their syllabi. Rewriting − a common process approach technique − was applied to reinforce grammar learning in A’s and N´s classes. In relation to A’s writing part of her course, the genre abstract was approached as a linguistic structure format. One could argue that this contextualization could be provided in the classes. However, even if it was the case, not addressing this information at all in the interviews or in the feedback provided indicates the lower status this information has in the writing courses compared to language work. Although A and N taught genres in their courses, they focused on the linguistic structures used in these genres. The following excerpts show how the instructors understood the concept of genre: a sequence of moves and a format to follow decontextualized of a broader social practice: AI2: (...) I work with phrases that marked the movements the moves of the abstract so when I correct I try to do the same in order to see for instance in this study they investigated it’s typical of purpose of the abstract the analysis included so it’s typical of the methodology and results and things like that so I try to see the movements according to the sequence they have studied (…) for your reader to understand it better you should follow the sequence we have learned … Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 410 NI1: (…) a kind of genre... of course… it was a genre… I didn’t know it was a genre, but you had to follow a format you know there was not much move away from the format required by the teacher As seen in table 2, N also teaches some genres: NI2: There were 2 letters [recommendation and cover letters] but I gave them , because it’s a matter of copy in the correct phrase I gave them a large list of reminders (…)should have some creativity involved but not a lot this is a genre that you should, there are some things that are accepted and not accepted so know how to start, dear, very truly yours not best wishes, best regards because this is formal ok NI2: This assignment is writing their own résumé in English … and as a résumé is a writing that requires some technique I teach them previously what they should include,(…) I teach them how to write a résumé that could be used in any field of work – alright?! Write an objective, write with an aim so I give examples I use a blackboard and they copy the information that I write about the Education How they should write about Education How they say I have a BA, a BSC how they write about their work experience, post and responsibilities the period I write all the items including in the résumé Although it was not possible to examine further N’s knowledge on genre theories in the data collection, this excerpt reveals that the social aspect of the concept is overlooked to give prominence to its linguistic features which becomes the way to address the text. It is noteworthy the use of “proper sequence” by A and “matter of copy in the correct phrase” by N. They reveal a view of genre as formulaic texts (JOHNS, 2011; TARDY, 2016) deprived of a rhetorical dimension - social purpose, audience, sociohistorical context (PERALES-ESCUDERO; ELSEN; CRUZ, 2017). Furthermore, the correction of the students’ productions in these genres focused on translation (N) or grammar issues (A and N). The social context that affects the conventions of the genre was not addressed either. f Based on their background information, it is possible to hypothesize they have not been familiar with the concept of genre and the concept of language it entails. For this reason, the teachers’ association of a new concept (genre) with an old one (writing as language focused) turned the former into a formula. hi These findings, despite being limited, pinpoint the need for structured and continuous EAP teacher education in which genre knowledge could be developed among these instructors. I: is it something from a scientific journal or …? A: Not from scientific journal ... scientific magazine … it was a text commenting on a piece of research .. and then they had to write an abstract based on the data presented in this article AI2: The moves if they are correct and first of all he had to write this abstract based on his reading so he was interested to understand how he could get the main ideas because in this article, the ideas were not very clear the moves of the abstract so they had to read and try to put in a proper sequence and then to show to see if he was using the patterns of the abstract and also his English usage , grammar and vocabulary and he had all kinds of14 14 This exploratory study unfortunately did not investigate further the reasons for the participants´ actions. In this example, if we could ask the instructor the reasons behind this task could provide further information about her understanding of genre. Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 411 As seen in table 2, N also teaches some genres: As Worden (2018) remarks, genre knowledge as part of teachers’ content knowledge has a crucial impact on the students’ literacy experiences that could break this vicious circle of perpetuating a structuralist view of language. In other words, teacher education could promote significant changes in the literacy practices of teachers and, by extension, of students. Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 412 In the excerpts above, it is interesting to notice that the instructors seem to perceive the features of the genres taught – the sequence, the format, the linguistic structures used, the (lack of ) creativity – as given. They accept these features and pass them along without connecting them to a broader social context and the function the genre plays in it – which are the very reasons to explain the features they are teaching in the first place. As can be seen in table 2, R did not use genres in her classes. Her course had two parts: an extensive focus on grammar followed by the discussion of students´ texts by the whole class and guided by her. The text required was the one students were writing at that moment for their graduate degree. RI2: (…)… 1, 2 pages of your writing shows how you write so you don’t have to show me 10 pages of your writing, you are going to repeat your mistakes it’s like recurring mistakes like collocations and etc The above excerpt also illustrates R’s approach to EAP writing: the text, regardless of any social context, is a manifestation of grammar which should be corrected. As seen in research question 1, this excerpt illustrates the teacher’s belief about academic writing as a set of linguistic structures and as a product-focused process. Another aspect of their approach to writing instruction was the role of rewriting. The instructors conceived it either as a second chance given to students, who performed poorly in the assignment, in the case of A or as a common procedure to help students incorporate the feedback received, in the case of A and N. Either way the use of this technique is not surprising considering the teachers’ language and product-based view of academic writing instruction and their focus on error correction and the development of proofreading skills. Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 As seen in table 2, N also teaches some genres: The process writing manifested in this context represents a remedial resource for the students to achieve a better final product rather than the main venue for them to achieve it: AI1: (…) I usually mark the errors if the problem is concerning the content ‘link of ideas then I comment but if it´s a language mistake and I point out and ask them to think about to rewrite or then tell me what the problems were Interviewer: So your feedback then would happen during the process, the students would have a chance to rewrite? Interviewer: So your feedback then would happen during the process, the students would have a chance to rewrite? Interviewer: So your feedback then would happen during the process, the students would have a chance to rewrite? Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 413 NI1: First I’d ask them to retake the exercise again, or do everything again, because and they have 1 week or 2 to give back to me, and they usually do NI1: First I’d ask them to retake the exercise again, or do everything again, because and they have 1 week or 2 to give back to me, and they usually do The process also indicates a wish that cannot be realized due to some constraints such as lack of time: Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 RI1: (…) it is a pity we don’t have much time to have a second draft AI1: (…) when you ask them to rewrite they have to be committed to the task sometimes they aren’t. Not because they don’t want but because they don’t have time available to they have lots of assignments here at their undergraduate courses here at [their university] so it depends basically on that We can see that the rewriting promoted involves students’ making punctual linguistic corrections indicated by A, who likes to use symbols to assist students to identify and understand their mistakes (“I like to work with rubrics ... I think they are very helpful”) (see excerpt below). Rather than consisting of larger modifications at the macrostructural level or in the exposition of ideas, rewriting is an opportunity for the student to show the instructor that he/she figured out the mistake. Similarly the use of rewriting is influenced by the instructors’ beliefs about academic writing. N does not use rubrics (“it is so beautiful, but it doesn’t really work I mean it takes a long time to give the feedback “); she explicitly corrects the language issues of the texts. In sum, no rewriting of the texts is needed as the correction is clear and on the spot; students would just have to copy the text incorporating the corrections. Similarly to genre approach to writing, a process writing approach technique can become a tool to reinforce grammar accuracy and a confirmation of the product-based approach to writing: knowing grammar means knowing how to write (ZAMEL,1976). Differently from A and N, R does not ask for rewriting in her classes. The feedback is given mainly by the tutor either before the whole class as general comments or to specific students: AI1: when I was correcting their homework the material that I asked them to do sometimes there were mistakes then I usually come to the board and explain and sometimes when a friend that uses an expression or mix a mistake they don’t understand they ask me and I always Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 414 explain to them you do this you do that sometimes when they speak to I explain and I always use the board and explain to the class as a whole. NI2: Everybody reads ... 5 minutes to take a look they read as we move along they suggest ways to correct sometimes NI2: Everybody reads ... 5 minutes to take a look they read as we move along they suggest ways to correct sometimes From the discussion above it is possible to see that these EAP instructor’s beliefs about academic writing and its instruction are highly affected by a structuralist view of language reduced to grammar and vocabulary without any social contextualization and present in the pedagogical practice of their instructors. The new concepts that emerged along their practice (pedagogical approaches, genre, rewriting, translation) were adapted to conform to this language view. RI1: (…) it is a pity we don’t have much time to have a second draft RI1: Yeah I do that they discuss the texts in class and later on to the class I give general feedback very good you should pay attention to this or the mistakes that you present on the paper and later on after the class I finish I give more details to the students AI1: I guess … basically the students … the time available … the students´ commitment … I would say …because when you ask them 4.4 Other factors influencing the academic writing instruction Besides beliefs about academic writing, other factors influencing the teachers’ academic writing instruction were identified: students’ motivation and time for the learning process: I: But does that [making the lists N suggested] happen, from your practice, your experience... in your EAP classes (…) does that happen, do the students do that? NI1: (…) the students are worried about their proficiency … the language proficiency exam … to be admitted in the graduate course… many do … they show me … not in their writing courses, because I mean … they don’t have time N compares how reading course students are more committed than writing course ones due to the urgent need to pass an English proficiency test to be admitted in the graduate school. For this reason, students would dedicate more to these courses than to writing. Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 415 to rewrite … they have to be committed to the task … sometimes they aren’t…. not because they don’t want… but because they don’t have time available to (…) to rewrite … they have to be committed to the task … sometimes they aren’t…. not because they don’t want… but because they don’t have time available to (…) NI1: (…) you give them .. you ask them to correct, and they don’t give the exercise back …and when they do … it has been 3 weeks, it makes them no more sense… to work on that… NI1: (…) you give them .. you ask them to correct, and they don’t give the exercise back …and when they do … it has been 3 weeks, it makes them no more sense… to work on that… The two excerpts above show how time management is an issue for the students and how it affects the quality of the teachers’ work. Also, students seem not to fully understand the extra amount of class work that writing courses entail probably due to their lack of experience with it in Brazil. Feedback provision and rewriting in this case do not seem to be related to writing improvement in the students’ perception. h This lack of understanding about learning how to write is also present below and it affects the organization of the writing learning process. I: Did you have a chance to explain to him [his problems in the text]? Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 I: Did you have a chance to explain to him [his problems in the text]? AI1: No … I didn’t because he missed one class and then he appeared and I was to talk to him, but then he arrived late and then I don’t remember what happened but I couldn’t talk to him that day and he said he was travelling again, because he was attending another conference in his area I don’t remember where is .. he told he would come, but he didn’t …I didn’t have the opportunity to talk and even ask him to rewrite his abstracts … Cheating is also another example of students’ lack of commitment to learning how to write that can be a consequence of lack of time and of awareness of what this learning involves: A: (I2: R(a student) she’s (nationality) and she’s about to finish her dissertation she didn’t come, because she went to a conference 3 weeks ago and she told she would travel this week I don’t know if she returned to her country to spend Christmas and the holidays or not but her abstract is very well-written … I underlined the moves (…) But I got all the pieces of writing and she makes a lot of mistakes so I think this one has already been corrected I: You see a difference from the language present here and from another [piece of writing]? I: You see a difference from the language present here and from another [piece of writing]? Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 416 A: Yeah but I can’t tell her I can’t tell her: you didn’t do this. Maybe she has done but somebody has already corrected… A I2: C [a student] presented an abstract that she wrote to me last semester … when she was writing her dissertation. To sum up, students’ lack of time and commitment to the extra-curricular writing courses have a negative impact on the teachers’ work, and obviously, the quality of students’ learning. Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 5 Conclusion Studies on teachers’ beliefs often remark their influence on teaching. This analysis reveals that the instructors’ beliefs concerning academic writing as product and language-focused may have affected their teaching of this skill; for example, through types of written assignments and feedback that did not focus on the specificity of disciplinary discourse or the social context of the genres. Moreover, their little experience with academic writing as social practice may have contributed to the adoption of a more structuralist view of language and writing. f Despite the small sample of the study, the method proved effective to elicit data on beliefs as the interviews focused on instructors’ learning and teaching histories and on the actual feedback present on the assignments rather than explicitly on the topic. The analysis call attention to the need of structured teacher development to assist these professionals so as to better support the academic community in their internationalization demands.ih EAP instruction requires a considerable level of specificity. The academic/ specific component appears in these courses by means of the academic genres chosen to be taught (abstract or texts they are writing) but without a clear connection to social practices. As pointed out by the literature (ALEXANDER, 2007; HUTTNER ET AL, 2009; WU; BADGER, 2009), specificity in academic discourse has also been a challenge for these EAP educators. Such a structuralist view may have increased this challenge. hi y g This view of language also made the teachers redefine concepts from other perspectives like rewriting – from process approach – and genre – from a more social based view of language functioning. As can be seen, these instructors’ adaptation to new demands of EAP courses in Brazil – from reading to writing for publication – can be rather challening. With proper EAP education this burden could be reduced. They could also promote more integration with the Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 417 discourse community members to properly address specificity allowing the students to understand language functioning and the specificity of their areas in the three levels pointed out by Fergunson (1997): sociological, epistemological and linguistic. 5 Conclusion Sustainable professional development could create zones of proximal development (VYGOTSKY, 1987) which could help these instructors move from experienced-based beliefs – derived from past and often times frustrating experiences which also lack good role models – to more scientific concepts of EAP practice such as genre, process writing and feedback provision. Moreover, this education could engage instructors in self-reflection about these beliefs and challenge them in close connection with practice (NEGUERUELA AZAROLA, 2011). p The combination identified here (little background in academic writing with scarce or no transformative interventions in the form of continuous teacher education led to the teachers’ ‘fossilization’ of apparent grounding beliefs (in this case language). These beliefs molded updated concepts acquired along their teaching trajectories. However, this incorporation of new concepts does not necessarily lead to any deeper pedagogical changes. Thus, further studies are needed to verify the impact of the lack of teacher education on EAP teachers’ cognition. p g Despite its small sample, the testimonies of these three instructors might shed light on writing instruction practices of other Brazilian current instructors with a similar profile: teachers who graduated before the internationalization move at Brazilian universities, which brought the urgent need for writing and publishing in English, and with a background in EAP focused on reading skills or on general English courses. They ring a bell on how these in-service teachers respond to the new linguistic needs of academia without adequate teacher education support. These needs require updated views of language and of writing instruction, which can be adequately provided by structured EAP teacher development. In other words, the analysis reveals that continuous EAP teacher education is crucial to promote development and reconfiguration of beliefs that can foster better EAP teaching. EAP teaching is the firm basis of any internationalization policy for higher education. ABREU-E-LIMA, D.M.de et al. O programa Inglês sem Fronteiras e a política de incentivo à internacionalização do ensino superior brasileiro. In: SARMENTO, S.; ABREU-E-LIMA, D.; MORAES FILHO, W.B.(Eds ). Do Inglês sem Fronteiras ao Idiomas sem Fronteiras. Belo Horizonte: Editora UFMG. 2016. p. 19-46. REFERENCES ABREU-E-LIMA, D.M.de et al. O programa Inglês sem Fronteiras e a política de incentivo à internacionalização do ensino superior brasileiro. In: SARMENTO, S.; ABREU-E-LIMA, D.; MORAES FILHO, W.B.(Eds ). Do Inglês sem Fronteiras ao Idiomas sem Fronteiras. Belo Horizonte: Editora UFMG. 2016. p. 19-46. Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 418 ALEXANDER, O. Groping in the dark or turning on the light: routes into teaching English for academic purposes. In: LYNCH, T.; NORTHCOTT,J. (Eds),Educating legal English specialists and teacher education in teaching EAP. Proceedings of IALS teacher education symposia, 2004 and 2006. Institute for Applied Language Studies, University of Edinburgh. 2007 ALEXANDER, O. Exploring teacher beliefs in teaching EAP at low proficiency levels. Journal of English for Academic Purposes, v. 11, n.2, p. 99-111.2011. BASTURKMEN, H. LSP teacher education: review of literature and suggestion for the research agenda. Ibérica,v. 28, n. 1 p. 17-33.2014. BARCELOS, A.M. F. Researching beliefs about SLA: A critical review. In:KALAJA,P; BARCELOS,A.M.F. ( Eds.), Beliefs about SLA: New research approaches. Dordrecht: Kluwer Academic Press, p. 7–33, 2003. BARCELOS, A. M. F.; KALAJA, P;. Beliefs in second language acquisition: Teacher. In: Chapelle, C. (Ed). The Encyclopedia of Applied Linguistics, Londres: Blackwell, p.491- 496, 2013. BERGMAN, L. Supporting academic literacies: university teachers in collaboration for change. Teaching in Higher Education, v.21, n.5, p.516-531.2016. CAMPION, G.C. ‘The learning never ends’: exploring teachers´ views on the transition from General English to EAP. Journal of English for Academic Purposes, v. 23, n. 1 p. 59- 70, 2016. CHARLES, M. English for academic purposes. In: PALTRIDGE, B; STARFIELD, S. (Eds.). The Handbook of English for Specific Purposes. Londres: John Wiley & Sons. p. 137-153, 2014. CRISTÓVÃO, V.L.L.; VIEIRA,I.R. Letramentos em língua portuguesa e inglesa na educação superior brasileira: marcos e perspectivas. Ilha do Desterro, v. 69, n.3, p. 209- 221, 2016. CROSS, D.I . Alignment, cohesion, and change: examining mathematics teachers´ belief sttuctures and their influence on instructional practice. Journal of Mathematics Teacher Education v.12, n.5, p. 325-346, 2009. DUDLEY-EVANS, T.; ST JOHN, M.. Developments in English for Specific Purposes. Cambridge: Cambridge University Press, 1998.301p. FARRELL, T.S.C.; IVES, J.. Exploring teachers´ beliefs and classroom practices through reflective practice: a case study. Language Teaching Research v.19, n.5, p. 594-610, 2015. FARRELL, T.S.C.; BENNIS, K.. Reflecting on ESL Teacher Beliefs and Classroom Practices: A Case Study. RELC Journal v. 44, n. 2, p.163-176, 2013. Rev. Bras. Linguíst. Apl., v. REFERENCES 22, n. 2, p. 395-422, 2022 419 FERGUSON, G. Teacher education and LSP: the role of specialised knowledge. In:. HOWARD, R.; BROWN,G. (Eds). Teacher Education for Language for Specific Purposes. Bristol.PA: Multilingual Matters. 1997, p.80-89. FERREIRA, M.M. Perspectivas de estudo sobre letramento acadêmico em inglês e influências no Brasil. In: ARAÚJO,J. (Ed). Gêneros e Letramentos em Múltiplas Esferas de Atividade. Campinas: Pontes. 2016, p.171-193. FINARDI, K. R.; PORCINO, M. C.. O papel do inglês na formação e na internacionalização da educação no Brasil. Horizontes de Linguística Aplicada, v. 14, n. 1, p. 109-134, 2015. FLOWERDEW, J.; PEACOCK, M. Issues in EAP: a preliminary perspective. In: J. FLOWERDEW,J.; PEACOCK,M. (Eds), Research perspectives on English for Academic Purposes. Cambridge: Cambridge University Press. 2001. p..8-24. HOLMES, J.; CELANI, A. A. Sustainability and local knowledge: The case of the Brazilian ESP Project 1980–2005. English for Specific Purposes v.25, n.1, p.109-122, 2006. HUTCHINSON, T.; A. WATERS. English for Specific Purposes: A Learning-Centred Approach. Cambridge: Cambridge University Press.1987. 185p. HSIEH, H-F.; SHANNON, S. E. Three approaches to qualitative content analysis. Qualitative Health Research v.15. n.9, p. 1277-1288, 2005. HUTTNER,J.; SMIT, U. ; MEHLMAUER-LARCHER,B. (2009). ESP teacher education at the interface of theory and practice: introducing a model of mediated corpus-based genre analysis. System v.37, n,1, p. 99-109,2009. HYLAND, K. Specificity revisited: How far should we go now?. English for specific purposes, v. 21, n. 4, p. 385-395, 2002. HYLAND, K. AND HAMP-LYONS, L. EAP: issues and directions. Journal of English for Academic Purposes v. 1 n.1 p.1-12, 2002. GIRI, M.M.; MARTINS, C.B.M.J. Os processos formativos do professor de Língua Inglesa no curso de Letras da UTFPR e no Programa Idiomas sem Fronteiras. Revista Letras Raras, v.6, n.1, p.58-82, 2017. JOHNS, A. M. (2011). The future of genres in L2 writing: fundamental but contested instructional decisions. Journal of Second Language Writing v.20, n.1, p.56-68, 2011. NEGUERUELA-AZAROLA, E. Beliefs as conceptualizing activity: A dialectical approach for the second language classroom. System, v. 39, n. 3, p. 359-369, 2011. PALTRIDGE, B; STARFIELD, S. (Ed.). The Handbook of English for Specific Purposes. Londres: John Wiley & Sons, 2014.560p. Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 420 PERALEZ-ESCUDERO; ELSEN, P.B.; CRUZ, M.R.R. Variation in pre-service EFL Teachers’ Implicit Theories of Reading: A Qualitative Study. MEXTESOL Journal v.41, n.4 ,p.1-18, 2017. RICHARDS, J. C.; GALLO, P. B.; RENANDYA, W. A. Exploring teachers’ beliefs and the processes of change. Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 ZAMEL, V. (1976). Teaching composition in the ESL classroom: what we can learn from research in the teaching of English. TESOL Quaterly, v. 10, n.1, p.67-76, 1976. ZHENG, H. A review of research on EFL pre-service teachers´ beliefs and practices. Journal of Cambridge Studies v.4, n.1, p.73- 81, 2009. REFERENCES PAC journal, v. 1, n. 1, p. 41-58, 2001. SKOTT, J. The promises, problems and prospects of research on teachers´ beliefs. In: FIVES, H. ; GILL, M.G. (Eds).International Handbook of Research on Teachers´ beliefs. , New York: Routledge. 2015. p.13-30. TAGLE, T. ET AL. Pre-service EFL teachers´ beliefs about teaching writing: a case study in two Chilean Universities. Electronic Journal of Foreign Language Teaching v.14, n.2, p. 187-200, 2017. TAILLEFER, G. “CLIL in higher education: the (perfect?) crossroads of ESP and didactic reflection”. ASp 63, 31-53, 2013. TARDY, C.M.. Beyond Conventions: genre innovation in academic writing. Ann Arbor: University of Michigan Press.2016.198p. TENG, L.S. Changes in teachers’ beliefs after a professional development project for teaching writing: two Chinese cases. Journal of Education for Teaching v. 42, n.1, p.106- 109, 2017. TSUI, A.B.M. (1996). Learning how to teach ESL writing. In: FREEMAN, D.; RICHARDS, J.C.(Eds). Teacher learning in language teaching. Cambridge: Cambridge TSUI, A.B.M. (1996). Learning how to teach ESL writing. In: FREEMAN, D.; RICHARDS, J.C.(Eds). Teacher learning in language teaching. Cambridge: Cambridge University Press.1996. p.97-119. TORNER, G. et al. Understanding teachers´ actions in the classroom by applying Schoenfeld´s theory Teaching in Context: reflecting on goals and beliefs. In: SRIRAMAN, B.; ENGLISH, L. (Eds) Theories of Mathematics Education. Heidelberg: Springer, 2010.p.401-420. VYGOTSKY, L. S. The collected works of L. S. Vygotsky: Vol 1. New York: Plenum, 1987.350p. WOODS, D. Teacher cognition in language teaching: Cambridge, Cambridge University Press. 1996.316p. WORDEN, D. Mediation and development of a novice L2 writing teacher’s pedagogical content knowledge of genre. Journal of English for Academic Purposes v.34, n.1, p.12-27, 2018. Rev. Bras. Linguíst. Apl., v. 22, n. 2, p. 395-422, 2022 421 WU, H. AND BADGER, R. G.. In a strange and uncharted land: ESP teachers´ strategies for dealing with unpredicted problems in subject knowledge during class. English for Specific Purposes v. 28 n. 1, p. 19-32, 2009. ZAMEL, V. (1976). Teaching composition in the ESL classroom: what we can learn from research in the teaching of English. TESOL Quaterly, v. 10, n.1, p.67-76, 1976. ZHENG, H. A review of research on EFL pre-service teachers´ beliefs and practices. Journal of Cambridge Studies v.4, n.1, p.73- 81, 2009. 422
https://openalex.org/W2747254626	https://europepmc.org/articles/pmc5570904?pdf=render	English	null	A Strategy for Nonmigrating Highly Plasticized PVC	Scientific reports	2,017	cc-by	5,152	A Strategy for Nonmigrating Highly Plasticized PVC OPEN Jun Yuan & Bin Cheng Nonmigrating highly plasticized PVC was prepared based on a new compound that acts as a plasticizer that was derived from di(2-ethylhexyl) 4-hydrophthalate and chlorinated paraffin-52. The as-prepared PVC has a plasticizing efficiency as high as DOP and its migration is totally suppressed. Unlike other reported methods, this approach increases the interaction between phthalate and PVC to suppress its migration, not simply to enlarge its molecular size (or molecular weight). This methodology is highly versatile for producing the desired non-leaching PVC with a permanent plasticizer effect. Received: 17 May 2017 Accepted: 2 August 2017 Published: xx xx xxxx Poly(vinyl chloride) (PVC), as the second largest polymeric material on the market1, is widely used in all areas of human activity, for example, building materials, medical devices, food packaging, clothing and toys. However, PVC is a strongly polar polymer in which force between molecules is very strong. To show plasticity, PVC must be heated to a certain temperature, which is challenging for PVC moulding products. Therefore, in PVC materi- als, plasticization is necessary to reduce softening temperature, decrease melt viscosity and increase mobility to improve its processing properties and product flexibility2. Plasticization can be achieved internally by introducing into the original polymer a comonomer, which reduces crystallizability and increases chain flexibility, or exter- nally by compounding PVC with a low molecular weight compound. The low molecular weight compound was defined as a plasticizer by The Council of the International Union of Pure and Applied Chemistry. Internally plas- ticized PVC can maintain its performance over long-term use because there is no plasticizer migration. However, internal plasticization is less efficient and generally has an unsatisfactorily narrow use temperature range. Using external plasticizers, it is very convenient to select from a variety of plasticizers depending on the desired prop- erties and processability of PVC. Plasticizer is not only a processing aid but also an important component to determine the performance and application of PVC. Therefore, plasticizers are the most important commercial application of PVC. However, plasticizers can leach out of flexible PVC, changing the performance of PVC with age and contaminating the environment. Phthalate esters are still the most powerful plasticizers and dominate the plasticizer market due to their great plasticizing effect and low-cost, although phthalate plasticizers can migrate to the surface, leading to material performance degradation and a negative influence on human health3–16. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 17 May 2017 Accepted: 2 August 2017 Published: xx xx xxxx SCieNtifiC RePortS \| 7: 9277 \| DOI:10.1038/s41598-017-10159-7 A Strategy for Nonmigrating Highly Plasticized PVC OPEN 1H NMR spectrum of DOP-OH. Figure 2. 1H NMR spectrum of DOP-OH. Figure 2. 1H NMR spectrum of DOP-OH. Figure 2. 1H NMR spectrum of DOP-OH. plasticizer efficiency, and vice versa36. No better methods involving migration/plasticizing efficiency balance for plasticizers have been documented. h d h d hl d ffi ( ) d l In this study, we attached DOP to chlorinated paraffin (CP), acting as a secondary plasticizer in PVC, to pre- pare a new compound giving PVC highly plasticization without migration. Unlike other reported methods, our approach covalently attaches phthalate to chlorinated paraffin, a low molecular weight compound with a structure similar to PVC. We intend to increase the interaction between DOP and PVC to suppress migration, rather than increasing molecular size (or molecular weight). A Strategy for Nonmigrating Highly Plasticized PVC OPEN In response to consumer and regulatory pressures, developing nonmigrating or phthalate-free PVC is very active in past few years. Recently, some other plasticizers instead of phthalate plasticizers, such as biocompatible material and oligomer17–27, have been reported without toxicity. However, compared to phthalate plasticizers, majority of them have a poor plasticizing effect and the plasticizer migration still cannot be avoided. Therefore, phtha- lates might be still used, but its leaking or migration should be prevented. A coating on the surface of PVC was reported to isolate plasticizer from the environment and prevent the leaking of phthalate from PVC plastics28–31. However, the surface coating costs are too high to be commercialized. Another potential approach is covalently attaching phthalate molecules to the main chain of polymers to suppress its migration. For example, Rebecca Braslau et al.32 attached phthalate molecules to all-carbon polymer backbone to develop new polymeric phthalates via (co)polymerization of 4-vinyl phthalate ester. Unfortunately, they did not try them to PVC. In general, their large molecule size hinders diffusion in PVC to suppress migration. However, the rate of diffusion of the plasti- cizer is one of the most important factors determining plasticizer efficiency33, 34. Polymeric plasticizer generally has low plasticizing efficiency. Navarro et al.35 replaced the chlorine on the PVC backbone via nucleophilic substi- tution of thiol groups attached to the benzene ring of di(2-ethylhexyl)phthalate (also known as dioctyl phthalate, DOP, the most common plasticizer), to give totally nonleachable PVC. Unfortunately, although the modified PVC came from a common plasticizer DOP, it had very poor plasticization performance and its glass transition temperature cannot fall below 20 °C without a ratio of attached DOP to PVC below 1.7:1. This is because DOP molecules have been fixed on the PVC backbone and lack the freedom to solvate and desolvate various attach- ments on the PVC molecule. While a low rate of diffusion provides high plasticizer permanence, it results in lower Key Laboratory of Beijing City on Preparation and Processing of Novel Polymer Materials, Beijing University of Chemical Technology, Beijing, 100029, China. Correspondence and requests for materials should be addressed to B.C. (email: chengb@mail.buct.edu.cn) SCieNtifiC RePortS \| 7: 9277 \| DOI:10.1038/s41598-017-10159-7 1 tificreports/ Figure 1. Synthetic Route of the DOP-like Plasticizer. Figure 2. 1H NMR spectrum of DOP-OH. www.nature.com/scientificreports/ Figure 1. Synthetic Route of the DOP-like Plasticizer. Figure 1. Synthetic Route of the DOP-like Plasticizer. Figure 1. Synthetic Route of the DOP-like Plasticizer. Figure 2. Results h Synthesis of the DOP-like plasticizer. As shown in Fig. 1, a commercially available 4-hydroxyphthalic acid was converted into acyl chloride with thionyl chloride, which was then alcoholised with iso-octanol with N,N-dimethyl formamide (DMF) as an acid scavenger. Unreacted 4-hydroxyphthalic acid and iso-octanol was sequentially removed with sodium bicarbonate solution and ethylene glycol. Purification was achieved through column chromatography and the yield was 93%. The 1H-NMR spectrum of DOP-OH is shown in Fig. 2. g p y yh p g As shown in Fig. 3, DOP-OH has a similar structure to DOP, and therefore they may have similar plasti- cization properties. Chlorinated paraffin with chlorination levels of 52% (CP-52) was selected as an alkyl chloride because its chlorine content was similar to PVC. The purified, final DOP-like plasticizer from di(2-ethylhexyl)4-hydrophthalate and chlorinated paraffin (DOP-O-CP52) had a yield of 20.1%. As shown in Fig. 4, the proton peak of aromatic (7.0 to 8.0 ppm) and aliphatic (0.5 to 1.8 ppm) groups should be attributed to DOP-OH attached to CP-52 in DOP-O-CP52. Peaks near 4.3 ppm were from DOP-OH. Glass Transition Temperature. As shown in Fig. 5, glass temperatures (Tgs) of DOP-plasticized and DOP-O-CP52-plasticized PVC were studied with respect to the ratio of plasticizers to PVC. Both Tgs were gradu- ally reduced with the increased addition of plasticizer. DOP-O-CP52 has almost the same plasticizing efficiency as DOP within the ratio range (from 0 to 0.3) of plasticizer to PVC, which indicated the novel DOP-like plasticizer SCieNtifiC RePortS \| 7: 9277 \| DOI:10.1038/s41598-017-10159-7 2 www.nature.com/scientificreports/ Figure 3. Chemical structures of DOP and DOP-OH. Figure 3. Chemical structures of DOP and DOP-OH. Figure 3. Chemical structures of DOP and DOP-OH. could have the same performance as DOP in many applications. Further increases in the ratio of plasticizer over PVC led to decreased plasticizing efficiency of DOP-O-CP52 compared to DOP. However, the system with 1:1 plasticizer over PVC had a Tg below 0 °C, indicating that the system was completely flexible at room temperature and could be used for soft (flexible) PVC products. Migration. To observe the migration of plasticizers, the migration behaviour of the new DOP-like plasticizer in PVC was tested through extraction experiments with n-heptane at room temperature35. As a comparison, migration of DOP was also measured under the same conditions. Discussion High plasticizing efficiency of DOP for PVC is attributed to DOP’s good compatibility with PVC and its high rate of diffusion in PVC, which result from the interaction between DOP and PVC molecules. For plasticiza- tion, intermolecular forces between plasticizer molecules must be as strong as those between the plasticizer and the polymer to be plasticized36, 37. The stronger interaction between plasticizer molecule and PVC molecule, the greater the compatibility If intermolecular forces between the plasticizer molecules themselves is stronger than between plasticizer and polymer, the plasticizer will be less compatible with polymer, possibly losing plas- ticization. Although it has limited compatibility with PVC, chlorinated paraffin as a secondary plasticizer may have a stronger interaction with PVC than other secondary plasticizers because it has a chemical structure similar to PVC. In this study, DOP and chlorinated paraffin were chemically combined into a new compound, DOP-O-CP52. Enlarged molecular size will reduce its diffusion rate, resulting in the decreased plasticizing effi- ciency of DOP in the compound. However, besides DOP, the chlorinated paraffin parts of the new compound will also interact with PVC to mask more sites of attachment between PVC molecules and hinder forces holding PVC chains together, partially compensating the decreased plasticizing efficiency. This may why DOP-O-CP52 has such amazing plasticizing efficiency. With higher DOP-O-CP52 levels in PVC, CP52 in DOP-O-CP52 will form microclusters owing to their lower compatibility with PVC, to retard the movement of DOP-O-CP52 in PVC, resulting in a decline in plasticizing efficiency. fi Figure 6 shown the extraction of plasticized PVCs. Flexible samples with 0.32:1 plasticizer over PVC were selected to ensure a high migration rate of plasticizers. These results indicated that the new DOP-like plasticizer in PVC was very stable. Compared to DOP, the larger molecular size of DOP-O-CP52 could obstruct its migration. More importantly, there was a stronger dipole-dipole interaction between PVC and DOP-O-CP52. In addition to DOP, there is also CP52 in DOP-O-CP52 which can interact with PVC molecules. Therefore, polar parts in DOP-O-CP52 were much larger than in DOP, and CP52 appears to play a key role. Combining CP52 and DOP into one molecule undoubtedly increases DOP’s interaction with PVC, resulting in DOP’s high permanence in PVC. However, stronger interactions between plasticizer and PVC will reduce its rate of diffusion in PVC, leading to lower plasticizing efficiency. A compromise is achieved using DOP-O-CP52, yielding PVC with no migra- tion and high plasticization. Results h A small piece of plasticized PVC was placed in a b l h h l d ll f h h l d h l Figure 4. 1H NMR spectra of CP-52 and DOP-O-CP52. Figure 5. Variation of glass temperature with content of DOP (black stars) and DOP-O-CP52 (red blocks). Figure 4 1H NMR spectra of CP 52 and DOP O CP52 Figure 4. 1H NMR spectra of CP-52 and DOP-O-CP52. Figure 4. 1H NMR spectra of CP-52 and DOP-O-CP52. Figure 5. Variation of glass temperature with content of DOP (black stars) and DOP-O-CP52 (red blocks). Figure 5. Variation of glass temperature with content of DOP (black stars) and DOP-O-CP52 (red blocks). Figure 5. Variation of glass temperature with content of DOP (black stars) and DOP-O-CP52 (red blocks). could have the same performance as DOP in many applications. Further increases in the ratio of plasticizer over PVC led to decreased plasticizing efficiency of DOP-O-CP52 compared to DOP. However, the system with 1:1 plasticizer over PVC had a Tg below 0 °C, indicating that the system was completely flexible at room temperature and could be used for soft (flexible) PVC products. ould have the same performance as DOP in many applications. Further increases in the ratio of plasticizer over VC led to decreased plasticizing efficiency of DOP-O-CP52 compared to DOP. However, the system with 1:1 lasticizer over PVC had a Tg below 0 °C, indicating that the system was completely flexible at room temperature nd could be used for soft (flexible) PVC products. Migration. To observe the migration of plasticizers, the migration behaviour of the new DOP-like plasticizer in PVC was tested through extraction experiments with n-heptane at room temperature35. As a comparison, migration of DOP was also measured under the same conditions. A small piece of plasticized PVC was placed in a bottle with n-heptane as extraction solvent and a small amount of 1, 6-hexamethylene diisocyanate as the internal SCieNtifiC RePortS \| 7: 9277 \| DOI:10.1038/s41598-017-10159-7 3 www.nature.com/scientificreports/ Figure 6. Extraction of plasticized PVC strips with heptane at room temperature (PVC/DOP (black blocks) and PVC/DOP-O-CP52 (solid red circles)). Figure 6. Extraction of plasticized PVC strips with heptane at room temperature (PVC/DOP (black blocks) and PVC/DOP-O-CP52 (solid red circles)). reference. The solution was sampled over time. The amount of plasticizer in the solution was determined with IR spectroscopy based on the calibration curve. As shown in Fig. Results h 6, the DOP-plasticized PVC lost almost all DOP within 4 h, but DOP-O-CP52 did not lose any DOP, even after a 30 h extraction. reference. The solution was sampled over time. The amount of plasticizer in the solution was determined with IR spectroscopy based on the calibration curve. As shown in Fig. 6, the DOP-plasticized PVC lost almost all DOP within 4 h, but DOP-O-CP52 did not lose any DOP, even after a 30 h extraction. Discussion CP52 appears to be a better choice for DOP. The interaction between CP52 and PVC is appropriately strong to maintain both the plasticization and nonmigration of DOP-O-CP52. Chlorinated Paraffins (CPs) are a family of complex mixtures. The chlorination degree and structure of CPs can be widely var- ied, and thus the interaction between CPs and PVC can be regulated. It is speculated that covalently combining phthalates and CP with appropriate chlorination degree and structure can yield a large number of plasticizers with high plasticizing efficiency and the desired nonmigrating performance of PVC.fih fi Overall, a compound combining di(2-ethylhexyl)phthalate and chlorinated paraffin-52 was prepared. This new compound ensured the plasticity of PVC with as high plasticizing efficiency as DOP, without DOP leaking or migration. This approach covalently attaches phthalate to chlorinated paraffin, a low molecular weight compound with a structure similar to PVC. Both plasticization and nonimmigration were maintained. Based on phthalates and chlorinated paraffins with a broad range of chlorination degrees and structures, the approach is highly versa- tile for producing desired PVC with a permanent plasticizer effect and no leaching. Methods M t i l Materials. PVC was from Sumitomo Chemical (Japan), and 4-hydroxyphthalic acid with 98% purity % was from Wuxi Discovery Medical Technology. Thionyl chloride (SOCl2) was analytical grade from Tianjin Damao Chemical Reagent Factory. Iso-octanol was analytical from Tianjin Fuchen Chemical Reagents Factory. SCieNtifiC RePortS \| 7: 9277 \| DOI:10.1038/s41598-017-10159-7 4 www.nature.com/scientificreports/ Chlorinated paraffin-52 was an experimental reagent purchased from Chengdu Kelon Chemical Reagent Factory. Sodium bicarbonate, ethylene glycol and DMF were analytical grade from Sinopharm Chemical Reagent Co., Ltd. Chlorinated paraffin-52 was an experimental reagent purchased from Chengdu Kelon Chemical Reagent Factory. Sodium bicarbonate, ethylene glycol and DMF were analytical grade from Sinopharm Chemical Reagent Co., Ltd. Preparation of the Plasticizer. The 4-Hydroxyphthalic acid (2.2750 g, 0.0125 mol) was weighed into a three-necked flask (100 mL), and DMF (0.3 g) was added. SOCl2 (29.750 g, 0.250 mol, 20 equiv) was dropped with ice cooling through a constant-pressure dropping funnel. The system was heated to 60 °C for an hour. The excess SOCl2 was distilled away under reduced pressure, and iso-octanol (3.9000 g, 0.0300 mol, 2.4 equiv) was added. After the reaction was heated at 65 °C for 2 hours, the reaction solution was washed successively with dis- tilled water, saturated sodium bicarbonate, distilled water, ethylene glycol and distilled water. After the solution was dried over anhydrous magnesium sulphate, a pale yellow liquid was obtained. It was purified by column chromatography on silica gel with petroleum ether/ethyl acetate (v/v = 5/1.5) as the eluent. The pure product (4.720 g) was obtained with a yield of 93.0%. Rf = 0.52 (petroleum ether/ethyl acetate, 5:1.5). 1H NMR (400 MHz, CDCl3) δ: 7.68 (d, 1H, J = 8.4 Hz, Ar-H), 7.00 (d, 1H, J = 2.0 Hz, Ar-H), 6.90 (dd, 1H, J = 8.4 and 2.4 Hz, Ar-H), 4.09–4.25 (m, 4H, 2 × COOCH2), 3.58 (s, 1H, -OH), 1.59–1.72 (m, 2H, 2 × (CH2)2CH-CH2), 1.26–1.44 (m, 16H, 8 × CH2), 0.86–0.95 (m, 12H, 4 × CH3). CP-52 (0.6030 g) and the prepared di(2-ethylhexyl)-4-hydrophthalate hydroxyl compounds (3.2480 g, 8 mmol) were dissolved in 50 ml of cyclohexanone. After potassium carbonate was added, the reaction was per- formed at 65 °C in an N2 atmosphere for 24 hours. The reaction solution was washed successively with distilled water, ethylene glycol, N,N-dimethylformamide (DMF) and distilled water. After the solution was dried over anhydrous magnesium sulphate, a yellow liquid was obtained. Methods M t i l The crude product was purified by column chro- matography on silica gel with petroleum ether/ethyl acetate (v/v = 5/1.5) as the eluent. The pure product was obtained (0.653 g, 20.1%). Rf = 0.72 (Petroleum ether/ethyl acetate, 5:1.5). Preparation of Plasticized PVC Specimens. The plasticized PVC specimens with different amounts of plasticizer (DOP or DOP-O-CP52) were prepared. PVC powder and plasticizer were dissolved in THF, and THF was evaporated to obtain a film, which was further dried under low vacuum. For extraction experiments, 0.50 g of PVC powder and 0.16 g of plasticizer (DOP or DOP-O-CP52) were dissolved in 30 ml of THF, and THF was evaporated. After drying under vacuum, the film was cut into 15 mm × 15 mm pieces. Extraction Experiment. 0.206 g of a plasticized PVC specimen was accurately weighed and transferred into a 10 ml volumetric flask, half-filled with n-heptane. The internal standard 1,6-hexamethylene diisocy- anate(0.020 g) was added. Volume was made up to the mark with n-heptane. The flask was shaken from time to time for a predetermined time period. Two to four drops of the solution were sandwiched between two KBr windows and fixed on a holder for IR measurement. The amount of DOP extracted was determined using a cali- bration curve. Characterization. The 1H NMR spectrum data of the bis(2-ethylhexyl)-4-hydroxy phthalate(DOP-OH), the ester of di(2-ethylhexyl)-4-hydrophthalate and chlorinated paraffin were recorded on an AVANCE III 400 MHz spectrometer with chloroform-D as the solvent. The infrared spectrum to determine the extraction DOP was recorded on the Fourier Transform Infrared Spectrometer 8700. Tg was measured on a Perkin-Elmer differential scanning calorimeter DSC-8000. The sample (approximately 5 mg) was scanned from room temperature (25 °C) to 150 °C at 10 °C/min in a nitrogen atmosphere and quenched to −70 °C at a cooling rate of 150 °C/min, then maintained at −70 °C for 5 min. The Tg value was reported from the second run, and the midpoint of the corre- sponding DSC curve was obtained. SCieNtifiC RePortS \| 7: 9277 \| DOI:10.1038/s41598-017-10159-7 References 1. Braun, D. Recycling of PVC. Prog. Polym. Sci. 27(10), 2171–2195 (2002).h y g g y 2. Rahman, M. & Brazel, C. S. The plasticizer market: an assessment of traditional plasticizers and research trends to meet new challenges. Prog. Polym. Sci. 29(12), 1223–1248 (2004). h p challenges. Prog. Polym. Sci. 29(12), 1223–1248 (2004). g g y ( ) ( ) 3. Gayathri, N. S., Dhanya, C. R., Indu, A. R. & Kurup, P. A. Changes in some hormones by low doses of di (2-ethyl hexyl) phthalate (DEHP), a commonly used plasticizer in PVC blood storage bags & medical tubing. Indian Journal of Medical Research. 119(4), 139–144 (2004).ft g g y 3. Gayathri, N. S., Dhanya, C. R., Indu, A. R. & Kurup, P. A. Changes in some hormones by low doses of di (2-ethyl hexyl) phthalate (DEHP), a commonly used plasticizer in PVC blood storage bags & medical tubing. Indian Journal of Medical Research. 119(4), 139–144 (2004). ( ) 4. Lee, K. Y. et al. Diverse developmental toxicity of di-n-butyl phthalate in both sexes of rat offspring after maternal exposure during the period from late gestation through lactation. Toxicology. 203(1), 221–238 (2004).hhf 4. Lee, K. Y. et al. Diverse developmental toxicity of di-n-butyl phthalate in both sexes of rat offspring after maternal exposure during the period from late gestation through lactation. Toxicology. 203(1), 221–238 (2004).hhf p g g gy 5. Thomas, J. A., Thomas, M. J. & Gangolli, S. D. Biological effects of di-(2-ethylhexyl) phthalate and other phthalic acid esters. CRC critical reviews in toxicology. 13(4), 283–317 (1984). gy ( ), ( ) 6. Janjua, N. R. et al. Systemic uptake of diethyl phthalate, dibutyl phthalate, and butyl paraben following whole-body topical application and reproductive and thyroid hormone levels in humans. Environmental science & technology. 41(15), 5564–5570 (2007). 6. Janjua, N. R. et al. Systemic uptake of diethyl phthalate, dibutyl phthalate, and butyl paraben following whole-body topica application and reprod cti e and th roid hormone le els in h mans Environmental science & technology 41(15) 5564 5570 (2007) application and reproductive and thyroid hormone levels in humans. Environmental science & technology. 41(15), 5564–5570 (2007). 7. Heudorf, U., Mersch-Sundermann, V. & Angerer, J. Phthalates: toxicology and exposure. International journal of hygiene and environmental health. 210(5), 623–634 (2007).ft ( ) ( ) 8. Nagao, T. et al. References Effect of butyl benzyl phthalate in Sprague-Dawley rats after gavage administration: a two-generation reproductive study. Reproductive Toxicology. 14(6), 513–532 (2000).f ( ) ( ) 8. Nagao, T. et al. Effect of butyl benzyl phthalate in Sprague-Dawley rats after gavage administration: a two-generation reprodu study. Reproductive Toxicology. 14(6), 513–532 (2000).f y p gy 9. Lamb, J. C., Chapin, R. E., Teague, J., Lawton, A. D. & Reel, J. R. Reproductive effects of four phthalic acid esters in the mouse. Toxicology and applied pharmacology. 88(2), 255–269 (1987). y p gy 9. Lamb, J. C., Chapin, R. E., Teague, J., Lawton, A. D. & Reel, J. R. Reproductive effects of four phthalic acid esters in the mouse. Toxicology and applied pharmacology. 88(2), 255–269 (1987). gy pp p gy 10. Meeker, J. D., Sathyanarayana, S. & Swan, S. H. Phthalates and other additives in plastics: human exposure and associated health outcomes. Philosophical Transactions of the Royal Society B: Biological Sciences. 364(1526), 2097–2113 (2009). gy pp p gy 10. Meeker, J. D., Sathyanarayana, S. & Swan, S. H. Phthalates and other additives in plastics: human exposure and associated health outcomes. Philosophical Transactions of the Royal Society B: Biological Sciences. 364(1526), 2097–2113 (2009). p f y y g ( ) ( ) 11. Rusyn, I. & Corton, J. C. Mechanistic considerations for human relevance of cancer hazard of di (2-ethylhexyl) phthalate. Mut Research/Reviews in Mutation Research. 750(2), 141–158 (2012). p f y y g ( ) ( ) 1. Rusyn, I. & Corton, J. C. Mechanistic considerations for human relevance of cancer hazard of di (2-ethylhexyl) phthalate. Mutation Research/Reviews in Mutation Research. 750(2), 141–158 (2012). 12. Gupta, R. K. et al. Di-(2-ethylhexyl) phthalate and mono-(2-ethylhexyl) phthalate inhibit growth and reduce estradiol levels of antral follicles in vitro. Toxicology and applied pharmacology. 242(2), 224–230 (2010).f 12. Gupta, R. K. et al. Di-(2-ethylhexyl) phthalate and mono-(2-ethylhexyl) phthalate inhibit growth and reduce estradiol levels of antral follicles in vitro. Toxicology and applied pharmacology. 242(2), 224–230 (2010).f 13. Reinsberg, J. et al. Effect of mono-(2-ethylhexyl) phthalate on steroid production of human granulosa cells. Toxicology and applied pharmacology. 239(1), 116–123 (2009).h 13. Reinsberg, J. et al. Effect of mono-(2-ethylhexyl) phthalate on steroid production of human granulosa cells. Toxicology and applied pharmacology. 239(1), 116–123 (2009).h p gy ( ), ( ) 4. Kluwe, W. M. et al. The carcinogenicity of dietary di (2‐ethylhexyl) phthalate (DEHP) in Fischer 344 rats and B6C3F1 mice. Author Contributions B.C. conceived the idea and J.Y. conducted the experiments. All authors contributed to designing experiments, preparing the manuscript, suggesting modifications and analysing the data. B.C. conceived the idea and J.Y. conducted the experiments. All authors contributed to designing experiments, preparing the manuscript, suggesting modifications and analysing the data. B.C. conceived the idea and J.Y. conducted the experiments. All authors contributed to designing experiments, preparing the manuscript, suggesting modifications and analysing the data. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 15. Melnick, R. L. Is peroxisome proliferation an obligatory precursor step in the carcinogenicity of di (2-ethylhexyl) phthalate (DE Environmental Health Perspectives. 109(5), 437 (2001). 15. Melnick, R. L. Is peroxisome proliferation an obligatory precursor step in the carcinogenicity of di (2-ethylhexyl) phthalate (DEHP). Environmental Health Perspectives. 109(5), 437 (2001). 16 Chipman J K Mally A & Edwards G O Disruption of gap junctions in toxicity and carcinogenicity Toxicological Sciences 71(2) p 16. Chipman, J. K., Mally, A. & Edwards, G. O. Disruption of gap junctions in toxicity and carcinogenicity. Toxicological Sciences. 7 146–153 (2003).l ( ) 7. Choi, J. & Kwak, S. Y. Hyperbranched poly (ε-caprolactone) as a nonmigrating alternative plasticizer for phthalates in flexible PVC Environmental science & technology. 41(10), 3763–3768 (2007).i gy ( ) ( ) 8. Faria-Machado, A. F., da Silva, M. A., Vieira, M. G. A. & Beppu, M. M. Epoxidation of Modified Natural Plasticizer Obtained from Rice Fatty Acids and Application on Polyvinylchloride Films. J. Appl. Polym. Sci. 127(5), 3543–3549 (2013). y pp y y pp y 19. Chen, J. et al. Synthesis and application of environmental soybean oil-based epoxidized glycidyl ester plasticizer for poly(vinyl chloride). Eur. J. Lipid Sci. Technol. 119(5), doi:10.1002/ejlt.201600216 (2017). p j 20. Vieira, M. G. A., SilvaI, M. Ada, MaçumotoI, A. C. G., Santos, L. Odos & Beppu, M. M. Synthesis and application of n polymeric plasticizer obtained through polyesterification of rice fatty acid. Materials Research. 17(2), 386–391 (2014). y g yi y 21. Chiellini, F., Ferri, M., Morelli, A., Dipaola, L. & Latini, G. Perspectives on alternatives to phthalate plasticized poly (vinyl chlo in medical devices applications. Prog. Polym. Sci. 38(7), 1067–1088 (2013). 22. Fernandez, S., Kunchandy, S. & Ghosh, S. Linseed oilplasticizer based natural rubber/expandable graphite vulcanizates: Synthesis and characterizations. J. Polym. Environ. 23, 1–8 (2015).l and characterizations. J. Polym. Environ. 23, 1–8 (2015). y 23. Chavan, P. & Gogate, R. Ultrasound assisted synthesis ofepoxidized sunflower oil and application as plasticizer. J. Ind.Eng. Chem. 21, 842–850 (2014). 4. Choi, W., Chung, J. W. & Kwak, S. Y. Unentangled Star-Shape Poly(ε-caprolactone)s as Phthalate-Free PVC Plasticizers Designed fo N T i i d I d Mi i R i ACS A l M t I t f 6(14) 11118 11128 (2014) 24. Choi, W., Chung, J. W. & Kwak, S. Y. Unentangled Star-Shape Poly(ε-caprolactone)s as Phthalate-Free PVC Plasticizer Non-Toxicity and Improved Migration Resistance. ACS Appl. Mater. Interfaces. References Journa of Toxicology and Environmental Health, Part A Current Issues. 10(4–5), 797–815 (1982). SCieNtifiC RePortS \| 7: 9277 \| DOI:10.1038/s41598-017-10159-7 5 SCieNtifiC RePortS \| 7: 9277 \| DOI:10.1038/s41598-017-10159-7 www.nature.com/scientificreports/ & Reineckeet, H. Phthalate plasticizers covalently bound to PVC: plasticization with suppressed migration. Macromolecules. 43(5), 2377–2381 (2010).h 36. Daniels, P. H. A Brief Overview of Theories of PVC Plasticization and Methods Used to Evaluate PVC-Plasticizer Interaction. J. Addit. Technol. 15, 219–223 (2009). 37. Immergut, E. H. & Mark, H. F. Principles of Plasticization. Plasticization and Plasticizer Processes. 1–26 (1965). 37. Immergut, E. H. & Mark, H. F. Principles of Plasticization. Plasticization and Plasticizer Processes. 1–26 (1965). Acknowledgementsh Acknowledgements The authors think Rui Hua (PerkinElmer lab in Beijing) for DSC measurements. www.nature.com/scientificreports/ 6(14), 11118–11128 (2014).l , , g, J K k, g p y( p ) Non-Toxicity and Improved Migration Resistance. ACS Appl. Mater. Interfaces. 6(14), 11118–11128 (2014).l 25. Erythropel, H. C. et al. Designing green plasticizers: Influence of molecule geometry a effectiveness of diester plasticizers in PVC blends. Polymer. 89, 18–27 (2016). 25. Erythropel, H. C. et al. Designing green plasticizers: Influence of molecule geometry and alkyl chain length on the plasticizing effectiveness of diester plasticizers in PVC blends. Polymer. 89, 18–27 (2016).l f y 26. Erythropel, H. C., Dodd, P., Leask, R. L., Maric, M. & Cooper, D. G. Designing green plasticizers: influence of alkyl chain le biodegradation and plasticization properties of succinate based plasticizers. Chemosphere. 91(3), 358–365 (2013). g p p p p p 7. Kastner, J., Cooper, D. G., Marić, M., Dodd, P. & Yargeau, V. Aqueous leaching of di-2-ethylhexyl phthalate and “green” plasticizer from poly (vinyl chloride). Science of the Total Environment. 432, 357–364 (2012). 28. Barreto, M. C. et al. Reduction of Plasticizer Leaching from PVC by Barrier Coatings Deposited Using DBD Processes at Atmospheric Pressure. Plasma Process. Polym. 9, 1208–1214 (2012). Barreto, M. C. et al. Reduction of Plasticizer Leaching from PV 28. Barreto, M. C. et al. Reduction of Plasticizer Leaching from PVC by Barrier Coati Atmospheric Pressure. Plasma Process. Polym. 9, 1208–1214 (2012). Atmospheric Pressure. Plasma Process. Polym. 9, 1208–1214 (2012) p y 29. Levin, G. Stabilized PVC products and their production. U.S. Patent 5,209,931, May 11, 1993. p p y 30. Levin G. Prevention of plasticizer migration from PVC products. U.S. Patent 4,806,393, Feb. 21, 1989. 30. Levin G. Prevention of plasticizer migration from PVC produc 1. Messori, M. et al. Prevention of plasticizer leaching from PVC medical devices by using organic–inorganic hybrid coatings. Polymer 45(3), 805–813 (2004).f 2. Braslau, R., Schaffner, F. & Earla, A. Polymeric Phthalates: Potential Nonmigratory Macromolecular Plasticizers. Journal of Polyme Science. Polymer Chemistry. 51, 1175–1184 (2013). y y 33. Gooch, J. W. Polymeric Plasticizer 564 (Springer New York, 2011). y p g 34. Tarvainen, M., Sutinen, R., Somppi, M., Paronen, P. & Poso, A. Predicting Plasticization Efficiency from Three-Dimensional Molecular Structure of a Polymer Plasticizer. Pharmaceutical Research. 18(12), 1760–1766 (2001). 5. Navarro, R., Pérez, P. M., Gómez Tardajos, M. & Reineckeet, H. Phthalate plasticizers covalently bound to PVC: plasticization with suppressed migration. Macromolecules. 43(5), 2377–2381 (2010).h 35. Navarro, R., Pérez, P. M., Gómez Tardajos, M. 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https://openalex.org/W3030955863	https://pure.ulster.ac.uk/ws/files/79474277/energies_779490.pdf	English	null	Energy Storage on a Distribution Network for Self-Consumption of Wind Energy and Market Value	Energies	2,020	cc-by	17,429	General rights Th i h General rights The copyright and moral rights to the output are retained by the output author(s), unless otherwise stated by the document licence. Unless otherwise stated, users are permitted to download a copy of the output for personal study or non-commercial research and are permitted to freely distribute the URL of the output. They are not permitted to alter, reproduce, distribute or make any commercial use of the output without obtaining the permission of the author(s). If the document is licenced under Creative Commons, the rights of users of the documents can be found at https://creativecommons.org/share-your-work/cclicenses/. If the document is licenced under Creative Commons, the rights of users of the documents can be found at https://creativecommons.org/share-your-work/cclicenses/. Energy Storage on a Distribution Network for Self-Consumption of Wind Energy and Market Value Energy Storage on a Distribution Network for Self-Consumption of Wind Energy and Market Value Ademulegun, O., Keatley, P., Bani Mustafa, M., & Hewitt, N. (2020). Energy Storage on a Distribution Network for Self-Consumption of Wind Energy and Market Value. Energies, 13(11), 1-17. Article 2688. https://doi.org/10.3390/en13112688 Ademulegun, O., Keatley, P., Bani Mustafa, M., & Hewitt, N. (2020). Energy Storage on a Distribution Network for Self-Consumption of Wind Energy and Market Value. Energies, 13(11), 1-17. Article 2688. https://doi.org/10.3390/en13112688 Link to publication record in Ulster University Research Portal Published in: Energies Publication Status: Published (in print/issue): 26/05/2020 DOI: 10.3390/en13112688 Document Version Author Accepted version Published in: Energies Publication Status: Published (in print/issue): 26/05/2020 DOI: 10.3390/en13112688 Document Version Author Accepted version Document Version Author Accepted version General rights The copyright and moral rights to the output are retained by the output author(s), unless otherwise stated by the document licence. 45 4 Centre for Sustainable Technologies, University of Ulster, Jordanstown, BT37 0QB, Northern Ireland, U nj.hewitt@ulster.ac.uk * Correspondence: ademulegun-o@ulster.ac.uk; Tel.: +44-7747-238-873 Abstract: Wind energy could be generated and captured with a storage device within the customer 15 premises for local utilization of the wind energy and for the provision of various services across the 16 electricity supply chain. To assess the benefits of adding a storage device to an electricity 17 distribution network that has two wind turbines with a base load of 500 kW and a typical peak load 18 under 1,500 kW, a 2MW/4MWh storage is installed. To observe the effects of adding the storage 19 device to the network, a technical analysis is performed using the NEPLAN 360 modelling tool 20 while an economic analysis is carried out by estimating the likely payback period on investment. A 21 storage potential benefit analysis suggests how changes in integration policies could affect the 22 utility of adding the storage device. With the addition of the storage device, self-consumption of 23 wind energy increased by almost 10%. The profitability of the project increased when the device is 24 also deployed to provide stacked services across the electricity supply chain. Some policies that 25 permit the integration of devices into the grid could increase the profitability of storage projects. 26 Keywords: distributed energy resources; economics of storage; energy storage; self-consumption of 27 wind; storage services; wind energy 28 29 Energies 2020, 13, x; doi: FOR PEER REVIEW Take down policy a e do po cy The Research Portal is Ulster University's institutional repository that provides access to Ulster's research outputs. Every effort has been made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the Research Portal that you believe breaches copyright or violates any law, please contact pure-support@ulster.ac.uk Download date: 24/10/2024 Article 1 Energy Storage on A Distribution Network for 2 Self-Consumption of Wind Energy and Market Value 3 Oluwasola O. Ademulegun 1,, Patrick Keatley 2, Motasem Bani Mustafa 3 and Neil J. Hewitt 4 4 1 Centre for Sustainable Technologies, University of Ulster, Jordanstown, BT37 0QB, Northern Ireland, UK; 5 ademulegun-o@ulster.ac.uk 6 2 Centre for Sustainable Technologies, University of Ulster, Jordanstown, BT37 0QB, Northern Ireland, UK; 7 p.keatley@ulster.ac.uk 8 3 Centre for Sustainable Technologies, University of Ulster, Jordanstown, BT37 0QB, Northern Ireland, UK; 9 bani_mustafa-m@ulster.ac.uk 10 4 Centre for Sustainable Technologies, University of Ulster, Jordanstown, BT37 0QB, Northern Ireland, UK; 11 nj.hewitt@ulster.ac.uk 12 Correspondence: ademulegun-o@ulster.ac.uk; Tel.: +44-7747-238-873 13 Received: date; Accepted: date; Published: date 14 Abstract: Wind energy could be generated and captured with a storage device within the customer 15 premises for local utilization of the wind energy and for the provision of various services across the 16 electricity supply chain. To assess the benefits of adding a storage device to an electricity 17 distribution network that has two wind turbines with a base load of 500 kW and a typical peak load 18 under 1,500 kW, a 2MW/4MWh storage is installed. To observe the effects of adding the storage 19 device to the network, a technical analysis is performed using the NEPLAN 360 modelling tool 20 while an economic analysis is carried out by estimating the likely payback period on investment. A 21 storage potential benefit analysis suggests how changes in integration policies could affect the 22 utility of adding the storage device. With the addition of the storage device, self-consumption of 23 wind energy increased by almost 10%. The profitability of the project increased when the device is 24 also deployed to provide stacked services across the electricity supply chain. Some policies that 25 permit the integration of devices into the grid could increase the profitability of storage projects. 26 Keywords: distributed energy resources; economics of storage; energy storage; self-consumption of 27 wind; storage services; wind energy 28 29 1. Take down policy Introduction 30 The need for low-carbon energy systems in achieving energy sustainability has encouraged the 31 adoption of different techniques for increasing cleaner energy generation and utilization through 32 Distributed Energy Resources (DER). For instance, in the UK where a net-zero emission target has 33 been set [1] and in Northern Ireland where an increasing level of System Non-Synchronous 34 Penetration (SNSP) is to be achieved on the electricity grid [2], it is desirable to generate clean energy 35 from renewables like wind turbines. The variable nature of the renewables reduces their 36 effectiveness where the stability and reliability of the electricity grid is to be maintained. To address 37 the challenges in the variability of the renewables for a resilient grid, some solutions have been 38 d l d d id t d th f t d i [3 4] 39 www.mdpi.com/journal/energies Energies 2020, 13, x; doi: FOR PEER REVIEW www.mdpi.com/journal/energies Energy Storage on A Distribution Network for 2 Self-Consumption of Wind Energy and Market Value 3 Oluwasola O. Ademulegun 1,, Patrick Keatley 2, Motasem Bani Mustafa 3 and Neil J. Hewitt 4 4 1 Centre for Sustainable Technologies, University of Ulster, Jordanstown, BT37 0QB, Northern Ireland, UK; 5 ademulegun-o@ulster.ac.uk 6 2 Centre for Sustainable Technologies, University of Ulster, Jordanstown, BT37 0QB, Northern Ireland, UK; 7 p.keatley@ulster.ac.uk 8 3 Centre for Sustainable Technologies, University of Ulster, Jordanstown, BT37 0QB, Northern Ireland, UK; 9 bani_mustafa-m@ulster.ac.uk 10 4 Centre for Sustainable Technologies, University of Ulster, Jordanstown, BT37 0QB, Northern Ireland, UK; 11 nj.hewitt@ulster.ac.uk 12 Correspondence: ademulegun-o@ulster.ac.uk; Tel.: +44-7747-238-873 13 Received: date; Accepted: date; Published: date 14 Abstract: Wind energy could be generated and captured with a storage device within the customer 15 premises for local utilization of the wind energy and for the provision of various services across the 16 electricity supply chain. To assess the benefits of adding a storage device to an electricity 17 distribution network that has two wind turbines with a base load of 500 kW and a typical peak load 18 under 1,500 kW, a 2MW/4MWh storage is installed. To observe the effects of adding the storage 19 device to the network, a technical analysis is performed using the NEPLAN 360 modelling tool 20 while an economic analysis is carried out by estimating the likely payback period on investment. A 21 storage potential benefit analysis suggests how changes in integration policies could affect the 22 utility of adding the storage device. With the addition of the storage device, self-consumption of 23 wind energy increased by almost 10%. The profitability of the project increased when the device is 24 also deployed to provide stacked services across the electricity supply chain. Some policies that 25 permit the integration of devices into the grid could increase the profitability of storage projects. 26 Keywords: distributed energy resources; economics of storage; energy storage; self-consumption of 27 wind; storage services; wind energy 28 29 1. Introduction 30 The need for low-carbon energy systems in achieving energy sustainability has encouraged the 31 adoption of different techniques for increasing cleaner energy generation and utilization through 32 Distributed Energy Resources (DER). For instance, in the UK where a net-zero emission target has 33 been set [1] and in Northern Ireland where an increasing level of System Non-Synchronous 34 Penetration (SNSP) is to be achieved on the electricity grid [2], it is desirable to generate clean energy 35 from renewables like wind turbines. The variable nature of the renewables reduces their 36 effectiveness where the stability and reliability of the electricity grid is to be maintained. To address 37 the challenges in the variability of the renewables for a resilient grid, some solutions have been 38 proposed, namely demand-side energy management and the use of energy storage devices [3,4]. 39 Integrating renewables and energy storage devices into the grid comes with challenges and 40 opportunities. The opportunities include optimal power management and economic benefits [5], 41 better utilization of relatively cheap renewable resources [6], increased consumption of the energy 42 produced from renewable sources [6], less pollution from energy production activities, and 43 reduction of the curtailments and constraints of renewables [7]. The storage could also be deployed 44 for stacked services in multi-use purposes [8,9]. 45 Energies 2020, 13, x; doi: FOR PEER REVIEW www.mdpi.com/journal/energies gy g Self-Consumption of Wind Energy and Market Value 3 Oluwasola O. Ademulegun 1,, Patrick Keatley 2, Motasem Bani Mustafa 3 and Neil J. Hewitt 4 4 1 Centre for Sustainable Technologies, University of Ulster, Jordanstown, BT37 0QB, Northern Ireland, UK; 5 ademulegun-o@ulster.ac.uk 6 2 Centre for Sustainable Technologies, University of Ulster, Jordanstown, BT37 0QB, Northern Ireland, UK; 7 p.keatley@ulster.ac.uk 8 3 Centre for Sustainable Technologies, University of Ulster, Jordanstown, BT37 0QB, Northern Ireland, UK; 9 bani_mustafa-m@ulster.ac.uk 10 4 Centre for Sustainable Technologies, University of Ulster, Jordanstown, BT37 0QB, Northern Ireland, UK; 11 nj.hewitt@ulster.ac.uk 12 Correspondence: ademulegun-o@ulster.ac.uk; Tel.: +44-7747-238-873 13 Received: date; Accepted: date; Published: date 14 Abstract: Wind energy could be generated and captured with a storage device within the customer 15 premises for local utilization of the wind energy and for the provision of various services across the 16 electricity supply chain. To assess the benefits of adding a storage device to an electricity 17 distribution network that has two wind turbines with a base load of 500 kW and a typical peak load 18 under 1,500 kW, a 2MW/4MWh storage is installed. To observe the effects of adding the storage 19 device to the network, a technical analysis is performed using the NEPLAN 360 modelling tool 20 while an economic analysis is carried out by estimating the likely payback period on investment. A 21 storage potential benefit analysis suggests how changes in integration policies could affect the 22 utility of adding the storage device. With the addition of the storage device, self-consumption of 23 wind energy increased by almost 10%. The profitability of the project increased when the device is 24 also deployed to provide stacked services across the electricity supply chain. Some policies that 25 permit the integration of devices into the grid could increase the profitability of storage projects. 26 Keywords: distributed energy resources; economics of storage; energy storage; self-consumption of 27 wind; storage services; wind energy 28 29 1. Introduction 30 The need for low-carbon energy systems in achieving energy sustainability has encouraged the 31 adoption of different techniques for increasing cleaner energy generation and utilization through 32 Distributed Energy Resources (DER). For instance, in the UK where a net-zero emission target has 33 been set [1] and in Northern Ireland where an increasing level of System Non-Synchronous 34 Penetration (SNSP) is to be achieved on the electricity grid [2], it is desirable to generate clean energy 35 from renewables like wind turbines. The variable nature of the renewables reduces their 36 effectiveness where the stability and reliability of the electricity grid is to be maintained. To address 37 the challenges in the variability of the renewables for a resilient grid, some solutions have been 38 proposed, namely demand-side energy management and the use of energy storage devices [3,4]. 39 Integrating renewables and energy storage devices into the grid comes with challenges and 40 opportunities. The opportunities include optimal power management and economic benefits [5], 41 better utilization of relatively cheap renewable resources [6], increased consumption of the energy 42 produced from renewable sources [6], less pollution from energy production activities, and 43 reduction of the curtailments and constraints of renewables [7]. The storage could also be deployed 44 for stacked services in multi-use purposes [8,9]. 1. Introduction 30 The need for low-carbon energy systems in achieving energy sustainability has encouraged the 31 adoption of different techniques for increasing cleaner energy generation and utilization through 32 Distributed Energy Resources (DER). For instance, in the UK where a net-zero emission target has 33 been set [1] and in Northern Ireland where an increasing level of System Non-Synchronous 34 Penetration (SNSP) is to be achieved on the electricity grid [2], it is desirable to generate clean energy 35 from renewables like wind turbines. The variable nature of the renewables reduces their 36 effectiveness where the stability and reliability of the electricity grid is to be maintained. To address 37 the challenges in the variability of the renewables for a resilient grid, some solutions have been 38 proposed, namely demand-side energy management and the use of energy storage devices [3,4]. 39 Integrating renewables and energy storage devices into the grid comes with challenges and 0 opportunities. The opportunities include optimal power management and economic benefits [5], 1 better utilization of relatively cheap renewable resources [6], increased consumption of the energy 2 produced from renewable sources [6], less pollution from energy production activities, and 3 reduction of the curtailments and constraints of renewables [7]. The storage could also be deployed 4 for stacked services in multi-use purposes [8,9]. 5 www.mdpi.com/journal/energies Energies 2020, 13, x; doi: FOR PEER REVIEW Energies 2020, 13, x FOR PEER REVIEW 2 of 18 The challenges in the integration include complex nature of the real benefits of storage, the locational nature of the values for renewables and storage [10], the dynamics of storage economics, and certain inconsistencies in policies that could discourage innovation. The peculiarities in the characteristics of the aggregate power system within a region (the structure of the grid, the fuel mix of the grid, the load profile of attached loads to the grid, the point on the grid where DER are to be located, the availability of different energy sources, and the electricity market at the location) make the value derivable from installing DER rather unique, typically varying from location to location [10]. In [11], the market designs for and the characteristics of different ancillary services are described with emphasis on the increasing role of DER in providing the ancillary services that have historically been provided by conventional synchronous generators. 1. Introduction 30 The procurement schemes and the emerging ancillary services that may be offered by the distributed resources are also described. The roles that DER may play in decarbonization within the distribution network through the provision of ancillary services have been described in [12]. In [13], a multi-source energy storage model that consists of a conventional energy storage, a multi-energy flow resources, and a demand response resource, at the demand and the supply sides, has been described for achieving economic self-management of energy through an intelligent control management method. The integrated distributed energy system was deployed to deal with the variability in loads and renewable supply. In [14], an energy management system that maximizes renewable energy utilization while providing certain ancillary services using heat pump and a thermal energy storage system has been reported to help achieve cost saving, reduction of purchased energy imbalance from the grid, more reliable use of the heat pump, and a more stable surrounding temperature. This work investigates the use of an energy storage device for increasing self-consumption of 68 wind energy and providing market services within a distribution network having features given in 69 [15,16]. It is well known that energy storage techniques could be used to capture renewable energy 70 for a later use. However, there is a knowledge gap in ascertaining the real value of deploying the 71 storage at the specific locations having unique network, market, and policy characteristics. 72 Moreover, as reported in [17], it is often uneconomical to deploy storage devices at high investment 73 costs when the other possible storage application revenues are not considered. The work explores 74 the other value streams that could make deploying the storage device more profitable at the 75 distribution network. The addition of the storage device is modelled and technically analyzed using 76 the NEPLAN 360 software while the economic feasibility of the storage project is assessed by 77 estimating the likely payback period on investment. 78 The local network is a campus site where the base load is 500 kW while the typical peak load is 79 below 1,500 kW. The distribution network has two behind-the-meter (BTM) wind turbines which are 80 connected to an alternating current electricity grid through an 11kV substation. Currently, any 81 excess energy production from the turbines is fed to the grid at a price fixed by the utility. 1. Introduction 30 Instead of 82 feeding the excess locally generated wind energy to the grid, the work examines installing a 83 2MW/4MWh storage device to capture the excess energy – to increase self-consumption of wind 84 energy while also using some capacity of the storage device for providing certain ancillary services 85 to the grid. As reported in [18], wind turbines could be deployed for providing grid services; in this 86 work, only the storage device is deployed for the grid services. To see how changes in policies could 87 impact the profitability of the project, a potential benefit analysis for adding the device is done using 88 an existing market structure. 89 2.1. Description of Distribution Network 91 To investigate how the energy storage device could be used to increase local consumption of 92 wind energy and provide certain ancillary services, a model of the distribution network is created 93 Energies 2020, 13, x FOR PEER REVIEW 3 of 18 using the NEPLAN 360 software. There are ten substations that feed different loads on-site. There are 94 two grid-connected wind turbines running on-site. 95 The site is connected to the electricity grid via an 11kV feeder. From a typical one-year data of 96 the site, a total energy of 3,720,642 kWh was imported from the grid. A total energy of 3,042,075 kWh 97 was generated from the wind turbines. Whereas, 601,780 kWh – representing about 20% of the total 98 energy generated from wind – was exported to the grid. The total annual energy consumption 99 within the same one-year period was 6,189,647 kWh. The load profile depicts that of a campus site 100 where the base load is 500 kW and the typical peak load is less than 1,500 kW, Figure 1. 101 using the NEPLAN 360 software. There are ten substations that feed different loads on-site. There are 94 two grid-connected wind turbines running on-site. 95 102 Figure 1. One Year (365-day) Load Profile of Site 103 Figure 1. One Year (365-day) Load Profile of Site Figure 1. One Year (365-day) Load Profile of Site A high voltage connection agreement puts the maximum energy that may be exported from the 104 site to the grid (the maximum export capacity) at 1,242 kW; the maximum energy that may be 105 imported from the grid to the site (the maximum import capacity) is 2,500 kW. 106 The line diagram of Figure 2 and Equation 1 both describe the initial configuration of the 107 distribution network. 108 109 Figure 2. Line Diagram of Distribution Network 110 Figure 2. Line Diagram of Distribution Network Figure 2. 2.1. Description of Distribution Network 91 Line Diagram of Distribution Network 0 (1) 111 111 (1) where L denotes the total power consumed in the aggregated system load, Z represents the total 112 power expended in system impedances, T1 represents the power supplied from the turbine number 113 where L denotes the total power consumed in the aggregated system load, Z represents the total 112 power expended in system impedances, T1 represents the power supplied from the turbine number 113 where L denotes the total power consumed in the aggregated system load, Z represents the total 112 power expended in system impedances, T1 represents the power supplied from the turbine number 113 Energies 2020, 13, x FOR PEER REVIEW 4 of 18 Energies 2020, 13, x FOR PEER REVIEW 4 of 18 one, Ggrid represents the energy from the power grid, and T2 represents the power supplied from the 114 turbine number two. 115 The BTM energy storage device is installed to capture any excess energy generation from the 116 117 one, Ggrid represents the energy from the power grid, and T2 represents the power supplied from the 114 turbine number two. 115 The BTM energy storage device is installed to capture any excess energy generation from the 116 wind turbines T1 and T2. The network elements of the site are depicted in Figure 3. 117 118 Figure 3. Arrangement of Network Elements 119 118 119 Figure 3. Arrangement of Network Elements Meanwhile, the loads in the local network are constantly linked to the grid for continuous 0 power supply irrespective of the power output of the wind turbines. Rather than feeding the excess wind energy from the turbines to the grid, a storage device is installed on the network to take up the 2 excess wind energy for later consumption on-site. 3 The data of the aggregate power produced from the turbines and a data of the maximum power 124 demanded for the one-year period are used as the typical energy profiles of the site. During this 125 period, the base load swung around 500 kW and the peak demand was 1,376 kW. The generation 126 profiles of the wind turbines, the local load profile, and the total exported electricity data are used in 127 estimating a suitable storage portfolio that could help in achieving the objectives of maximizing 128 self-consumption of wind energy and providing market services. 2.1. Description of Distribution Network 91 In other words, the power profiles 129 of site within the same period (the power demand, the power generation, and the electricity 130 import-export profiles) are used in ascertaining a suitable storage device – a storage technology that 131 could meet the charge-discharge characteristics required. A cost analysis is carried out on some of 132 the applicable storage technologies. 133 2.2. Storage Technologies 134 It is usually possible to find more than one suitable storage device for any storage project. The 135 final device selection could be made based on any specific storage, utility, or user requirements. The 136 account of the characteristics of different storage technologies, including the storage that may be 137 suitable in a BTM application, are given in [19,20]. The technical characteristics of the different 138 energy storage technologies and applications are presented in [21,22]. Some storage technologies 139 possess interesting characteristics. Considering batteries for example: they are modular – they could 140 be combined in modules to form small, medium, and large power banks. Such modularity of 141 batteries and some other storage devices makes them rather suitable in BTM and customer-premise 142 storage applications. Moreover, the battery could be sized to meet the exact user requirements, 143 optimizing the use of resources. The other factors that are considered in selecting the storage device 144 Energies 2020, 13, x FOR PEER REVIEW 5 of 18 for the BTM application include power requirement, charge-discharge requirement, duration of 145 service required, operating temperature, space and location requirements, maintenance needs, 146 maturity of the storage technology, and cost. 147 Some of the established storage options are considered for the project and a few of the most 148 suitable technologies meeting the desired needs are selected for economic analysis; for example, 149 flywheel storage and lithium ion (Li-ion) battery are considered. 150 Power Flow Analysis for Determining the Effect of Storage To observe how the installation of the storage device will affect the distribution network, a 152 power flow analysis is done. The network is considered operationally stable before the installation of 153 the device. After installing the storage device, the network is observed to verify that installing the 154 device has not compromised the stability and reliability of the distribution network. 155 the device. After installing the storage device, the network is observed to verify that installing the 154 device has not compromised the stability and reliability of the distribution network. 2.2. Storage Technologies 134 155 Given that the real and the reactive power flowing into a bus i of a network is P and Q 156 respectively, the static load flow equations used for network analysis could be expressed as: 157 (2a) 158 Given that the real and the reactive power flowing into a bus i of a network is P and Q 156 respectively, the static load flow equations used for network analysis could be expressed as: 157 (2a) 158 (2b) 159 (2a) 159 (2b) where Vk is the voltage at bus k, Yik is the mutual admittance between the ith node and a kth node, n 160 is the number of buses within the network, θ represents the phase angle between current and 161 voltage, δ represents the load angle, and Vi represents the bus voltage. 162 where Vk is the voltage at bus k, Yik is the mutual admittance between the ith node and a kth node, n 160 is the number of buses within the network, θ represents the phase angle between current and 161 voltage, δ represents the load angle, and Vi represents the bus voltage. 162 Appendix contains a derivation of the load flow equations. The non-linear static load flow 163 equations are solved numerically. The NEPLAN 360 modeller has a library of numerical solutions for 164 technical power flow analysis. The modeller takes the network elements and their electrical 165 parameters as inputs, uses a numerical method to analyse the power network, and outputs the 166 electrical signals (current, voltage, power) at the network nodes and within the elements. It also 167 indicates whether the numerical model converges or not and indicates where any excess power 168 flows occur. With a model of the distribution network created, running a power flow reveals the 169 changes to the network as a result of installing the storage device. 170 The diagram of Figure 4 describes the final configuration of the distribution network. 172 The diagram of Figure 4 describes the final configuration of the distribution network. 172 172 173 Figure 4. Adding Storage to Distribution Network 174 Figure 4. Adding Storage to Distribution Network 174 Figure 4. Adding Storage to Distribution Network Figure 4. Adding Storage to Distribution Network The switch Sw1 links the distribution network to the grid. Equations (3a) and (3b) describe how the 175 switch Sw1 is to be operated. 2.2. Storage Technologies 134 206 2.5.1. Benefits Through Self-consumption of Wind Energy 207 A benefit analysis is carried out to ascertain the gains in installing the storage device for 208 increasing self-consumption of wind energy. The costs of energy storage systems are not fixed. 209 Because of the dynamic nature of storage economics, in estimating the cost of storage, hypothesised 210 (3a) (3b) 177 (3a) (3b) (3b) The switch Sw2 determines the time that the storage device E is to be charged or discharged; it is 183 operated according to a control rule set at the Cnode. Equation 4 describes the operation of the switch 184 Sw2 and the control at the Cnode. 185 (4) (4) where T2 represents the energy feed from the turbine number two, T1 represents the energy feed 188 from the turbine number one, TimeTariff is the instantaneous price of electricity, Eservices is the aggregate 189 ancillary service demand on the storage device, ESOC is any specified charging state of the device, 190 E(min) represents the implied device discharge limit, “AND” is a summing logic, Sw2 represent switch 191 two and “OR” is also a logical expression. 192 where T2 represents the energy feed from the turbine number two, T1 represents the energy feed 188 from the turbine number one, TimeTariff is the instantaneous price of electricity, Eservices is the aggregate 189 ancillary service demand on the storage device, ESOC is any specified charging state of the device, 190 E(min) represents the implied device discharge limit, “AND” is a summing logic, Sw2 represent switch 191 two and “OR” is also a logical expression. 192 193 194 that is, ; for any discharge-limit instances t = 1, 2, 3, …, n 195 Switches Sw1 and Sw2 operate to ensure that the storage device is charged with a power supply 196 from the wind turbines only. The switches ensure that the device is discharged to maximize 197 self-consumption of the on-site-generated wind energy while also securing certain capacity of the 198 device for the provision of any commitment to ancillary services. 199 Switches Sw1 and Sw2 operate to ensure that the storage device is charged with a power supply 196 from the wind turbines only. The switches ensure that the device is discharged to maximize 197 self-consumption of the on-site-generated wind energy while also securing certain capacity of the 198 device for the provision of any commitment to ancillary services. 2.2. Storage Technologies 134 176 The switch Sw1 links the distribution network to the grid. Equations (3a) and (3b) describe how the 175 switch Sw1 is to be operated. 176 switch Sw1 is to be operated. 176 switch Sw1 is to be operated. 176 Energies 2020, 13, x FOR PEER REVIEW 6 of 18 (3a) 177 (3b) 178 where L denotes the energy demand by system load, Z represents the total energy expended in the 179 system impedance, T1 represents the energy feed from the turbine number one, T2 represents the 180 energy feed from the turbine number two, E(min) represents the implied device discharge limit, and 181 Sw1 represents switch one. 182 The switch Sw2 determines the time that the storage device E is to be charged or discharged; it is 183 operated according to a control rule set at the Cnode. Equation 4 describes the operation of the switch 184 Sw2 and the control at the Cnode. 185 186 (4) 187 where T2 represents the energy feed from the turbine number two, T1 represents the energy feed 188 from the turbine number one, TimeTariff is the instantaneous price of electricity, Eservices is the aggregate 189 ancillary service demand on the storage device, ESOC is any specified charging state of the device, 190 E(min) represents the implied device discharge limit, “AND” is a summing logic, Sw2 represent switch 191 two and “OR” is also a logical expression. 192 193 194 that is, ; for any discharge-limit instances t = 1, 2, 3, …, n 195 Switches Sw1 and Sw2 operate to ensure that the storage device is charged with a power supply 196 from the wind turbines only. The switches ensure that the device is discharged to maximize 197 self-consumption of the on-site-generated wind energy while also securing certain capacity of the 198 device for the provision of any commitment to ancillary services. 199 2.5. Assessing the Benefit of Storage 200 A feature assessment of some storage technologies discussed in [19,20,21,22] is done to identify 201 some of the storage options that could meet the defined objectives of maximizing self-consumption 202 of wind energy and providing ancillary services. A cost analysis is carried out on the identified 203 devices. The profitability of adding the storage device is determined taking the likely storage cost 204 ranges, storage efficiencies, storage capacity, the electricity market, and the potential additional 205 storage services as key parameters. 2.2. Storage Technologies 134 Taking into cognizance the high likelihood of changes in the prices of some of 213 the storage technologies and with a broad study of the inconsistencies in price quotes from literature 214 and industry – for example, consider the different prices specified for the same storage technology 215 plus notes on cost inconsistencies in [14,19,22–32] – the most likely cost range for each of the storage 216 technologies is heuristically selected for analysis. 217 While the analysis is not claiming that any storage option is currently economical under the 218 existing market arrangement, the analysis aims to identify the cost point at which the storage 219 becomes economically feasible with respect to the distribution network and to reveal where changes 220 in market conditions or storage costs could impact the profitability of the storage project. The cost 221 range also makes it possible to apply the results of the analysis within any reasonable future changes 222 to the economics of storage. 223 Using an existing market system, the benefits of installing the storage device for increasing 224 self-consumption of wind energy is analysed. In the market, the price of import electricity and the 225 price of export electricity are in the ratio of 7 to 3 typically, the price of import electricity being often 226 higher: when the import electricity price is at £7/kWh, the exported electricity price will be around 227 £3/kWh. The prices could vary in different economic settings but have consistent relations – based 228 on the historical analysis of the site export-import payment data and in [33]. 229 The benefit through self-consumption of wind energy is based on the difference between the 230 import and export electricity prices; the prices are fixed within days but could change when the 231 utility decides to review rates to reflect new economics. The total recoverable energy is obtained by 232 multiplying the captured (used to be exported) energy by the storage efficiency. The market value of 233 the recovered energy is obtained by multiplying the total recoverable energy by the market price. 234 The gross annual gain is the difference between the market value of the recovered energy at the 235 import electricity price and the market value of the exported energy at the export electricity price. 2.2. Storage Technologies 134 In the market, the price of import electricity and the 225 price of export electricity are in the ratio of 7 to 3 typically, the price of import electricity being often 226 higher: when the import electricity price is at £7/kWh, the exported electricity price will be around 227 £3/kWh. The prices could vary in different economic settings but have consistent relations – based 228 on the historical analysis of the site export-import payment data and in [33]. 229 The benefit through self-consumption of wind energy is based on the difference between the 230 import and export electricity prices; the prices are fixed within days but could change when the 231 utility decides to review rates to reflect new economics. The total recoverable energy is obtained by 232 multiplying the captured (used to be exported) energy by the storage efficiency. The market value of 233 the recovered energy is obtained by multiplying the total recoverable energy by the market price. 234 The gross annual gain is the difference between the market value of the recovered energy at the 235 import electricity price and the market value of the exported energy at the export electricity price. 236 2.5.2. Benefits Through Market Services 237 In another case, in addition to helping to increase self-consumption of wind energy, certain 238 capacity of the storage device is committed to providing some services to the electricity grid through 239 DS3/ISEM [34,35,36] – DS3 is a programme developed to increase the penetration of renewables like 240 wind on the power network, whereas ISEM is a cross-border electricity market that allows the 241 interconnection of grids for wholesale electricity trading. 242 The values from the actual provision of the ancillary services are not included because the 243 actual provision of the services is usually within very short times [18] and the exact amount of the 244 services provided may not be pre-determinable since the services are demanded by the electricity 245 prices are used to reduce the effect of random errors that could arise from the use of a static price 211 quote. Using a price quote given at a time for an analysis invalidates any result from the analysis in a 212 new economic setting. 2.2. Storage Technologies 134 199 A feature assessment of some storage technologies discussed in [19,20,21,22] is done to identify 201 some of the storage options that could meet the defined objectives of maximizing self-consumption 202 of wind energy and providing ancillary services. A cost analysis is carried out on the identified 203 devices. The profitability of adding the storage device is determined taking the likely storage cost 204 ranges, storage efficiencies, storage capacity, the electricity market, and the potential additional 205 storage services as key parameters. 206 A benefit analysis is carried out to ascertain the gains in installing the storage device for 208 increasing self-consumption of wind energy. The costs of energy storage systems are not fixed. 209 Because of the dynamic nature of storage economics, in estimating the cost of storage, hypothesised 210 Energies 2020, 13, x FOR PEER REVIEW 7 of 18 prices are used to reduce the effect of random errors that could arise from the use of a static price 211 quote. Using a price quote given at a time for an analysis invalidates any result from the analysis in a 212 new economic setting. Taking into cognizance the high likelihood of changes in the prices of some of 213 the storage technologies and with a broad study of the inconsistencies in price quotes from literature 214 and industry – for example, consider the different prices specified for the same storage technology 215 plus notes on cost inconsistencies in [14,19,22–32] – the most likely cost range for each of the storage 216 technologies is heuristically selected for analysis. 217 While the analysis is not claiming that any storage option is currently economical under the 218 existing market arrangement, the analysis aims to identify the cost point at which the storage 219 becomes economically feasible with respect to the distribution network and to reveal where changes 220 in market conditions or storage costs could impact the profitability of the storage project. The cost 221 range also makes it possible to apply the results of the analysis within any reasonable future changes 222 to the economics of storage. 223 Using an existing market system, the benefits of installing the storage device for increasing 224 self-consumption of wind energy is analysed. 2.2. Storage Technologies 134 It is 274 assumed that committing the storage device to providing the ancillary services comes at zero or 275 insignificant extra cost. 276 For this case of presenting the device for both maximizing self-consumption of wind energy 263 and committing to providing certain ancillary services in stack, a new economic analysis is 264 performed. The new analysis is to reveal how the commitment of the device to providing stacked 265 market services impacts the profitability of the storage project. The total DS3 service provided is the 266 summation of the storage eligible DS3 service suite of Table 1 – at the aggregated standard rate of 267 £10.47/MWh. 268 2.2. Storage Technologies 134 The products 253 are described by the transmission network operators – EirGrid and SONI in [37,39] – with rates 254 defined for specified times in [39]. The suite of services that a typical storage device could provide is 255 summarised in Table 1 [40,41,42]. 256 Table 1. Storage Eligible DS3 Service Suite with Base Rates in £/MWh (2019-2020) While ancillary services were traditionally provided by equipment connected to the 258 transmission network; in certain instances, the services could be provided through devices 259 connected to the distribution network – this will usually depend on locational service needs, existing 260 interconnection policies, and requires planning and coordination of network operations. The storage 261 device could be restricted within certain limits in providing the services [42,43]. 262 While ancillary services were traditionally provided by equipment connected to the 258 transmission network; in certain instances, the services could be provided through devices 259 connected to the distribution network – this will usually depend on locational service needs, existing 260 interconnection policies, and requires planning and coordination of network operations. The storage 261 device could be restricted within certain limits in providing the services [42,43]. 262 For this case of presenting the device for both maximizing self-consumption of wind energy 263 and committing to providing certain ancillary services in stack, a new economic analysis is 264 performed. The new analysis is to reveal how the commitment of the device to providing stacked 265 market services impacts the profitability of the storage project. The total DS3 service provided is the 266 summation of the storage eligible DS3 service suite of Table 1 – at the aggregated standard rate of 267 £10.47/MWh. 268 20% of the storage capacity is committed within less than 2% of total lifespan of the storage 269 device at the first instance, for the estimation of Gain 1 and the payback Period 1. The same storage 270 capacity is committed for 25% of the device lifespan at the second instance, for the estimation of the 271 Gain 2 and the payback Period 2. The ancillary service gain is a product of the committed capacity 272 and the aggregated value, £10.47/MWh. The new annual gains are estimated as the sum of the gain 273 from self-consumption of wind energy and the gain from the provision of ancillary services. 2.2. Storage Technologies 134 236 In another case, in addition to helping to increase self-consumption of wind energy, certain 238 capacity of the storage device is committed to providing some services to the electricity grid through 239 DS3/ISEM [34,35,36] – DS3 is a programme developed to increase the penetration of renewables like 240 wind on the power network, whereas ISEM is a cross-border electricity market that allows the 241 interconnection of grids for wholesale electricity trading. 242 The values from the actual provision of the ancillary services are not included because the 243 actual provision of the services is usually within very short times [18] and the exact amount of the 244 services provided may not be pre-determinable since the services are demanded by the electricity 245 grid only during special operating conditions, maintaining the stability of the grid. The value 246 accounted for here are only for the service “commitment,” and not for the actual performance: the 247 value derivable from connecting the storage device to the grid and making certain capacities 248 available for charging or discharging in supporting the grid during operational emergencies. 249 The services that the storage devices could provide are selected and aggregated from the DS3 250 service suite given in [36]. The service suite helps in maintaining the stability and reliability of the 251 grid as non-synchronous power sources increase with the integration of the variable renewables. 252 8 of 18 Energies 2020, 13, x FOR PEER REVIEW The service products are required to guarantee a qualitative performance of the grid. The products 253 are described by the transmission network operators – EirGrid and SONI in [37,39] – with rates 254 defined for specified times in [39]. The suite of services that a typical storage device could provide is 255 summarised in Table 1 [40,41,42]. 256 Table 1. Storage Eligible DS3 Service Suite with Base Rates in £/MWh (2019-2020) 257 Products Abbreviation Storage Eligible Payment Rate (£/MWh) Fast Frequency Response FFR Yes 1.98 Primary Operating Reserve POR Yes 2.97 Ramping Margin 1 RM1 Yes 0.11 Ramping Margin 3 RM3 Yes 0.16 Ramping Margin 8 RM8 Yes 0.15 Replacement Reserve (De-Synchronised) RRD Yes 0.51 Replacement Reserve (Synchronised) RRS Yes 0.23 Secondary Operating Reserve SOR Yes 1.80 Tertiary Operating Reserve 1 TOR1 Yes 1.42 The service products are required to guarantee a qualitative performance of the grid. 2.5.3. Potential Benefit Across Electricity Supply Chain 277 This section examines the value of the storage device installed on the described distribution 278 network in general, not only the device deployed to capture the wind energy produced by BTM 279 turbines. To account for the full range of values that could be derived from any typical installation, a 280 potential benefit analysis is carried out for the entire stack of services that the storage device could 281 potentially offer across the electricity supply chain. 282 Energies 2020, 13, x FOR PEER REVIEW 9 of 18 Energies 2020, 13, x FOR PEER REVIEW 9 of 18 In accounting for the potential storage benefits, with assumptions where required, the following 283 approximate daily storage service values are estimated: 284 • DS3 Services: the total suite of the DS3 service that the storage device commits to is £10/MWh, 285 the size of the device deployed is 2MW/4MWh, 40% of the device capacity has been committed 286 to providing the services, the storage system has 85% roundtrip efficiency – the storage has 287 minimal energy losses while charging and discharging. 288 • Increased Wind Self-consumption: the size of the storage device is 2MW/4MWh, the device is 289 85% efficient (roundtrip), the site data – containing the import and the export electricity prices, 290 the energy exports from the wind turbines, the energy generated by the turbines, and the total 291 load energy required – are used in calculating the gross annual gain from self-consumption of 292 wind energy. The daily potential gain is estimated by dividing the gross annual gain by the 293 number of days in a year. 294 • Time-of-Use-Bill-Management: the size of the storage device is 2MW/4MWh; the device is 85% 295 efficient (roundtrip), the site data are used in calculating the mean daily import; using the Power 296 NI – an electricity supplier – Economy 7 (2-Rate) meter plan [44], a third of the total electricity 297 required is set to be imported at a low rate period (at nights) while the remaining electricity is 298 imported at a high rate period (during the day). 2.5.3. Potential Benefit Across Electricity Supply Chain 277 299 • Demand Response of Load Shifting: the size of the storage device is 2MW/4MWh; the device is 300 85% efficient (roundtrip), the site data are used in calculating the mean daily import; using the 301 SSE Airtricity (an electricity supplier) KeyPad Powershift meter plan, a third of the total 302 electricity required for the day is imported within the “low” rate period – between 1:00 and 9:00 303 [45,46] while the remaining electricity is imported at the “normal” rate period during the day. 304 Some of the storage services highlighted are mutually exclusive; for example, while the storage 305 device has been deployed for increasing self-consumption of wind energy and providing certain 306 levels of ancillary services, the device may no longer be fully utilisable for 307 time-of-use-bill-management at the same time. While inadequate policies may not allow some 308 storage benefits to be realizable now, the potential benefit analysis is to indicate storage-utilisation 309 possibilities and reveal the changes in policies that could monetise additional storage values at the 310 distribution network. 311 Other potential storage values could be estimated for specific sites within the distribution 312 network. Meanwhile, any given application could require using a storage device with specific 313 characteristics. 314 3. Results and Discussion 315 While the on-site loads are supplied with the power generation from the wind turbines and the 316 grid, the installed storage device takes up any excess wind energy generation from the turbines as 317 the load flow converges while the network elements operate within safe limits, illustrated for a 318 typical windy day in Figure 5. 319 In accounting for the potential storage benefits, with assumptions where required, the following 283 approximate daily storage service values are estimated: 284 In accounting for the potential storage benefits, with assumptions where required, the following 283 approximate daily storage service values are estimated: 284 • DS3 Services: the total suite of the DS3 service that the storage device commits to is £10/MWh, 85 the size of the device deployed is 2MW/4MWh, 40% of the device capacity has been committed 86 to providing the services, the storage system has 85% roundtrip efficiency – the storage has 87 minimal energy losses while charging and discharging. 2.5.3. Potential Benefit Across Electricity Supply Chain 277 88 • Increased Wind Self-consumption: the size of the storage device is 2MW/4MWh, the device is 289 85% efficient (roundtrip), the site data – containing the import and the export electricity prices, 290 the energy exports from the wind turbines, the energy generated by the turbines, and the total 291 load energy required – are used in calculating the gross annual gain from self-consumption of 292 wind energy. The daily potential gain is estimated by dividing the gross annual gain by the 293 number of days in a year. 294 • Time-of-Use-Bill-Management: the size of the storage device is 2MW/4MWh; the device is 85% 295 efficient (roundtrip), the site data are used in calculating the mean daily import; using the Power 296 NI – an electricity supplier – Economy 7 (2-Rate) meter plan [44], a third of the total electricity 297 required is set to be imported at a low rate period (at nights) while the remaining electricity is 298 imported at a high rate period (during the day). 299 • Demand Response of Load Shifting: the size of the storage device is 2MW/4MWh; the device is 300 85% efficient (roundtrip), the site data are used in calculating the mean daily import; using the 301 SSE Airtricity (an electricity supplier) KeyPad Powershift meter plan, a third of the total 302 electricity required for the day is imported within the “low” rate period – between 1:00 and 9:00 303 [45,46] while the remaining electricity is imported at the “normal” rate period during the day. 304 Other potential storage values could be estimated for specific sites within the distribution 312 network. Meanwhile, any given application could require using a storage device with specific 313 characteristics. 314 3. Results and Discussion 315 With the load demand rising 324 from the base point at 500 kW, the loads are served from the turbines (with the excess wind 325 generation and low demand at this time) and the storage device is discharged to meet the additional 326 demand until at around the 4:30 hour when the energy generation from the turbines increases, the 327 load demands being fully served and the excess wind energy charging the device through to around 328 the 5:40 hour. As the load demand increases through the day, more energy is imported from the grid 329 to supplement the energy generation from the turbines while the storage device is kept at a state of 330 charge. At about the 20:00 hour, the wind energy generation drops; the battery is discharged as 331 much as possible while the deficit in energy supply is met by the grid – the import from the grid 332 moving close to 400 kW. 333 The profile indicates that the deployed storage device could be subject to daily multiple rounds 334 of discharge cycles to achieve a maximum self-consumption of wind energy. This suggests that the 335 deployed storage device should have the capability for several rounds of deep discharging. 336 Within the one-year period under consideration, while a 3,720,642 kWh of energy at a market 337 value of £446,4777.04 (3,720,642 kWh * £0.12/kWh) was imported from the grid, a total energy of 338 601,780 kWh at a market value of £31,593.45 (601,780 kWh * £0.0525/kWh) was exported to the grid. 339 The total recoverable energy is obtained by multiplying the captured (used to be exported) energy 340 (601,780 kWh) by the storage efficiency. The market value of the recovered energy has been obtained 341 by multiplying the total recoverable energy by the market price of £0.12/kWh – the import and the 342 export electricity prices are approximated from the historical analysis of the export and the import 343 payments data. In [33], a similar price relation between the export electricity price and the import 344 electricity price for grid-connected wind turbines on the foregoing distribution network may be 345 seen. The gross annual gain shows the difference in market value at the import electricity price of 346 £0.12/kWh and at the export electricity price of £0.0525/kWh, Table 2. 3. Results and Discussion 315 While the on-site loads are supplied with the power generation from the wind turbines and the 316 grid, the installed storage device takes up any excess wind energy generation from the turbines as 317 the load flow converges while the network elements operate within safe limits, illustrated for a 318 typical windy day in Figure 5. 319 10 of 18 10 of 18 Energies 2020, 13, x FOR PEER REVIEW 320 Figure 5. Energy Profiles for an Illustrative Day 321 Figure 5. Energy Profiles for an Illustrative Day Figure 5. Energy Profiles for an Illustrative Day The energy profile reveals the charge-discharge characteristics, suggesting applicable storage 322 device, Figure 5. Between midnight (00:00 hour) and evening (18:00 hour), the aggregate power from 323 the two wind turbines was close to 600 kW – a typically windy day. With the load demand rising 324 from the base point at 500 kW, the loads are served from the turbines (with the excess wind 325 generation and low demand at this time) and the storage device is discharged to meet the additional 326 demand until at around the 4:30 hour when the energy generation from the turbines increases, the 327 load demands being fully served and the excess wind energy charging the device through to around 328 the 5:40 hour. As the load demand increases through the day, more energy is imported from the grid 329 to supplement the energy generation from the turbines while the storage device is kept at a state of 330 charge. At about the 20:00 hour, the wind energy generation drops; the battery is discharged as 331 much as possible while the deficit in energy supply is met by the grid – the import from the grid 332 moving close to 400 kW. 333 The energy profile reveals the charge discharge characteristics, suggesting applicable storage 322 device, Figure 5. Between midnight (00:00 hour) and evening (18:00 hour), the aggregate power from 323 the two wind turbines was close to 600 kW – a typically windy day. 3. Results and Discussion 315 For example, some battery 360 performances may degrade while operating outside recommended temperature ranges. The mean 361 annual temperature at this site over centuries have ranged from 8.50C to 10.00C, with a record 362 extreme maximum temperature at 32.30C and minimum temperature at -9.00C [47,48]. The storage 363 technologies selected can operate well within the site temperature range. 364 In other words, the storage technologies selected have typical roundtrip efficiencies above 65%, 365 could meet the charge-discharge characteristics required, are mature or demonstrated technologies, 366 have reasonable cost trends, have operating temperature features that make them appropriate at the 367 site, are applicable at the point of the distribution network, and that could serve both as load and as 368 generator. Of the considered storage technologies, flywheel storage, lithium ion battery, sodium ion 369 (Na-ion) battery, and zinc-bromine (Zn-Br) flow battery are found to meet the storage requirements 370 [19,20,21,22]. 371 Considering the changes to the energy mix of the site: with the storage, no on-site generated 372 wind energy is supplied to the grid – the storage captures the excess wind energy for 373 self-consumption on-site. As depicted in Figure 6, the percentage of the wind energy in the energy 374 mix at the location moved from 39.47% in Figure 6(a) to 48.32% in Figure 6(b) – an almost 10% 375 increase in self-consumption of wind energy. The other part of the energy mix came from a grid 376 supply with an average energy mix containing about 55% of the total energy generation coming 377 from fossil fuel sources [15]. 378 The quantity of the recoverable energy is more when using a storage a device of higher 349 efficiency – as less of the excess wind energy is wasted through the charge-discharge cycles with the 350 higher efficiency storage system; for example, while a total energy of 571,691.00 kWh is recoverable 351 when using a 95% efficient storage system, only a 421,246.00 kWh of energy is recoverable when 352 using a 70% efficient storage system. In the existing market in which the import electricity price is 353 £0.12/kWh and the export electricity price is £0.0525/kWh – taken as typical prices – the gross annual 354 gain and the percentage of energy serving the loads from the storage device are more while using 355 the high-efficiency storage system, Table 2. 3. Results and Discussion 315 347 The profile indicates that the deployed storage device could be subject to daily multiple rounds 334 of discharge cycles to achieve a maximum self-consumption of wind energy. This suggests that the 335 deployed storage device should have the capability for several rounds of deep discharging. 336 11 of 18 Energies 2020, 13, x FOR PEER REVIEW Table 2. Effect of Storage Efficiency on Total Recoverable Energy 48 Efficiency of Storage System (%) Total Recoverable Energy (kWh) Market Value of Recovered Energy at £0.12/kWh (£) Gross Annual Gain at £ (0.12-0.0525) /kWh (£) Self-consumption of Wind Energy (%) 95 571,691.00 68,602.92 37,009.47 48.89 90 541,602.00 64,992.24 33,398.79 48.40 85 511,513.00 61,381.56 29,788.11 47.91 80 481,424.00 57,770.88 26,177.43 47.42 75 451,335.00 54,160.20 22,566.75 46.93 70 421,246.00 50,549.52 18,956.07 46.45 348 Table 2. Effect of Storage Efficiency on Total Recoverable Energy Table 2. Effect of Storage Efficiency on Total Recoverable Energy The quantity of the recoverable energy is more when using a storage a device of higher 349 efficiency – as less of the excess wind energy is wasted through the charge-discharge cycles with the 350 higher efficiency storage system; for example, while a total energy of 571,691.00 kWh is recoverable 351 when using a 95% efficient storage system, only a 421,246.00 kWh of energy is recoverable when 352 using a 70% efficient storage system. In the existing market in which the import electricity price is 353 £0.12/kWh and the export electricity price is £0.0525/kWh – taken as typical prices – the gross annual 354 gain and the percentage of energy serving the loads from the storage device are more while using 355 the high-efficiency storage system, Table 2. The result of Table 2 suggests that, to derive more gain 356 from deploying a storage device for increasing self-consumption of the locally generated wind 357 energy, a storage technology having a higher efficiency should be used. 358 Another important storage characteristic that should be considered is the operating 359 temperature of the storage device in respect of its environment. For example, some battery 360 performances may degrade while operating outside recommended temperature ranges. The mean 361 annual temperature at this site over centuries have ranged from 8.50C to 10.00C, with a record 362 extreme maximum temperature at 32.30C and minimum temperature at -9.00C [47,48]. The storage 363 technologies selected can operate well within the site temperature range. 3. Results and Discussion 315 364 In other words, the storage technologies selected have typical roundtrip efficiencies above 65%, 365 could meet the charge-discharge characteristics required, are mature or demonstrated technologies, 366 have reasonable cost trends, have operating temperature features that make them appropriate at the 367 site, are applicable at the point of the distribution network, and that could serve both as load and as 368 generator. Of the considered storage technologies, flywheel storage, lithium ion battery, sodium ion 369 (Na-ion) battery, and zinc-bromine (Zn-Br) flow battery are found to meet the storage requirements 370 [19,20,21,22]. 371 Considering the changes to the energy mix of the site: with the storage, no on-site generated 372 wind energy is supplied to the grid – the storage captures the excess wind energy for 373 self-consumption on-site. As depicted in Figure 6, the percentage of the wind energy in the energy 374 mix at the location moved from 39.47% in Figure 6(a) to 48.32% in Figure 6(b) – an almost 10% 375 increase in self-consumption of wind energy. The other part of the energy mix came from a grid 376 supply with an average energy mix containing about 55% of the total energy generation coming 377 from fossil fuel sources [15]. 378 The quantity of the recoverable energy is more when using a storage a device of higher 349 efficiency – as less of the excess wind energy is wasted through the charge-discharge cycles with the 350 higher efficiency storage system; for example, while a total energy of 571,691.00 kWh is recoverable 351 when using a 95% efficient storage system, only a 421,246.00 kWh of energy is recoverable when 352 using a 70% efficient storage system. In the existing market in which the import electricity price is 353 £0.12/kWh and the export electricity price is £0.0525/kWh – taken as typical prices – the gross annual 354 gain and the percentage of energy serving the loads from the storage device are more while using 355 the high-efficiency storage system, Table 2. The result of Table 2 suggests that, to derive more gain 356 from deploying a storage device for increasing self-consumption of the locally generated wind 357 energy, a storage technology having a higher efficiency should be used. 358 Another important storage characteristic that should be considered is the operating 359 temperature of the storage device in respect of its environment. 3. Results and Discussion 315 Zn-Br Flow battery may not last for up to 10 years. 397 Figure 6. Energy Mix of Site In analysing the value derived from deploying the storage device for self-consumption of wind 381 energy: the total storage capacity cost is a total system cost – covering any cost associated with the 382 acquisition, the installation, and the usage of the storage (including fixed cost, variable cost, capital 383 cost, initial cost, maintenance cost, and any complementary costs). The cost ranges are heuristic 384 test-case selections. The cost options help to see where the profitability of the storage project lies for 385 different storage cost parameters that could typify varying market conditions, using a payback 386 period estimation within the life span of the storage device. 387 Each of the storage technologies has been assigned a nominal storage efficiency; the values are 388 the overall roundtrip efficiencies of the whole system of storage. The typical lifespan of a flywheel 389 storage is taken to be above 20 years, the lithium ion and the sodium ion batteries are taken to have 390 lifespans between 10 to 15 years, and the Zinc-bromine flow battery is considered to have lifespan of 391 between 5 to 10 years [19,22]. The lifespans of the storage technologies are included to show where 392 the technologies could make economic sense around the hypothesised prices. The payback period is 393 the ratio of the cost of the total storage system to the gross annual gain of storage, Table 3. 394 395 Table 3. minimum of 10-year frame. Zn-Br Flow battery may not last for up to 10 years. As most power equipment usually last for over 40 years, it is customary to evaluate new equipment within a 396 3. Results and Discussion 315 The result of Table 2 suggests that, to derive more gain 356 from deploying a storage device for increasing self-consumption of the locally generated wind 357 energy, a storage technology having a higher efficiency should be used. 358 Another important storage characteristic that should be considered is the operating 359 temperature of the storage device in respect of its environment. For example, some battery 360 performances may degrade while operating outside recommended temperature ranges. The mean 361 annual temperature at this site over centuries have ranged from 8.50C to 10.00C, with a record 362 extreme maximum temperature at 32.30C and minimum temperature at -9.00C [47,48]. The storage 363 technologies selected can operate well within the site temperature range. 364 In other words, the storage technologies selected have typical roundtrip efficiencies above 65%, 365 could meet the charge-discharge characteristics required, are mature or demonstrated technologies, 366 have reasonable cost trends, have operating temperature features that make them appropriate at the 367 site, are applicable at the point of the distribution network, and that could serve both as load and as 368 generator. Of the considered storage technologies, flywheel storage, lithium ion battery, sodium ion 369 (Na-ion) battery, and zinc-bromine (Zn-Br) flow battery are found to meet the storage requirements 370 [19,20,21,22]. 371 Considering the changes to the energy mix of the site: with the storage, no on-site generated 372 wind energy is supplied to the grid – the storage captures the excess wind energy for 373 self-consumption on-site. As depicted in Figure 6, the percentage of the wind energy in the energy 374 mix at the location moved from 39.47% in Figure 6(a) to 48.32% in Figure 6(b) – an almost 10% 375 increase in self-consumption of wind energy. The other part of the energy mix came from a grid 376 supply with an average energy mix containing about 55% of the total energy generation coming 377 from fossil fuel sources [15]. 378 12 of 18 Energies 2020, 13, x FOR PEER REVIEW 379 Figure 6. Energy Mix of Site 380 379 Figure 6. 3. Results and Discussion 315 Energy Mix of Site 380 In analysing the value derived from deploying the storage device for self-consumption of wind 381 energy: the total storage capacity cost is a total system cost – covering any cost associated with the 382 acquisition, the installation, and the usage of the storage (including fixed cost, variable cost, capital 383 cost, initial cost, maintenance cost, and any complementary costs). The cost ranges are heuristic 384 test-case selections. The cost options help to see where the profitability of the storage project lies for 385 different storage cost parameters that could typify varying market conditions, using a payback 386 period estimation within the life span of the storage device. 387 Each of the storage technologies has been assigned a nominal storage efficiency; the values are 388 the overall roundtrip efficiencies of the whole system of storage. The typical lifespan of a flywheel 389 storage is taken to be above 20 years, the lithium ion and the sodium ion batteries are taken to have 390 lifespans between 10 to 15 years, and the Zinc-bromine flow battery is considered to have lifespan of 391 between 5 to 10 years [19,22]. The lifespans of the storage technologies are included to show where 392 the technologies could make economic sense around the hypothesised prices. The payback period is 393 the ratio of the cost of the total storage system to the gross annual gain of storage, Table 3. 394 Table 3. Deployment of Storage Device to Store Excess Wind Energy Only 395 Selected Energy Storage Technologies and Costs (£/kW; £/kWh) Total Storage Capacity Cost (£ Million) Nominated Storage Efficiency (%) Life Span (Years) Gross Annual Gain (£) Payba Perio (Year Flywheel at £120/kW; at £80/kWh 0.56 90 20+ 33,398.79 16.8 Flywheel at £1,880/kW; at £1,715/kWh 10.62 90 20+ 33,398.79 318.0 Li-ion Battery at £110/kW, at £70/kWh 0.50 85 10-15 29,788.11 16.8 Li-ion Battery at £1,580/kW, at £1,510/kWh 9.20 85 10-15 29,788.11 308.8 Na-ion Battery at £90/kW, at £60/kWh 0.42 80 10-15 26,177.43 16.0 Na-ion Battery at £1,200/kW, at £1,100/kWh 6.80 80 10-15 26,177.43 259.8 Zn-Br Flow Battery at £105/kW, at £65/kWh 0.47 75 5-10 22,566.75 20.8 Zn-Br Flow Battery at £1,150/kW, at £800/kWh 5.50 75 5-10 22,566.75 243.7 As most power equipment usually last for over 40 years, it is customary to evaluate new equipment within a 396 minimum of 10-year frame. As most power equipment usually last for over 40 years, it is customary to evaluate new eq 6 3. Results and Discussion 315 Deployment of Storage Device to Store Excess Wind Energy Only 395 Selected Energy Storage Technologies and Costs (£/kW; £/kWh) Total Storage Capacity Cost (£ Million) Nominated Storage Efficiency (%) Life Span (Years) Gross Annual Gain (£) Payback Period (Years) Flywheel at £120/kW; at £80/kWh 0.56 90 20+ 33,398.79 16.8 Flywheel at £1,880/kW; at £1,715/kWh 10.62 90 20+ 33,398.79 318.0 Li-ion Battery at £110/kW, at £70/kWh 0.50 85 10-15 29,788.11 16.8 Li-ion Battery at £1,580/kW, at £1,510/kWh 9.20 85 10-15 29,788.11 308.8 Na-ion Battery at £90/kW, at £60/kWh 0.42 80 10-15 26,177.43 16.0 Na-ion Battery at £1,200/kW, at £1,100/kWh 6.80 80 10-15 26,177.43 259.8 Zn-Br Flow Battery at £105/kW, at £65/kWh 0.47 75 5-10 22,566.75 20.8 Zn-Br Flow Battery at £1,150/kW, at £800/kWh 5.50 75 5-10 22,566.75 243.7 As most power equipment usually last for over 40 years, it is customary to evaluate new equipment within a 396 minimum of 10-year frame. Zn-Br Flow battery may not last for up to 10 years. 397 Table 3. Deployment of Storage Device to Store Excess Wind Energy Only 396 397 396 397 Energies 2020, 13, x FOR PEER REVIEW 13 of 18 The results of Table 3 suggest that, with the current market conditions, the deployment of the 398 2MW/4MWh energy storage device for self-consumption of wind energy could become 399 economically feasible at the storage cost around £500,000. Given that the storage technologies have 400 similar costs, flywheel storage promises higher return on investment because of its longer lifespan, 401 inherent almost-unlimited cycles, and ruggedness in responding effectively to providing specialised 402 electricity grid services. However, its considerable self-discharge rate could make it a less desirable 403 choice for deferred self-consumption of stored energy [22]. Lithium ion battery could be a better 404 option for being a more mature technology, being less susceptible to self-discharge, being able to 405 withstand several rounds of deep discharging, and like most batteries, being able to respond in time 406 to providing grid services [19]. 407 While the results of Table 3 are for the case where the storage device has been deployed only for 408 increasing self-consumption of wind energy, Table 4 depicts the result of deploying the device for 409 providing certain DS3 market services in addition to increasing self-consumption of wind energy. 410 Table 4. 3. Results and Discussion 315 Deployment of Storage for Self-consumption of Wind Energy and Ancillary Services 411 Selected Energy Storage Technologies and Costs (£/kW; £/kWh) Ancillary Services Duration/ Lifespan (%) New Annual Gain 1 (£) New Payback Period 1 (Years) Ancillary Services Duration/ Lifespan (%) New Payback Period 2 (Years) Flywheel at £120/kW; at £80/kWh 0.42 36,150.31 15.5 25 2.8 Flywheel at £1,880/kW; at £1,715/kWh 0.42 36,150.31 293.8 25 53.9 Li-ion Battery at £110/kW, at £70/kWh 0.56-0.83 32,126.90 15.6 25 3.9 Li-ion Battery at £1,580/kW, at £1,510/kWh 0.56-0.83 32,126.90 286.4 25 72.3 Na-ion Battery at £90/kW, at £60/kWh 0.56-0.83 28,048.42 15.0 25 3.5 Na-ion Battery at £1,200/kW, at £1,100/kWh 0.56-0.83 28,048.42 242.4 25 57.7 Zn-Br Flow Battery at £105/kW, at £65/kWh 0.83-1.7 23,970.04 19.6 25 6.3 Zn-Br Flow Battery at £1,150/kW, at £800/kWh 0.83-1.7 23,970.04 229.5 25 74.2 Table 4. Deployment of Storage for Self-consumption of Wind Energy and Ancillary Services Table 4. Deployment of Storage for Self-consumption of Wind Energy and Ancillary Services With the storage deployed for the multipurpose of increasing self-consumption of wind energy 412 and providing the ancillary services, the results indicate a shorter payback period on investment, 413 suggesting increased profitability. The total DS3 service provided has been taken from the storage 414 eligible DS3 service suite of Table 1. The storage capacity is committed within less than 2% of total 415 lifespan of the storage device at the first instance: estimates the new annual Gain 1 and the new 416 payback Period 1. The same capacity is committed for 25% of the device total lifespan at the second 417 instance: estimates a new Gain 2 and the new payback Period 2, Table 4. The new annual gain is the 418 sum of the gain from self-consumption of wind energy and the gain from the provision of ancillary 419 services. 420 The payback periods are shorter when the storage device is committed for longer duration. This 421 suggests that, when deploying a storage device at the distribution network, it could be more 422 profitable to commit the device to providing ancillary services to an extent permissible and that does 423 not pose risk to the security of other investments serving the grid. 424 With the storage deployed for the multipurpose of increasing self-consumption of wind energy 412 and providing the ancillary services, the results indicate a shorter payback period on investment, 413 suggesting increased profitability. 3. Results and Discussion 315 The total DS3 service provided has been taken from the storage 414 eligible DS3 service suite of Table 1. The storage capacity is committed within less than 2% of total 415 lifespan of the storage device at the first instance: estimates the new annual Gain 1 and the new 416 payback Period 1. The same capacity is committed for 25% of the device total lifespan at the second 417 instance: estimates a new Gain 2 and the new payback Period 2, Table 4. The new annual gain is the 418 sum of the gain from self-consumption of wind energy and the gain from the provision of ancillary 419 services. 420 With the storage deployed for the multipurpose of increasing self-consumption of wind energy 412 and providing the ancillary services, the results indicate a shorter payback period on investment, 413 suggesting increased profitability. The total DS3 service provided has been taken from the storage 414 eligible DS3 service suite of Table 1. The storage capacity is committed within less than 2% of total 415 lifespan of the storage device at the first instance: estimates the new annual Gain 1 and the new 416 payback Period 1. The same capacity is committed for 25% of the device total lifespan at the second 417 instance: estimates a new Gain 2 and the new payback Period 2, Table 4. The new annual gain is the 418 sum of the gain from self-consumption of wind energy and the gain from the provision of ancillary 419 services. 420 The payback periods are shorter when the storage device is committed for longer duration. This 421 suggests that, when deploying a storage device at the distribution network, it could be more 422 profitable to commit the device to providing ancillary services to an extent permissible and that does 423 not pose risk to the security of other investments serving the grid. 424 14 of 18 Energies 2020, 13, x FOR PEER REVIEW 425 Figure 7. Potential Daily Revenue of Storage across Electricity Supply Chain 426 Figure 7. Potential Daily Revenue of Storage across Electricity Supply Chain Another picture is depicted in Figure 7, where the daily potential value that the deployed 427 energy storage system could offer to stakeholders across the electricity supply chain has been 428 estimated using the approximate data described in section 2.5.3. 3. Results and Discussion 315 While some of the potential values 429 such as demand charge reduction and increased wind self-consumption are concrete, others – such 430 as transmission and distribution deferrals – could be conceptual and often require favourable 431 integration policies and proper grid planning or coordination to become realizable. 432 Certain incentives could be available for generating and using more clean energy on-site; for 433 example, the revenue stream from the Renewable Obligation Certificate (ROC) that was in place to 434 promote renewable energy in Northern Ireland [33]. Similarly, some mechanisms that reduce 435 investment risks; for example, the Power Purchase Agreements (PPA) could serve to guarantee the 436 market for the storage services. The ROC and the PPA arrangements are typical market and 437 integration policies that could impact the value of any energy storage project. 438 Meanwhile, beyond the distribution network, some other storage benefits which are also 439 typically very site-specific could be derived while using the storage device for capturing or saving 440 energy for a later use. To mention a few: to manage the output of mass wind turbines where a 441 network congestion would have disallowed any further grid-integration of turbines, a storage 442 device could be installed for managed connection. The storage device could also be installed at the 443 higher voltage ends of the electricity network for energy arbitrage; for example, for bulk energy 444 trading during periods of high price volatility through the Irish ISEM intra-day market [35]. 445 Lastly, a country-wide analysis could be performed to see how storage systems could be 446 deployed to support renewables and bring optimal benefits to the customer, to the grid, and to the 447 utility; maximizing renewable energy generation in achieving key sustainability targets. 448 4. Conclusions 449 476 Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the 477 study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to 478 publish the results. 479 Appendix 480 Static Load Flow Equations: 481 Given that the net complex power flowing into a bus i of a network is 482 (A1) 483 where PD and QD are the real power demand and the reactive power demand respectively while PG 484 and QG are the real power generation and the reactive power generation within the bus respectively, 485 486 ; for i = 1, 2, 3, …, n 487 If n represents the number of buses within the network, the flow of current through the bus i is 488 ; for i = 1, 2, 3, …, n (A2) 489 where Vk is the voltage at bus k, Yik is the mutual admittance – the admittance between the ith and the 490 kth nodes; is the negative of the total admittances existing between the ith and kth nodes, 491 whereas Yik = Yki 492 Similarly, the complex power flowing into a bus i is given as 493 ; for i = 1, 2, 3, …, n (A3) 494 i h i l j f h fl i hi h i h b d i 495 self-consumption of wind energy makes the storage project more profitable – suggesting a 459 mechanism through which the storage system could be deployed to contribute to the on-going effort 460 of maximizing the utilization of clean energy for sustainable development. The profitability of the 461 storage system deployed at the distribution network is dependent on the aggregate storage cost, the 462 integration policies at the location, and the ability to deploy the device for stacked services. Through 463 favourable integration and environment-cautious policies, energy storage could provide customer 464 and ancillary services within the electricity supply chain. 4. Conclusions 449 Energy generation from wind turbines connected to the distribution network could contribute 450 to the effort of decarbonizing electricity systems. With storage devices, more of the on-site generated 451 wind energy could be captured for later energy consumption. For grid-connected systems, where 452 the market and integration policies permit it, the storage device could – in addition to providing 453 customer services – be committed to providing DS3 services of active and reactive power, ramping 454 margins, and reserves. When a 2MW/4MWh storage device was deployed at a distribution network 455 having two 800KW BTM wind turbines, a typical peak load under 1,500 kW and a base load around 456 500 kW, the percentage of self-consumption of wind energy rose from 39.47% to 48.32%. Deploying 457 the device for providing other market services in addition to helping to achieve increased 458 15 of 18 Energies 2020, 13, x FOR PEER REVIEW self-consumption of wind energy makes the storage project more profitable – suggesting a 459 mechanism through which the storage system could be deployed to contribute to the on-going effort 460 of maximizing the utilization of clean energy for sustainable development. The profitability of the 461 storage system deployed at the distribution network is dependent on the aggregate storage cost, the 462 integration policies at the location, and the ability to deploy the device for stacked services. Through 463 favourable integration and environment-cautious policies, energy storage could provide customer 464 and ancillary services within the electricity supply chain. 465 Author Contributions: Conceptualization, Oluwasola O. Ademulegun, Patrick Keatley, Neil J. Hewitt; 466 Methodology, Oluwasola O. Ademulegun; software, Oluwasola O. Ademulegun; writing—original draft 467 preparation and editing, Oluwasola O. Ademulegun; writing—review, Patrick Keatley, Motasem Bani Mustafa, 468 Neil J. Hewitt; supervision, Neil J. Hewitt, Patrick Keatley; funding acquisition, Neil J. Hewitt. All authors have 469 read and agreed to the published version of the manuscript. 470 Funding: This research was funded by the Science Foundation Ireland (SFI) and the Department for the 471 Economy (DfE) in Northern Ireland, grant number 92160R. 472 Acknowledgments: James Waide of the Physical Resources Department at Ulster University provided support 473 while collecting data. Paul Bell of the Utility Regulator Northern Ireland supported in information gathering. 474 The System Operator for Northern Ireland (SONI) and the Northern Ireland Electricity (NIE) Networks 475 provided support in data collection. References 507 The 508 Stationery Office Limited under the authority and superintendence of Jeff James, Controller of Her 509 Majesty’s Stationery Office and Queen’s Printer of Acts of Parliament, Draft Statutory Instrument, 2019. 510 1. UK Houses of Parliament. The Climate Change Act 2008 (2050 Target Amendment) Order 2019. The 508 Stationery Office Limited under the authority and superintendence of Jeff James, Controller of Her 509 Majesty’s Stationery Office and Queen’s Printer of Acts of Parliament, Draft Statutory Instrument, 2019. 510 2. SONI. Strategy 2020-25: Transform the Power System for Future Generations. Strategy 2020-2025 Report, 511 Available online: www.soni.ltd.uk, (accessed on 1 September 2019). 512 2. SONI. Strategy 2020-25: Transform the Power System for Future Generations. Strategy 2020-2025 Report, 511 Available online: www.soni.ltd.uk, (accessed on 1 September 2019). 512 3. Electric Power Research Institute. Time and Locational Value of DER: Methods and Applications. EPRI 513 Report 3002008410, 2016; pp. 1–8. 514 3. Electric Power Research Institute. Time and Locational Value of DER: Methods and Applications. EPRI 513 Report 3002008410, 2016; pp. 1–8. 514 4. Olinsky-Paul, Todd. Energy Storage: The New Efficiency - How states can use energy efficiency funds to 515 support battery storage and flatten costly demand peaks. Clean Energy Group Report, 2019; pp. 1–102. 516 5. Pietrosanti, Stefano; Holderbaum, William; Becerra, V. M. Optimal Power Management Strategy for 517 Energy Storage with Stochastic Loads. Energies, 2016, vol. 9, no. 3, pp. 1–17. 518 6. Finn, P.; Fitzpatrick, C. Demand side management of industrial electricity consumption: Promoting the 519 use of renewable energy through real-time pricing. Appl. Energy, 2014, vol. 113, pp. 1–11. 520 5 7. EirGrid and SONI. Annual Renewable Energy Constraint and Curtailment Report 2018. Report, Available 521 online: 522 http://www.eirgridgroup.com/site-files/library/EirGrid/Annual-Renewable-Constraint-and-Curtailment- 523 Report-2018-V1.0.pdf (accessed on 30 May 2019); pp. 1–26. 524 http://www.eirgridgroup.com/site-files/library/EirGrid/Annual-Renewable-Constraint-and-Curtailment- 523 Report-2018-V1.0.pdf (accessed on 30 May 2019); pp. 1–26. 524 8. Rocky Mountain Institute. The Economics of Battery Energy Storage - How Multi-use, Customer-sited 525 Batteries Deliver the Most Services and Value to Customers and Grid. Report, Available online: 526 https://rmi.org/wp-content/uploads/2017/03/RMI-TheEconomicsOfBatteryEnergyStorage-FullReport-FIN 527 AL.pdf, (accessed on 30 October 2019); pp. 14-16. 528 9. Hartmann, Bálint; Vokony, István; Sorés, Péter; Táczi, István. Service aspect assessment of energy storage 529 under the ownership of distribution system operators. Journal of Energy Storage, 2019, vol 25. 530 10. Fine, Steve; Martini, D. Paul; Succar, Samir; Robison, Matt. The Value in Distributed Energy: It’s All About 531 Location, Location, Location. References 507 1. UK Houses of Parliament. The Climate Change Act 2008 (2050 Target Amendment) Order 2019. The 508 Stationery Office Limited under the authority and superintendence of Jeff James, Controller of Her 509 Majesty’s Stationery Office and Queen’s Printer of Acts of Parliament, Draft Statutory Instrument, 2019. 510 2. SONI. Strategy 2020-25: Transform the Power System for Future Generations. Strategy 2020-2025 Report, 511 Available online: www.soni.ltd.uk, (accessed on 1 September 2019). 512 3. Electric Power Research Institute. Time and Locational Value of DER: Methods and Applications. EPRI 513 Report 3002008410, 2016; pp. 1–8. 514 4. Olinsky-Paul, Todd. Energy Storage: The New Efficiency - How states can use energy efficiency funds to 515 support battery storage and flatten costly demand peaks. Clean Energy Group Report, 2019; pp. 1–102. 516 5. Pietrosanti, Stefano; Holderbaum, William; Becerra, V. M. Optimal Power Management Strategy for 517 Energy Storage with Stochastic Loads. Energies, 2016, vol. 9, no. 3, pp. 1–17. 518 6. Finn, P.; Fitzpatrick, C. Demand side management of industrial electricity consumption: Promoting the 519 use of renewable energy through real-time pricing. Appl. Energy, 2014, vol. 113, pp. 1–11. 520 7. EirGrid and SONI. Annual Renewable Energy Constraint and Curtailment Report 2018. Report, Available 521 online: 522 http://www.eirgridgroup.com/site-files/library/EirGrid/Annual-Renewable-Constraint-and-Curtailment- 523 Report-2018-V1.0.pdf (accessed on 30 May 2019); pp. 1–26. 524 8. Rocky Mountain Institute. The Economics of Battery Energy Storage - How Multi-use, Customer-sited 525 Batteries Deliver the Most Services and Value to Customers and Grid. Report, Available online: 526 https://rmi.org/wp-content/uploads/2017/03/RMI-TheEconomicsOfBatteryEnergyStorage-FullReport-FIN 527 AL.pdf, (accessed on 30 October 2019); pp. 14-16. 528 9. Hartmann, Bálint; Vokony, István; Sorés, Péter; Táczi, István. Service aspect assessment of energy storage 529 under the ownership of distribution system operators. Journal of Energy Storage, 2019, vol 25. 530 10. Fine, Steve; Martini, D. Paul; Succar, Samir; Robison, Matt. The Value in Distributed Energy: It’s All About 531 Location, Location, Location. ICF International Whitepaper, 2015, pp. 1–11. 532 11. Oureilidis, Konstantinos; Kyriaki-Nefeli, Malamaki; Gallos, Konstantinos; Tsitsimelis, Achilleas; 533 Dikaiakos, Christos; Gkavanoudis, Spyros; Cvetkovic, Milos; Mauricio, Manuel Juan; Ortega, Maza Maria 534 Jose; Ramos, Luis Martinez Jose; Papaioannou, Georg; Demoulias, Charis. Ancillary Services Market 535 Design in Distribution Networks: Review and Identification of Barriers. Energies 2020, 13(4), 917; 536 https://doi.org/10.3390/en13040917. 537 12. Maza-Ortega, M. José; Mauricio, M. Juan; Barragán-Villarejo, Manuel; Demoulias, Charis; 538 1. UK Houses of Parliament. The Climate Change Act 2008 (2050 Target Amendment) Order 2019. 4. Conclusions 449 Strategy 2020-25: Transform the Power System for Future Generations. Strategy 2020-2025 Report, 511 Available online: www.soni.ltd.uk, (accessed on 1 September 2019). 512 3. Electric Power Research Institute. Time and Locational Value of DER: Methods and Applications. EPRI 513 Report 3002008410, 2016; pp. 1–8. 514 4. Olinsky-Paul, Todd. Energy Storage: The New Efficiency - How states can use energy efficiency funds to 515 support battery storage and flatten costly demand peaks. Clean Energy Group Report, 2019; pp. 1–102. 516 5. Pietrosanti, Stefano; Holderbaum, William; Becerra, V. M. Optimal Power Management Strategy for 517 Energy Storage with Stochastic Loads. Energies, 2016, vol. 9, no. 3, pp. 1–17. 518 6. Finn, P.; Fitzpatrick, C. Demand side management of industrial electricity consumption: Promoting the 519 use of renewable energy through real-time pricing. Appl. Energy, 2014, vol. 113, pp. 1–11. 520 7. EirGrid and SONI. Annual Renewable Energy Constraint and Curtailment Report 2018. Report, Available 521 online: 522 http://www.eirgridgroup.com/site-files/library/EirGrid/Annual-Renewable-Constraint-and-Curtailment- 523 Report-2018-V1.0.pdf (accessed on 30 May 2019); pp. 1–26. 524 8. Rocky Mountain Institute. The Economics of Battery Energy Storage - How Multi-use, Customer-sited 525 Batteries Deliver the Most Services and Value to Customers and Grid. Report, Available online: 526 https://rmi.org/wp-content/uploads/2017/03/RMI-TheEconomicsOfBatteryEnergyStorage-FullReport-FIN 527 AL.pdf, (accessed on 30 October 2019); pp. 14-16. 528 9. Hartmann, Bálint; Vokony, István; Sorés, Péter; Táczi, István. Service aspect assessment of energy storage 529 under the ownership of distribution system operators. Journal of Energy Storage, 2019, vol 25. 530 10. Fine, Steve; Martini, D. Paul; Succar, Samir; Robison, Matt. The Value in Distributed Energy: It’s All About 531 Location, Location, Location. ICF International Whitepaper, 2015, pp. 1–11. 532 11. Oureilidis, Konstantinos; Kyriaki-Nefeli, Malamaki; Gallos, Konstantinos; Tsitsimelis, Achilleas; 533 Dikaiakos, Christos; Gkavanoudis, Spyros; Cvetkovic, Milos; Mauricio, Manuel Juan; Ortega, Maza Maria 534 Jose; Ramos, Luis Martinez Jose; Papaioannou, Georg; Demoulias, Charis. Ancillary Services Market 535 Design in Distribution Networks: Review and Identification of Barriers. Energies 2020, 13(4), 917; 536 https://doi.org/10.3390/en13040917. 537 12. Maza-Ortega, M. José; Mauricio, M. Juan; Barragán-Villarejo, Manuel; Demoulias, Charis; 538 Gómez-Expósito, Antonio. Ancillary Services in Hybrid AC/DC Low Voltage Distribution Networks. 539 Energies 2019, 12(19), 3591; https://doi.org/10.3390/en12193591. 4. Conclusions 449 540 ; for i = 1, 2, 3, …, n (A4) 498 ; for i = 1, 2, 3, …, n (A4) (A4) Now, if the real and the imaginary sections of Equation (A4) are correlated, 499 ; ; for i = 1, 2, 3, …, n (A5) 500 ; ; for i = 1, 2, 3, …, n 500 ; ; for i = 1, 2, 3, …, n (A5) (A5) In polar form, ; ; and ; while θ represents the phase angle 501 between current and voltage, δ represents the load angle. 502 In polar form, ; ; and ; while θ represents the phase angle 501 between current and voltage, δ represents the load angle. 502 In polar form, ; ; and ; while θ represents the phase angle 501 between current and voltage, δ represents the load angle. 502 Substituting the polar expressions for and in Equation (A5); the real power and the 503 reactive power components of the static load flow equation are respectively, 504 Substituting the polar expressions for and in Equation (A5); the real power and the 503 reactive power components of the static load flow equation are respectively, 504 reactive power components of the static load flow equation are respectively, 504 reactive power components of the static load flow equation are respectively, 504 505 506 4. Conclusions 449 465 (A1) (A1) where PD and QD are the real power demand and the reactive power demand respectively while PG 484 and QG are the real power generation and the reactive power generation within the bus respectively, 485 6 ; for i = 1, 2, 3, …, n f n represents the number of buses within the network, the flow of current through the bus i is ; for i = 1, 2, 3, …, n (A2) (A2) where Vk is the voltage at bus k, Yik is the mutual admittance – the admittance between the ith and the 490 kth nodes; is the negative of the total admittances existing between the ith and kth nodes, 491 where Vk is the voltage at bus k, Yik is the mutual admittance – the admittance between the ith and the 490 kth nodes; is the negative of the total admittances existing between the ith and kth nodes, 491 Similarly, the complex power flowing into a bus i is given as 3 ; for i = 1, 2, 3, …, n (A3) 494 (A3) with representing a complex conjugate of the current flow within the ith bus, and representing 495 the bus voltage, 496 with representing a complex conjugate of the current flow within the ith bus, and representing 495 the bus voltage, 496 497 ; for i = 1, 2, 3, …, n 497 ; for i = 1, 2, 3, …, n Energies 2020, 13, x FOR PEER REVIEW 16 of 18 ; for i = 1, 2, 3, …, n (A4) 498 Now, if the real and the imaginary sections of Equation (A4) are correlated, 499 ; ; for i = 1, 2, 3, …, n (A5) 500 In polar form, ; ; and ; while θ represents the phase angle 501 between current and voltage, δ represents the load angle. 502 Substituting the polar expressions for and in Equation (A5); the real power and the 503 reactive power components of the static load flow equation are respectively, 504 505 506 References 507 1. UK Houses of Parliament. The Climate Change Act 2008 (2050 Target Amendment) Order 2019. The 508 Stationery Office Limited under the authority and superintendence of Jeff James, Controller of Her 509 Majesty’s Stationery Office and Queen’s Printer of Acts of Parliament, Draft Statutory Instrument, 2019. 510 2. SONI. References 507 Energy & Environmental Science Review, 2018, DOI: 10.1039/c 567 24. Li, Xin; Chalvatzis, J. Konstantinos; Stephanides, Phedeas. Innovative Energy Islands: Life-Cycl 568 Cost-Benefit Analysis for Battery Energy Storage. Sustainability, September 2018. 569 25. Barelli, Linda; Bidini, Gianni; Cherubini, Paolo; Micangeli, Andrea; Pelosi, Dario; Tacconelli, C 570 Hybridization of Energy Storage Technologies Can Provide Additional Flexibility and Competi 571 Microgrids in the Context of Developing Countries. energies, August 2019. 572 26. Bradbury, Kyle. Energy Storage Technology Review. Review, Available 573 https://www.kylebradbury.org/docs/papers/Energy-Storage-Technology-Review-Kyle-Bradbur 574 f, (accessed on 30 October 2019). 575 27. Lazard. Lazard's Levelized Cost of Storage Analysis – Version 3.0. Technical Report, Availa 576 https://www.lazard.com/media/450338/lazard-levelized-cost-of-storage-version-30.pdf, (access 577 October 2019). 578 28. ADB. Handbook on Battery Energy Storage System. Asian Development Bank B 579 978-92-9261-470-6 (print), 978-92-9261-471-3 (electronic), Publication Stock No. TCS189791-2, 580 2018, DOI: http://dx.doi.org/10.22617/TCS189791-2. 581 29. IRENA. Electricity Storage and Renewables: Costs and Markets To 2030. International Renewab 582 Agency, Abu Dhabi Report, ISBN 978-92-9260-038-9, 2017. 583 30. Electric Power Research Institute. Electricity Energy Storage Technology Options: Applications, 584 Benefits. A White Paper Primer 1020676, 2011; pp. 1–170. 585 31. U.S. Department of Energy. Grid Energy Storage. Report on grid energy storage, December 2013. 586 32. Goldie-Scot, Logan. A Behind the Scenes Take on Lithium-ion Battery Prices. BloombergN 587 Available online: https://about.bnef.com/blog/behind-scenes-take-lithium-ion-battery-prices/, 588 on 1 January 2020). 589 33. Invest Northern Ireland. Wind Power: A best practice guide for Northern Ireland business. 590 De elo e t Tea Te t Relay N b 1800102890698273 A il b 591 13. Wang, Weiliang; Wang, Dan; Liu, Liu; Jia, Hongjie; Zhi, Yunqiang; Meng, Zhengji; Du, Wei. Research on 541 Modeling and Hierarchical Scheduling of a Generalized Multi-Source Energy Storage System in an 542 Integrated Energy Distribution System. Energies, 2019, 12(2), 246; https://doi.org/10.3390/en12020246. 543 14. Bartolucci, Lorenzo; Cordiner, Stefano; Mulone, Vincenzo; Santarelli, Marina. Ancillary Services Provided 44 by Hybrid Residential Renewable Energy Systems through Thermal and Electrochemical Storage Systems. 45 Energies 2019, 12(12), 2429; https://doi.org/10.3390/en12122429. 46 15. EirGrid and SONI. All-Island Generation Capacity Statement: 2019-2028. Available online: 547 http://www.soni.ltd.uk/media/documents/EirGrid-Group-All-Island-Generation-Capacity-Statement-201 548 9-2028.pdf, (accessed on 1 January 2020); pp. 1–78, 2019. 549 16. Northern Ireland Electricity Networks. NIE Networks RP6 Business Plan 2017-2024. NIE Networks RP6 550 Report, 2017. 551 17. Schmidt, Oliver; Melchior, Sylvain; Hawkes, Adam; Staffell, Iain. Projecting the Future Levelized Cost of 552 Electricity Storage Technologies. Joule 2019, 3, 81–100; https://doi.org/10.1016/j.joule.2018.12.008. 553 18. Rebello, E.; Watson, D.; Rodgers, M. References 507 Ancillary services from wind turbines: automatic generation control 554 (AGC) from a single Type 4 turbine. Wind Energy Science 2020, 5, 225-236; 555 https://doi.org/10.5194/wes-5-225-2020. 556 19. Aneke, M.; Wang, M. Energy storage technologies and real-life applications – A state of the art review. 557 Appl. Energy, 2016, vol. 179, pp. 350–377. 558 20. Sabihuddin, Siraj; Kiprakis, E. Aristides; Mueller, Markus. A Numerical and Graphical Review of Energy 559 Storage Technologies. Energies, 8(1), 2015, 172-216; https://doi.org/10.3390/en8010172. 560 56 21. Wonga, Ai Ling; Ramachandaramurthy, K. Vigna; Taylora, Phil; Ekanayake, J.B.; Walker, L. Sara; 561 Padmanaban, Sanjeevikumar. Review on the optimal placement, sizing and control of an energy storage 562 system in the distribution network. Journal of Energy Storage, 2019, vol 21, 489–504. 563 22. Koohi-Fayegh, S.; Rosen, M.A. A review of energy storage types, applications and recent developments. 564 Journal of Energy Storage, 2020, vol 27, 101047. 565 23. Balducci, J. Patrick; Alam, M. J. E.; Hardy, D. Trevor; Wu, Di. Assigning value to energy storage systems at 566 multiple points in an electrical grid. Energy & Environmental Science Review, 2018, DOI: 10.1039/c8ee00569a. 567 24. Li, Xin; Chalvatzis, J. Konstantinos; Stephanides, Phedeas. Innovative Energy Islands: Life-Cycle 568 Cost-Benefit Analysis for Battery Energy Storage. Sustainability, September 2018. 569 24. Li, Xin; Chalvatzis, J. Konstantinos; Stephanides, Phedeas. Innovative Energy Islands: Life-Cycle 568 A E 569 24. Li, Xin; Chalvatzis, J. Konstantinos; Stephanides, Phedeas. Innovative Energy Islan Cost-Benefit Analysis for Battery Energy Storage. Sustainability, September 2018. 25. Barelli, Linda; Bidini, Gianni; Cherubini, Paolo; Micangeli, Andrea; Pelosi, Dario; Tacconelli, Carlo. How 570 Hybridization of Energy Storage Technologies Can Provide Additional Flexibility and Competitiveness to 571 Microgrids in the Context of Developing Countries. energies, August 2019. 572 26. Bradbury, Kyle. Energy Storage Technology Review. Review, Available online: https://www.kylebradbury.org/docs/papers/Energy-Storage-Technology-Review-Kyle-Bradbury-2010.pd f, (accessed on 30 October 2019). 27. Lazard. Lazard's Levelized Cost of Storage Analysis – Version 3.0. Technical Report, Available online: 6 https://www.lazard.com/media/450338/lazard-levelized-cost-of-storage-version-30.pdf, (accessed on 30 7 October 2019). 8 28. ADB. Handbook on Battery Energy Storage System. Asian Development Bank Book, ISBN 579 978-92-9261-470-6 (print), 978-92-9261-471-3 (electronic), Publication Stock No. TCS189791-2, December 580 2018, DOI: http://dx.doi.org/10.22617/TCS189791-2. 581 29. IRENA. Electricity Storage and Renewables: Costs and Markets To 2030. International Renewable Energy 582 Agency, Abu Dhabi Report, ISBN 978-92-9260-038-9, 2017. 583 0. Electric Power Research Institute. Electricity Energy Storage Technology Options: Applications, Costs Benefits. A White Paper Primer 1020676, 2011; pp. 1–170. 31. U.S. Department of Energy. References 507 ICF International Whitepaper, 2015, pp. 1–11. 532 11. Oureilidis, Konstantinos; Kyriaki-Nefeli, Malamaki; Gallos, Konstantinos; Tsitsimelis, Achilleas; 533 Dikaiakos, Christos; Gkavanoudis, Spyros; Cvetkovic, Milos; Mauricio, Manuel Juan; Ortega, Maza Maria 534 Jose; Ramos, Luis Martinez Jose; Papaioannou, Georg; Demoulias, Charis. Ancillary Services Market 535 Design in Distribution Networks: Review and Identification of Barriers. Energies 2020, 13(4), 917; 536 https://doi.org/10.3390/en13040917. 537 12. Maza-Ortega, M. José; Mauricio, M. Juan; Barragán-Villarejo, Manuel; Demoulias, Charis; 538 Gómez-Expósito, Antonio. Ancillary Services in Hybrid AC/DC Low Voltage Distribution Networks. 539 Energies 2019, 12(19), 3591; https://doi.org/10.3390/en12193591. 540 17 of 18 Energies 2020, 13, x FOR PEER REVIEW 13. Wang, Weiliang; Wang, Dan; Liu, Liu; Jia, Hongjie; Zhi, Yunqiang; Meng, Zhengji; Du, Wei. R 541 Modeling and Hierarchical Scheduling of a Generalized Multi-Source Energy Storage Sys 542 Integrated Energy Distribution System. Energies, 2019, 12(2), 246; https://doi.org/10.3390/en1202 543 14. Bartolucci, Lorenzo; Cordiner, Stefano; Mulone, Vincenzo; Santarelli, Marina. Ancillary Service 544 by Hybrid Residential Renewable Energy Systems through Thermal and Electrochemical Storag 545 Energies 2019, 12(12), 2429; https://doi.org/10.3390/en12122429. 546 15. EirGrid and SONI. All-Island Generation Capacity Statement: 2019-2028. Available 547 http://www.soni.ltd.uk/media/documents/EirGrid-Group-All-Island-Generation-Capacity-State 548 9-2028.pdf, (accessed on 1 January 2020); pp. 1–78, 2019. 549 16. Northern Ireland Electricity Networks. NIE Networks RP6 Business Plan 2017-2024. NIE Net 550 Report, 2017. 551 17. Schmidt, Oliver; Melchior, Sylvain; Hawkes, Adam; Staffell, Iain. Projecting the Future Leveliz 552 Electricity Storage Technologies. Joule 2019, 3, 81–100; https://doi.org/10.1016/j.joule.2018.12.008 553 18. Rebello, E.; Watson, D.; Rodgers, M. Ancillary services from wind turbines: automatic generati 554 (AGC) from a single Type 4 turbine. Wind Energy Science 2020, 5, 555 https://doi.org/10.5194/wes-5-225-2020. 556 19. Aneke, M.; Wang, M. Energy storage technologies and real-life applications – A state of the a 557 Appl. Energy, 2016, vol. 179, pp. 350–377. 558 20. Sabihuddin, Siraj; Kiprakis, E. Aristides; Mueller, Markus. A Numerical and Graphical Review 559 Storage Technologies. Energies, 8(1), 2015, 172-216; https://doi.org/10.3390/en8010172. 560 21. Wonga, Ai Ling; Ramachandaramurthy, K. Vigna; Taylora, Phil; Ekanayake, J.B.; Walker 561 Padmanaban, Sanjeevikumar. Review on the optimal placement, sizing and control of an ener 562 system in the distribution network. Journal of Energy Storage, 2019, vol 21, 489–504. 563 22. Koohi-Fayegh, S.; Rosen, M.A. A review of energy storage types, applications and recent deve 564 Journal of Energy Storage, 2020, vol 27, 101047. 565 23. Balducci, J. Patrick; Alam, M. J. E.; Hardy, D. Trevor; Wu, Di. Assigning value to energy storage 566 multiple points in an electrical grid. References 507 Grid Energy Storage. Report on grid energy storage, December 2013. 31. U.S. Department of Energy. Grid Energy Storage. Report on grid energy storage 586 32. Goldie-Scot, Logan. A Behind the Scenes Take on Lithium-ion Battery Prices. BloombergNEF Article, 587 Available online: https://about.bnef.com/blog/behind-scenes-take-lithium-ion-battery-prices/, (accessed 588 on 1 January 2020). 589 33. Invest Northern Ireland. Wind Power: A best practice guide for Northern Ireland business. Sustainable 590 Development – Team Text Relay, Number: 1800102890698273, Available on: 591 https://secure.investni.com/static/library/invest-ni/documents/wind-power-a-best-practice-guide-for-busi 592 nesses-in-northern-ireland.pdf, (accessed on 1 January 2020); pp 26–86. 593 Energies 2020, 13, x FOR PEER REVIEW 18 of 18 34. SEM Committee. Quick Guide to I-SEM. Available 594 https://www.semcommittee.com/sites/semc/files/media-files/ISEM%20quick%20g 595 (accessed on 1 January 2020); pp 1–11. 596 35. EirGrid. Quick Guide to the Integrated Single Electricity Market; the I-SEM Project ver 597 Available 598 http://www.eirgridgroup.com/__uuid/f110639e-9e21-4d28-b193-ed56ee372362/EirGrid-Gro 599 ck-Guide.pdf, (accessed on 1 January 2020). 600 36. EirGrid and SONI. FlexTech Consultation 2019, A Flexible Technology Integration Initiat 601 online: http://www.soni.ltd.uk/media/documents/FlexTech-Consultation_30092019.pdf, ( 602 January 2020); pp. 1–21. 603 37. EirGrid and SONI. DS3 System Services: Portfolio Capability Analysis. Ava 604 http://www.eirgrid.ie/site-files/library/EirGrid/DS3-System-Services-Portfolio-Cap 605 ysis.pdf, (accessed on 1 January 2020); pp. 1–15. 606 38. EirGrid and SONI. DS3 System Services Scalar Design. Availa 607 http://www.eirgridgroup.com/site-files/library/EirGrid/OPI_INN_DS3-System-Se 608 DesignFinal_231017.pdf, (accessed on 1 January 2020); pp. 1–64. 609 39. EirGrid and SONI. DS3 System Services Interim Tariffs DECISION PAPER. Ava 610 http://www.eirgridgroup.com/site-files/library/EirGrid/DS3-System-Services-Deci 611 n-Interim-Tariffs-FINAL.pdf, (accessed on 1 January 2020); pp. 36. 612 40. SONI. DS3 System Services Statement of Payments. Statement of Payment. Avai 613 http://www.soni.ltd.uk/media/documents/DS3-SS-Statement-of-Payments-2019-20.pdf, (a 614 January 2020). 615 41. EirGrid and SONI DS3 System Services Market Ruleset Recommendation Paper. Ava 616 http://www.eirgridgroup.com/site-files/library/EirGrid/DS3-System-Services-Market-Rules 617 dations-Paper-16052018.pdf, (accessed on 1 January 2020); pp 1–28. 618 42. EirGrid and SONI. DS3 System Services Tariffs for Regulated Arrangements. Ava 619 http://www.eirgridgroup.com/site-files/library/EirGrid/OPI_INV_DS3-System-Services-Tar 620 ted-Arrangements-FINAL-23.10.2017.pdf, (accessed on 1 January 2020); pp. 15–18. 621 43. EirGrid and SONI. Consultation on Connecting Further Generation in Northern 622 pp. 39–44. 623 44. Power NI. Unit Rate Prices. Plans & Discounts Article, Availa 624 https://powerni.co.uk/plan-prices/compare-our-plans/tariff-rates/, (accessed on 1 Jan 625 45. SSE Airtricity. Our Tariffs. Plans & Products Article, Availa 626 https://www.sseairtricity.com/uk/home/help-centre/our-tariffs, (accessed on 1 Janua 627 46. SSE Airtricity. 1 Year Keypad Electricity Tariffs. Tariff Quote Document, Ava 628 https://www.sseairtricity.com/assets/Tariffs/ElecNI/Oct-19/1YR-KEYPAD-9.pdf, ( 629 January 2020). 630 47. Kendon, Mike; McCarthy, Mark; Jevrejeva, Svetlana. State of the UK Climate 201 631 Hadley Centre & National Oceanography Centre Report, July 2014. 632 48. Met Office Hadley Centre. © 2020 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). References 507 UK Climate Projections: Headline Findings. Met 633 version 2, September 2019. 634 635 © 2020 by the authors. Submitted for possible open access publication un and conditions of the Creative Commons Attribution (CC (http://creativecommons org/licenses/by/4 0/) 34. SEM Committee. Quick Guide to I-SEM. Available online: 594 https://www.semcommittee.com/sites/semc/files/media-files/ISEM%20quick%20guide_1.pdf, 595 (accessed on 1 January 2020); pp 1–11. 596 35. EirGrid. Quick Guide to the Integrated Single Electricity Market; the I-SEM Project version, pp. 1–11, 597 Available online: 598 http://www.eirgridgroup.com/__uuid/f110639e-9e21-4d28-b193-ed56ee372362/EirGrid-Group-I-SEM-Qui 599 ck-Guide.pdf, (accessed on 1 January 2020). 600 http://www.eirgridgroup.com/__uuid/f110639e-9e21-4d28-b193-ed56ee372362/EirGrid-Group-I-SEM-Q ck-Guide.pdf, (accessed on 1 January 2020). 36. EirGrid and SONI. FlexTech Consultation 2019, A Flexible Technology Integration Initiative. Available 601 online: http://www.soni.ltd.uk/media/documents/FlexTech-Consultation_30092019.pdf, (accessed on 1 602 January 2020); pp. 1–21. 603 37. EirGrid and SONI. DS3 System Services: Portfolio Capability Analysis. Available online: 604 http://www.eirgrid.ie/site-files/library/EirGrid/DS3-System-Services-Portfolio-Capability-Anal 605 ysis.pdf, (accessed on 1 January 2020); pp. 1–15. 606 38. EirGrid and SONI. DS3 System Services Scalar Design. Available online: 607 http://www.eirgridgroup.com/site-files/library/EirGrid/OPI_INN_DS3-System-Services-Scalar- 608 DesignFinal_231017.pdf, (accessed on 1 January 2020); pp. 1–64. 609 39. EirGrid and SONI. DS3 System Services Interim Tariffs DECISION PAPER. Available online: 0 http://www.eirgridgroup.com/site-files/library/EirGrid/DS3-System-Services-Decision-Paper-o 1 n-Interim-Tariffs-FINAL.pdf, (accessed on 1 January 2020); pp. 36. 2 40. SONI. DS3 System Services Statement of Payments. Statement of Payment. Available online: http://www.soni.ltd.uk/media/documents/DS3-SS-Statement-of-Payments-2019-20.pdf, (accessed on 1 January 2020). 41. EirGrid and SONI DS3 System Services Market Ruleset Recommendation Paper. Available online: http://www.eirgridgroup.com/site-files/library/EirGrid/DS3-System-Services-Market-Ruleset-Recommen dations-Paper-16052018.pdf, (accessed on 1 January 2020); pp 1–28. 42. EirGrid and SONI. DS3 System Services Tariffs for Regulated Arrangements. Available online: http://www.eirgridgroup.com/site-files/library/EirGrid/OPI_INV_DS3-System-Services-Tariffs-for-Regula ted-Arrangements-FINAL-23.10.2017.pdf, (accessed on 1 January 2020); pp. 15–18. 43. EirGrid and SONI. Consultation on Connecting Further Generation in Northern Ireland. 2018; pp. 39–44. 44. Power NI. Unit Rate Prices. Plans & Discounts Article, Available online: https://powerni.co.uk/plan-prices/compare-our-plans/tariff-rates/, (accessed on 1 January 2020). 45. SSE Airtricity. Our Tariffs. Plans & Products Article, Available online: https://www.sseairtricity.com/uk/home/help-centre/our-tariffs, (accessed on 1 January 2020). 46. SSE Airtricity. 1 Year Keypad Electricity Tariffs. Tariff Quote Document, Available online: 628 https://www.sseairtricity.com/assets/Tariffs/ElecNI/Oct-19/1YR-KEYPAD-9.pdf, (accessed on 1 629 January 2020). 630 47. Kendon, Mike; McCarthy, Mark; Jevrejeva, Svetlana. State of the UK Climate 2014. Met Office 631 Hadley Centre & National Oceanography Centre Report, July 2014. 632 48. Met Office Hadley Centre. UK Climate Projections: Headline Findings. Met Office Report 633 version 2, September 2019. 634 635 © 2020 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). 636
W4385615786.txt	https://jurnal.permataindonesia.ac.id/index.php/JPI/article/download/73/57	id		Pengembangan Makanan Tape dengan Bahan Dasar dari Biji Buah Nangka (Beton) (Artocarpus heterophyllus Lam.) yang Dikombinasikan dengan Daun Kelor (Moringa Oleifera Lam.) sebagai Obat Antidiabetes	Jurnal Permata Indonesia	2,022	cc-by	1,988	JURNAL PERMATA INDONESIA Volume 10, Nomor 2, November 2019 ISSN 2086-9185 Halaman 1-5 Pengembangan Makanan Tape dengan Bahan Dasar dari Biji Buah Nangka (Beton) (Artocarpus heterophyllus Lam.) yang Dikombinasikan dengan Daun Kelor (Moringa Oleifera Lam.) sebagai Obat Antidiabetes Development of Food “Tape” with Basic Material from Jackfruit Seeds (Concrete) (Artocarpus heterophyllus Lam.) Combined with Moringa Oleifera Lam. Leaves as Antidiabetic Medicine Joko Santosoa, Dwi Ratnaningsihb a,b Poltekkes Permata Indonesia Yogyakarta ABSTRAK Tape Merupakan makanan tradisional yang digemari oleh berbagai macam kalangan masyarakat dan sudah menjadi kearifan lokal di Indonesia misalkan peuyem yang berasal dari bandung dan sudah menjadi icon kota tersebut. Sebagian besar tape dibuat dengan bahan baku dari singkong dan ketan. Tetapi makanan tape ini dianggap sebelah mata karena tape belum diketahui manfaat dari tape tersebut dan belum terbukti secara empiris. kemajuan di bidang teknologi pangan mendorong masyarakat untuk memanfaatkan biji nangka secara optimal. Biji nangka selama ini menjadi limbah di beberapa kalangan masyarakat sehingga biji nangka ini dapat diolah menjadi tape. Biji nangka mempunyai kandungan kimia yaitu karbohidrat dan protein. Daun kelor mempunyai kandungan kimia yaitu flavonoid, vitamin dan mineral yang sangat bermanfaat untuk obat diabetes. Sehingga peneliti tertarik untuk mengembangkan makanan tape dengan berbahan baku biji buah nangka (beton) dikombinasikan dengan daun kelor sehingga bermanfaat untuk kesehatan. Tujuan : Menciptakan dan mengembangkan produk kearifan lokal Indonesia terhadap makanan tape dengan berbahan baku biji nangka yang dikombinasikan dengan daun kelor sebagai obat antidiabetes dibuktikan secara empiris. Metode penelitian eksperimental pre-post test control group design loboratories. Penelitian ini mempunyai TKT (Tingkat Kesiapan Teknologi) tingkat 3 karena masih dalam tahapan pengembangan secara laboratorium dan siap menuju ke TKT lebih tinggi dan siap untuk menuju ke prototipe. Hasil : Tape biji nangka memiliki tekstur semi padat, aromatik dan rasa manis.Kesimpulan : Biji nangka bisa diolah menjadi makanan tape dan memiliki tekstur yang semi padat, aromatis dan rasanya manis serta memiliki kandungan protein yang tinggi yaitu 5,57% dan dapat bermanfaat sebagai obat Diabetes Melitus tipe 2. Kata Kunci : Tape ; Biji Buah Nangka (Beton) ; Daun Kelor ; Makanan Antidiabetes ABSTRACT Tape Is a traditional food that is favored by various types of people and has become a local wisdom in Indonesia, for example peuyem from Bandung and has become an icon of the city. Most of the tapes are made with raw materials from cassava and sticky rice. But this food tape is considered one eye because the tape has not known the benefits of the tape and has not been proven empirically. advances in the field of food technology are encouraging people to make optimum use of jackfruit seeds. Jackfruit seeds have been a waste in some communities so that these jackfruit seeds can be processed into tape. Jackfruit seeds have chemical contents, namely carbohydrates and protein. Moringa leaves have a chemical content that is flavonoids, vitamins and minerals that are very useful for diabetes medications. So that researchers are interested in developing food tape made from jackfruit seeds (concrete) combined with Moringa leaves so it is beneficial for health. Purpose: Creating and developing Indonesian local wisdom products for food tape made from jackfruit seeds which are combined with Moringa leaves as an antidiabetic drug is empirically proven. The experimental research method pre-post test control group design loboratories. This research has a TKT (Technology Readiness Level) level 3 because it is still in the laboratory development stage and is ready to go to a higher TKT and ready to go to the prototype. Results: Jackfruit seed tape has a semi-solid texture, aromatic and sweet taste. Conclusion: Jackfruit seeds can be processed into food tape and has a semi-solid texture, aromatic and sweet taste and has a high protein content that is 5.57% and can be useful as Diabetes Mellitus type 2 drug. Keywords: Tape; Jackfruit Seeds (Concrete); Moringa Leaves; Antidiabetic Food 1 Joko Santoso, Dwi Ratnaningsih \| Pengembangan Makanan Tape ……. PENDAHULUAN Indonesia adalah negara tropis yang banyak ditumbuhi oleh beranekaragam jenis tanaman, salah satunya adalah tanaman nangka. Tanaman nangka disebut juga dengan Artocarpus heterophyllus Lamk. Pemanfaatan tanaman nangka telah banyak dalam industri pangan. Namun belum semua bagian dari tanaman nangka ini yang dapat dikelolah secara optimal sebagai komoditi yang bernilai tinggi. Salah satunya adalah biji nangka, yang sering terbuang dan hanya menjadi limbah. Hanya pada sebagian masyarakat kecil ada yang mengonsumsi biji nangka ini dengan cara direbus, digoreng, disangrai, atau dikukus. Belum banyak masyarakat yang mengetahui pemanfaatan biji nangka serta kandungan gizi yang terkandung didalamnya. Permasalahan yang akan diteliti adalah banyaknya limbah biji nangka yang kurang banyak dimanfaatkan sehingga dapat dikembangkan menjadi makanan olahan yaitu tape biji daun nangka. Tape merupakan produk olahan dengan kearifan lokal masyarakat di Indonesia dan banyak diminati dari oleh semua kalangan masyarakat dikarenakan kenikmatan dan kelezatannya. Tape adalah produk yang dihasilkan dari proses fermentasi, di mana terjadi suatu perombakan bahan-bahan yang tidak sederhana. Zat pati yang ada dalam bahan makanan diubah menjadi bentuk yang sederhana yaitu gula, dengan bantuan suatu mikroorganisme yang disebut ragi atau khamir. Ragi tape adalah bahan yang dapat digunakan dalam pembuatan tape, baik dari singkong dan beras ketan. Selama ini biji nangka dimanfaatkan dengan direbus atau dibakar dan beberapa dikalangan masyarakat biji nangka ini menjadi limbah kemajuan di bidang teknologi pangan mendorong masyarakat untuk memanfaatkan biji nangka secara optimal. Adanya potensi gizi yang terkandung dalam tepung biji nangka, diharapkan pemanfaatannya dalam pembuatan olahan makanan dapat membantu meningkatkan konsumsi gizi yang lebih variatif bagi masyarakat luas. Biji nangka merupakan sumber kabohidrat (36,7g), protein (4,2 g) dan energi (165 kkal) sehingga dapat dimanfaatkan sebagai bahan pangan yang potensial (Astawan, 2008). Ekstraksi daun Moringa oleifera dengan metode maserasi dalam larutan etanol 70%, mengungkapkan bahwa terdapat flavonoid, tannin, anthraquinone, cardiac glycosides alkaloids, triterpenoid, saponin, dan reducing sugars menunjukkan bahwa flavonoid mempunyai efek hipoglikemik, meskipun efek hipoglikemik terpenoid tampak terlibat dalam menstimulasi sel β pankreas dan selanjutnya meningkatkan sekresi insulin (Tende, 2011). METODE PENELITIAN Metode penelitian eksperimental pre-post test control group design loboratories HASIL DAN PEMBAHASAN 1. Hasil data Tape Beton (biji nangka) Tabel 1. Hasil Data Pembuatan Tape Dari hasil data diatas dalam membuat tape biji nangka dibutuhkan biji nangka sebanyak 2 kg, berat ragi 6,42g dan lama pembuatan 48 jam. 2. Hasil data Tape biji nangka Tabel 2. Hasil Data Uji Tape Biji Nangka 2 Joko Santoso, Dwi Ratnaningsih \| Pengembangan Makanan Tape ……. Dari hasil data diatas adalah uji organoleptis tape biji nangka yang memiliki aroma khas aromatic, bentuk semi padat, rasa manis dan memiliki pH 6. 3. Hasil data pengujian kandungan tape biji nangka 4. Hasil uji kadar gula darah Tabel 4. Hasil Uji Kadar Gula Darah pada Mencit Tabel 3. Hasil Data Kandungan Tape Dari hasil pengujian kadar gula darah dihasilkan kelompok I 74,75, Kelompok II 84,65, Kelompok III 85,2, Kelompok IV 84,45 dan kelompok V 105,15. Data kandungan yang ada di tape biji nangka sebelum di tambahkan daun kelor adalah kadar gula 2,77% dan setelah ditambahkan daun kelor sebesar 5,86%. Gambar 3. Kadar Gula Darah Mencit PEMBAHASAN Biji nangka merupakan limbah yang jarang dimanfaatkan sebagai makanan olahan yang bermanfaat terutama dalam bidang kesehatan. Sehingga limbah biji nangka ini bisa dijadikan makanan olahan yang memiliki manfaat dan khasiat menurunkan kadar gula dalam darah atau sebagai obat diabetes melitus tipe 2 yaitu dibuat menjadi tape biji nangka. Tape biji nangka ini diharapkan bisa memperkaya kearifan lokal akan perkembangan pengembangan makanan terutama dalam pembuatan tape. Dalam pembuatan tape biji nangka dibutuhkan 2 kg biji nangka dan ragi sebanyak 6,42g dalam pembuatan tape. Lama pembuatan tape atau proses fermentasinya selama 48 jam karena dalam waktu ini dihasilkan tape yang memiliki uji organoleptis tape biji nangka yang memiliki aroma khas aromatik, bentuk semi padat, Gambar 1. Grafik Kadar Gula Tape Dari data diatas adalah kandungan yang ada di tape biji nangka sebelum di tambahkan daun kelor adalah kadar protein 4,59% dan setelah ditambahkan daun kelor sebesar 5,57%. Gambar 2. Grafik Kadar Protein Tape 3 Joko Santoso, Dwi Ratnaningsih \| Pengembangan Makanan Tape ……. rasa manis dan memiliki pH 6. Sehingga dihasilkan tape yang tepat dalam melakukan dalam proses fermentasi, hal ini juga tergantung dari besar kecilnya bahan yang digunakan karena semakin tebal atau besar bahan yang digunakan maka proses fermentasinya juga akan semakin lama waktu yang dibutuhkan. Kandungan kimia dalam tape biji nangka sebelum di tambahkan daun kelor adalah protein 4,59% dan kadar gula 2,77% dan hasil tape setelah dilakukan penambahan daun kelor adalah protein 5,57% dan kadar gula 5,86%. menurut Astawan (2008), pada setiap 100 gr biji nangka mengandung protein sebesar 4,2 g. Hal ini menandakan bahwa dalam pembuatan tape dengan menambahakan daun kelor ini maka dapat menambahkan kadar dan kandungan protein dan kadar gula dalam proses fermentasi karena kandungan dalam daun kelor ini banyak mengandung vitamin, mineral, protein dan karbohidrat. Setelah dihasilkan tape yang sesuai atau dengan istilah lain sesuai proses fermentasi, bentuk sediaan tapenya dan kandungkan kimia dalam tapenya maka perlu dilakukan uji khasiat dari tape sehingga uji ini diharapkan dapat menambah kemanfaatan dari tape, selain enak tapi meliliki khasiat juga untuk menurunkan kadar gula dalam darah. Dari hasil pengujian kadar gula darah dihasilkan kelompok I 74,75, Kelompok II 84,65, Kelompok III 85,2, Kelompok IV 84,45 dan kelompok V 105,15. Dari data ini dapat disimpulkan bahwa untuk semua kelompok baik itu kelompok I,II,III dan IV dapat menurunkan kadar gula dalam tubuh dibandingkan dengan kelompok kontrol yaitu kelompok V. Tetapi yang paling efektif dalam menurunkan kadar gula dalam darah adalah kelompok I karena kelompok I ini paling effektif disbanding dengan kelompok Kontrol positif (kelompok IV) yang diberikan obat kimia dan kelompok V (Aquadest). Hasil penelitian ini dihasilkan tape dari biji nangka yang memiliki rasa yang enak yaitu manis, bentuk yang semi padat dan tidak menimbulkan iritasi lambung karena pH nya 6 sehingga tidak terlalu asam di lambung. Hasil penelitian dalam pengujian tape biji nangka ini sudah teruji secara empiris yaitu dengan pengujian khasiat dari tape biji nangka ini sebagai obat diabetes mellitus tipe 2. Hal ini dikarenakan adanya penambahan daun kelor dalam tape biji nangka tersebut karena daun kelor memiliki kandungan flavonoid dimana flafonoid ini sebagai antioksidan dan mampu dapat menurunkan kadar gula dalam darah dengan menstimulasi dan memperbaiki sel beta pankreas. Sehingga produksi insulin dalam tubuh dapat terpenuhi kembali. KESIMPULAN 1. Biji nangka bisa diolah menjadi makanan tape dan memiliki tekstur yang semi padat, aromatis dan rasanya manis serta memiliki kandungan protein yang tinggi yaitu 5,57%. 2. Tape biji nangka dapat digunakan sebagai obat diabetes mellitus tipe 2 yaitu pada kelompok I (dosis 1g ekstrak tape biji nangka). UCAPAN TERIMAKASIH Kami ucapkan terimakasih kepada Kemenristek DRPM dana Hibah Penelitian Penelitian Dosen Pemula (PDP) tahun anggaran 2019, Kedokteran hewan UGM dan Politeknik Kesehatan Permata Indonesia Yogyakarta. DAFTAR PUSTAKA Astawan, Made. 2008. Sehat dengan Buah.Jakarta:Dian Rakyat Dian Rakyat Tahun 2008 tentang Sehat dengan buah. Jurnal Pharm Toxicol hal 2(1):1–4 Tahun 2011tentang Effect of ethanolic leaves extract of moringaoleifera on blood glucose levels of 4 Joko Santoso, Dwi Ratnaningsih \| Pengembangan Makanan Tape ……. streptozotocin-induced diabetics and normoglycemic wistar rats. Panduan Pelaksanaan Penelitian dan Pengabdian Kepada Masyarakat Di Perguruan Tinggi Edisi XI Tahun 2017 Direktorat Riset dan Pengabdian kepada Masyarakat, Direktorat Jenderal Penguatan Riset dan Pengembangan Kementerian Riset, Teknologi, dan Pendidikan Tinggi. Panduan Pelaksanaan Penelitian dan Pengabdian Kepada Masyarakat Di Perguruan Tinggi Edisi X Tahun 2015 Direktorat Riset dan Pengabdian kepada Masyarakat, Direktorat Jenderal Penguatan Riset dan Pengembangan Kementerian Riset, Teknologi, dan Pendidikan Tinggi. Peraturan Dirjen Penguatan Riset dan Pengembangan Nomor 603/E1.2/2016 tentang Pedoman Indikator Capaian Tingkat Kesiaterapan Teknologi. Tende, JA, I. Ezekiel., AAU Dikko and ADT Goji. 2011. Effect of Ethanolic Leaves Extract of Moringa oleifera on Blood Glucose Levels of StreptozocinInduced Diabetics and Normoglycemic Wistar Rats. British Journal of Pharmacology and Toxicology. 2(1). Universitas Islam Negri Malang Skripsi Tahun 2012 tentang Pengaruh Lama fermentasi Terhadap Alkohol Tape Singkong (Manihot Utilissima Pohl). Universitas Diponegoro Skripsi Tahun 2015 tentang Pengaruh Ekstrak daun Kelor (Moringa oleifera) pada ekspresi Insulitis tikusDiabetes Melitus Universitas Muhammadiyah Surakarta Skripsi Tahun 2014 tentang Uji Protein dan Organoleptik Tape dari Bahan Dasar Biji Nangka dengan Penambahan Ekstrak daun Katuk Sebagai Pewarna alami dab lama Fermentasi yang Berbeda. 5
https://openalex.org/W3115599035	https://www.nature.com/articles/s41598-020-80023-8.pdf	English	null	Long-term effects of cerebellar anodal transcranial direct current stimulation (tDCS) on the acquisition and extinction of conditioned eyeblink responses	Scientific reports	2,020	cc-by	10,355	Long‑term effects of cerebellar anodal transcranial direct current stimulation (tDCS) on the acquisition and extinction of conditioned eyeblink responses Otilia Kimpel1,2, Thomas Hulst1,3, Giorgi Batsikadze1, Thomas M. Ernst1, Michael A. Nitsche4,5, Dagmar Timmann1 & Marcus Gerwig1* Cerebellar transcranial direct current stimulation (tDCS) has been reported to enhance the acquisition of conditioned eyeblink responses (CR), a form of associative motor learning. The aim of the present study was to determine possible long-term effects of cerebellar tDCS on the acquisition and extinction of CRs. Delay eyeblink conditioning was performed in 40 young and healthy human participants. On day 1, 100 paired CS (conditioned stimulus)–US (unconditioned stimulus) trials were applied. During the first 50 paired CS–US trials, 20 participants received anodal cerebellar tDCS, and 20 participants received sham stimulation. On days 2, 8 and 29, 50 paired CS–US trials were applied, followed by 30 CS-only extinction trials on day 29. CR acquisition was not significantly different between anodal and sham groups. During extinction, CR incidences were significantly reduced in the anodal group compared to sham, indicating reduced retention. In the anodal group, learning related increase of CR magnitude tended to be reduced, and timing of CRs tended to be delayed. The present data do not confirm previous findings of enhanced acquisition of CRs induced by anodal cerebellar tDCS. Rather, the present findings suggest a detrimental effect of anodal cerebellar tDCS on CR retention and possibly CR performance. Transcranial direct current stimulation (tDCS) can alter cortical excitability and enhance neuronal plasticity1,2, an important physiological foundation of learning and memory formation. tDCS of primary motor cortex (M1) has been shown to improve motor learning3,4, and has become a promising option to enhance the beneficial effects of motor training in various neurological disorders5–8. f g g tDCS modulates learning-related plasticity not only in M1, but likely also in the cerebellar cortex9,10. Cer- ebellar tDCS was found to improve adaptive learning in arm reaching tasks in young and elderly healthy participants11–13. A more recent study in mice showed that the deletion of long-term potentiation (LTP) of Purkinje cells eliminates cerebellar tDCS effects on vestibulo-ocular reflex (VOR) habituation14, and thus delivers mechanistic information about the plasticity effects of cerebellar tDCS. Initial findings on the effects of cerebellar tDCS in cerebellar patients, however, are partly contradictory15–18. Cerebellar tDCS effects, moreover, show a significant degree of variability in healthy participants in reach adaptation tasks19. gi g y y p p p Delay eyeblink conditioning is a motor learning task that critically depends on the integrity of the cerebellum20–23. www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| (2020) 10:22434 Results U di Unconditioned responses. Group mean values ± standard deviation (SD) of unconditioned response (UR) onset in unpaired trials in the initial pseudo-conditioning phase were 45.2 ± 9.9 ms in the anodal group and 51.3 ± 15.1 ms in the sham group (p = 0.14; unpaired t test). Group mean values ± SD of UR peak time were 103.8 ± 13.1 ms in the anodal group, and 107.8 ± 7.2 ms in the sham group (p = 0.61). Group mean UR duration was 119.3 ± 34.8 ms in the anodal group and 117.8 ± 49.9 ms in the sham group (p = 0.91). None of the trials had to be excluded, and analysis was based on 10 URs in each of the participants. CR incidence. The main findings are illustrated in Fig. 1 showing eyeblink recordings in two individual par- ticipants following sham and anodal stimulation across the four days. On day 1, the sham-stimulated participant and the verum-stimulated participant acquired conditioned responses (CRs) to a similar extent. In paired trials on subsequent days, CRs occurred later and were of smaller size in the verum-stimulated participant compared to the sham-stimulated participant. Furthermore, in the extinction phase one month after stimulation (day 29) the number of CRs was reduced in the verum-stimulated participant compared to the sham-stimulated partici- pant. p Group mean percentage of CR incidences ± standard errors (SE) across the four days in the two stimulation groups are shown in Fig. 2. On day 1, CR incidences increased in both stimulation groups across blocks. In the last five acquisition blocks, CR incidences were numerically higher in the anodal cerebellar stimulation group compared to the sham group. Mean total CR incidence on day 1 was 58.5% ± 5.0% in the anodal group compared to 52.2% ± 5.0% in the sham group. Mixed model ANOVA revealed a significant block effect [F(9,342) = 36.7; p < 0.0001]. The stimulation by block effect [F(9,342) = 0.55; p = 0.84] and the stimulation effect were not signifi- cant [F(1,38) = 0.67; p = 0.41]. CR incidences showed a further increase on day 2 compared to day 1, with no further increases during day 8 and day 29 and no differences between stimulation groups. Across days 2, 8 and 29 the mean total CR incidences was 79.5% ± 4.9% in the anodal group, and 78.5% ± 4.9% in the sham group. Results U di Mixed model ANOVA revealed no significant effects of day [F(2,76) = 1.0; p = 0.37] and stimulation [F(1,38) = 0.025; p = 0.87], and no significant stimulation by day interaction effect [F(2,76) = 1.06; p = 0.35]. That is both stimulation groups showed no further increase of CR incidences across the three days irrespective of the stimulation modality. y p y Regarding extinction, Fig. 2 shows a decrease of CR incidences across extinction blocks 1–3 on day 29 in both stimulation groups. Mean CR incidence in the extinction phase was lower in the anodal group (22.3% ± 5.3%) than in the sham group (39.8% ± 5.3%). Mixed model ANOVA revealed a significant block effect [F(2,76) = 17.6; p < 0.0001] and a significant stimulation effect [F(1,38) = 5.4; p = 0.026]. The stimulation by block interaction effect was not significant [F(2,76) = 0.083; p = 0.92]. Post-hoc pairwise comparisons, revealed a significant mean difference between the anodal and sham stimulation groups in the first extinction block (p = 0.032; mean dif- ference of − 19.0% ± 8.6%) and the second extinction block (p = 0.047; mean difference of − 17.5% ± 8.6%). The mean difference of − 16.0% ± 8.6% in the third extinction block was not significant (p = 0.069). CR area. On day 1, mean percentage CR area was not different between groups (anodal group: 95.5% ± 1.7%; sham group: 92.9% ± 14.7%) (Fig. 3). CR area increased across blocks in both groups. Mixed model ANOVA showed a significant block effect [F(9,303.25) = 6.6; p < 0.0001]. The stimulation by block interaction effect was not significant [F(9,303.25) = 0.86; p = 0.56]. The stimulation effect (that is anodal vs. sham stimulation) was not significant [F(1,31.7) = 1.1; p = 0.29]. gi [ ( ) p ] Mean percentage CR area on days 2, 8 and 29 was smaller in the anodal group (145.2% ± 14.8%) compared to the sham group (187.3% ± 14.8%). CR area increased across days in both groups. The stimulation effect was close to significance [F(1,38) = 4.0; p = 0.052]. There was no significant effect of day [F(2,76) = 1.4; p = 0.25] and no significant stimulation by day interaction [F(2,76) = 0.31; p = 0.84]. gi y y p In extinction trials on day 29, CR area was smaller in the anodal compared with the sham group (91.9% ± 21.5% vs. www.nature.com/scientificreports/ cathodal stimulation impeded acquisition. The application of anodal tDCS during acquisition, however, led to significantly earlier CR onset, that is CRs were less appropriately timed. In a follow up study, we were unable to reproduce our initial findings25. Study designs, however, were not identical. In our first study, tDCS started at the beginning of the acquisition phase, whereas tDCS was started during an initial pseudo-conditioning phase and prior to the beginning of the acquisition phase in the second study. Furthermore, reinforcement rate was 100% in the first study, and 70% in the second study. Both aspects of the second study could have compromised tDCS effects. tDCS during pseudo-conditioning might have resulted in unforeseen learning during this stage, and interference in the acquisition stage, and the reduced reinforcement rate might have strengthened such an interference effect. The aim of the present study was to confirm our initial findings using the same experimental eyeblink conditioning set-up and paradigm, including the same reinforcement rate and onset of cerebellar tDCS. In the initial study a very long stimulation time of 42.9 min was used24. Because significant tDCS effects on CR acquisition were present within less than 10 min, decision was made to apply a more conventional stimulation time of 24.2 min. In addition to immediate effects, long-term effects of cerebellar tDCS on the acquisition and extinction of conditioned eyeblinks were also evaluated across multiple days. Long‑term effects of cerebellar anodal transcranial direct current stimulation (tDCS) on the acquisition and extinction of conditioned eyeblink responses In this task, an initially neutral conditioned stimulus (CS), commonly a tone, is repeatedly pre- sented with an unconditioned stimulus (US), for example an air-puff directed to the eye. After repeated CS–US pairings participants learn that the CS predicts the occurrence of the US, and close their eye after onset of the CS and prior to occurrence of the US. We found that cerebellar tDCS of the cerebellum modulates delay eyeblink conditioning in healthy participants24. Anodal tDCS resulted in faster and enhanced acquisition of CRs, whereas 1Department of Neurology, Essen University Hospital, University of Duisburg-Essen, Hufelandstrasse 55, 45147 Essen, Germany. 2Department of Endocrinology, University Hospital, University of Würzburg, Würzburg, Germany. 3Erasmus University College, Rotterdam, The Netherlands. 4Department of Psychology and Neurosciences, Leibniz Research Centre for Working Environment and Human Factors, Dortmund, Germany. 5Department of Neurology, University Medical Hospital Bergmannsheil, Bochum, Germany. email: marcus.gerwig@uni‑due.de \| https://doi.org/10.1038/s41598-020-80023-8 www.nature.com/scientificreports/ Results U di 156.6% ± 20.7%). CR area declined across extinction blocks in both groups. Mixed model ANOVA revealed a significant block effect [F(2,50.047) = 3.9; p = 0.027] and a significant stimulation effect [F(1,35.43) = 4.7; p = 0.037]. The stimulation by block effect was not significant [F(2,50.047) = 0.23; p = 0.79]. Post-hoc pairwise comparisons comparing the anodal and sham stimulation groups in each of the extinction blocks revealed a significant mean difference in the third block (p = 0.033; mean difference of − 74.1% ± 33.9%). The mean Scientific Reports \| (2020) 10:22434 \| https://doi.org/10.1038/s41598-020-80023-8 www.nature.com/scientificreports/ Figure 1. Individual examples of representative eyeblink recordings following sham and anod across the four recording days. Rectified and filtered EMG-data of the orbicularis oculi muscle EMG recordings of 10 consecutive trials are superimposed. Day 1 is shown on the top, day 29 stimulation) on the bottom. Duration of tDCS on day 1 is shaded in gray. Paired CS–US trials in black, extinction trials are shown in green. The solid vertical lines indicate the onset of the C respectively. Responses occurring within the 150 ms interval after CS onset (dotted line) were responses. See “Methods” for further details. Figure 1. Individual examples of representative eyeblink recordings following sham and anodal tDCS across the four recording days. Rectified and filtered EMG-data of the orbicularis oculi muscle are shown. EMG recordings of 10 consecutive trials are superimposed. Day 1 is shown on the top, day 29 (1 month after stimulation) on the bottom. Duration of tDCS on day 1 is shaded in gray. Paired CS–US trials are shown in black, extinction trials are shown in green. The solid vertical lines indicate the onset of the CS and US, respectively. Responses occurring within the 150 ms interval after CS onset (dotted line) were considered alpha- responses. See “Methods” for further details. https://doi.org/10.1038/s41598-020-80023-8 Scientific Reports \| (2020) 10:22434 \| www.nature.com/scientificreports/ Figure 2. Group mean percentage CR incidence and standard errors (SE) on days 1, 2, 8 (1 week) and 29 (1 month). Ten blocks a 10 paired CS–US trials were presented on day 1, five blocks a 10 paired CS–US trials on days 2, 8 and 29. In addition, 3 blocks a 10 CS-only extinction trials were presented on day 29. Blue filled circles = anodal group, filled black circles = sham group. indicates significant effects in post-hoc pairwise comparisons. Figure 2. Results U di Group mean percentage CR incidence and standard errors (SE) on days 1, 2, 8 (1 week) and 29 (1 month). Ten blocks a 10 paired CS–US trials were presented on day 1, five blocks a 10 paired CS–US trials on days 2, 8 and 29. In addition, 3 blocks a 10 CS-only extinction trials were presented on day 29. Blue filled circles = anodal group, filled black circles = sham group. * indicates significant effects in post-hoc pairwise comparisons. Figure 3. Group mean percentage CR area and standard errors (SE) on days 1, 2, 8 (1 week) and 29 (1 month). Ten blocks a 10 paired CS–US trials were presented on day 1, five blocks a 10 paired CS–US trials on days 2, 8 and 29. In addition, 3 blocks a 10 CS-only extinction trials were presented on day 29. Note that the number of participants per block varies because of lack of CRs in individual blocks. Blue filled circles = anodal group, filled black circles = sham group. * indicates significant effects in post-hoc pairwise comparisons. Figure 3. Group mean percentage CR area and standard errors (SE) on days 1, 2, 8 (1 week) and 29 (1 month). Ten blocks a 10 paired CS–US trials were presented on day 1, five blocks a 10 paired CS–US trials on days 2, 8 and 29. In addition, 3 blocks a 10 CS-only extinction trials were presented on day 29. Note that the number of participants per block varies because of lack of CRs in individual blocks. Blue filled circles = anodal group, filled black circles = sham group. * indicates significant effects in post-hoc pairwise comparisons. differences in the first extinction block (p = 0.053; − 62.8% ± 31.5%) and the second block (p = 0.093; − 57.2% ± 33.4%) were not significant. CR timing. Mean values of CR onset and peak time latencies across paired trials and extinction trials are shown in Fig. 4. On day 1, mean CR onset latencies were − 123.7 ± 7.5 ms (that is, prior onset of the US set as 0) in the anodal group and − 132.2 ± 7.5 ms in the sham group. In the very first block, CR onsets differed between the sham and anodal group, with CR onset being closer to US onset in the anodal group, therefore occurring later after CS onset compared to the sham group. Results U di CR onsets did not differ in subsequent blocks. Linear mixed model analysis revealed a significant block effect [F(9,300.2) = 2.8; p = 0.004]. The stimulation by block [F(9,300.2) = 1.0; p = 0.41] and the stimulation effects [F(1,38.6) = 0.64; p = 0.43] were not significant. p ]f [ ( ) p ] gi Considering days 2, 8 and 29, mean CR onset latencies were − 142.0 ± 7.3 ms in the anodal group and − 161.0 ± 7.3 ms in the sham group. CR onset latencies occurred numerically later in the anodal compared to the sham group. Analysis of CR onset latencies did not reveal a significant effect of day [F(2,76) = 1.0; p = 0.36]. The stimula- tion by day effect [F(2,76) = 0.50; p = 0.61] and the stimulation effect [F(1,38) = 3.2; p = 0.079] were not significant. In extinction trials mean CR onset latencies were − 147.1 ± 10.1 ms in the anodal group and − 159.8 ± 9.6 ms in the sham group. Analysis of CR onset latencies did not reveal significant block [F(2,60.83) = 0.2; p = 0.81], stimulation by block [F(2 60 83)=0 6; p=0 55] or stimulation effects [F(1 36 39)=0 83; p=0 37] p ]f [ ( ) p ] gi Considering days 2, 8 and 29, mean CR onset latencies were − 142.0 ± 7.3 ms in the anodal group and − 161.0 ± 7.3 ms in the sham group. CR onset latencies occurred numerically later in the anodal compared to the sham group. Analysis of CR onset latencies did not reveal a significant effect of day [F(2,76) = 1.0; p = 0.36]. The stimula- tion by day effect [F(2,76) = 0.50; p = 0.61] and the stimulation effect [F(1,38) = 3.2; p = 0.079] were not significant. In extinction trials mean CR onset latencies were 147 1 ± 10 1 ms in the anodal group and 159 8 ± 9 6 ms y yf [ ( ) p ]f [ ( ) p ] gi In extinction trials mean CR onset latencies were − 147.1 ± 10.1 ms in the anodal group and − 159.8 ± 9.6 ms in the sham group. Analysis of CR onset latencies did not reveal significant block [F(2,60.83) = 0.2; p = 0.81], stimulation by block [F(2,60.83) = 0.6; p = 0.55] or stimulation effects [F(1,36.39) = 0.83; p = 0.37]. Results U di y pf p CR peak time latencies showed similar results. On day 1, mean peak time latencies were − 88.7 ± 6.7 ms in the anodal group and − 93.3 ± 6.7 ms in the sham group. In the very first block, CR peak time occurred later after CS onset in the anodal compared to the sham group. In the second block CR peak time, however, occurred earlier in the anodal group. The block effect [F(9,301.04) = 0.99; p = 0.44], and the stimulation effect were not significant [F(1,39.02) = 0.24; p = 0.63]. The stimulation by block effect was significant [F(9,301.04) = 2.7; p = 0.005]. Pairwise comparisons between anodal and sham stimulation revealed a significant mean difference of 48.3 ± 16.7 ms in https://doi.org/10.1038/s41598-020-80023-8 Scientific Reports \| (2020) 10:22434 \| www.nature.com/scientificreports/ Figure 4. Group mean and standard errors (SE) of CR onset (A) and peak time (B) latencies on days 1, 2, 8 (1 week) and 29 (1 month). Ten blocks a 10 paired CS–US trials were presented on day 1, five blocks a 10 paired CS–US trials on days 2, 8 and 29. In addition, 3 blocks a 10 CS-only extinction trials were presented on day 29. Negative values refer to the time prior to the onset of the US (air-puff) set as 0 ms. Note that the number of participants per block varies because of lack of CRs in individual blocks. Blue filled circles = anodal group, filled black circles = sham group. * indicates significant effects in post-hoc pairwise comparisons. Figure 4. Group mean and standard errors (SE) of CR onset (A) and peak time (B) latencies on days 1, 2, 8 (1 week) and 29 (1 month). Ten blocks a 10 paired CS–US trials were presented on day 1, five blocks a 10 pai CS–US trials on days 2, 8 and 29. In addition, 3 blocks a 10 CS-only extinction trials were presented on day 29. Negative values refer to the time prior to the onset of the US (air-puff) set as 0 ms. Note that the number participants per block varies because of lack of CRs in individual blocks. Blue filled circles = anodal group, fil black circles = sham group. * indicates significant effects in post-hoc pairwise comparisons. Figure 4. Results U di Group mean and standard errors (SE) of CR onset (A) and peak time (B) latencies on days 1, 2, 8 (1 week) and 29 (1 month). Ten blocks a 10 paired CS–US trials were presented on day 1, five blocks a 10 paired CS–US trials on days 2, 8 and 29. In addition, 3 blocks a 10 CS-only extinction trials were presented on day 29. Negative values refer to the time prior to the onset of the US (air-puff) set as 0 ms. Note that the number of participants per block varies because of lack of CRs in individual blocks. Blue filled circles = anodal group, filled black circles = sham group. * indicates significant effects in post-hoc pairwise comparisons. Table 1. Spontaneous blink rates (blinks per minute ± SD) as assessed at the beginning and at the end of each day in the anodal and sham group. Blinks/min Anodal Sham Day 1 Beginning End 17.0 ± 4.0 17.0 ± 5.2 17.8 ± 4.1 17.9 ± 4.5 Day 2 Beginning End 17.0 ± 3.7 16.4 ± 3.2 17.8 ± 4.1 17.9 ± 4.2 Day 8 Beginning End 17.7 ± 4.1 18.1 ± 4.9 18.2 ± 5.1 18.0 ± 4.9 Day 29 Beginning End 17.2 ± 3.5 17.1 ± 3.0 17.7 ± 3.8 17.5 ± 3.3 Table 1. Spontaneous blink rates (blinks per minute ± SD) as assessed at the beginning and at the end of each day in the anodal and sham group. Table 1. Spontaneous blink rates (blinks per minute ± SD) as assessed at the beginning and at the end of each day in the anodal and sham group. Table 1. Spontaneous blink rates (blinks per minute ± SD) as assessed at the beginning and at the end of each day in the anodal and sham group. the first block (p = 0.004), and a significant mean difference of − 33.1 ± 13.8 ms in the second block (p = 0.018). Mean differences comparing blocks 3–10 were not significant (all p values > 0.2). C id i d 2 8 d 29 CR k i l i 98 4 6 7 i h d l d the first block (p = 0.004), and a significant mean difference of − 33.1 ± 13.8 ms in the second block (p = 0.018). Mean differences comparing blocks 3–10 were not significant (all p values > 0.2). Discussionh In the present study, neither immediate nor long-term effects on the acquisition of conditioned eyeblink responses were observed.hii gf q y p The present findings on conditioned eyeblink acquisition are in good accordance with previous findings of cerebellar tDCS effects on reach adaptation, another motor learning task which is cerebellar dependent. Here initial findings of Galea et al.11 were not reproduced in later studies18,27. Again, one may argue that these studies used different reach adaptation set-ups and paradigms. Lack of reproducibility, however, was shown also in a later study by Galea and colleagues themselves in a carefully performed study using the same set-up and paradigm19. y y g y p y g p p g In the present study anodal tDCS tended to impede performance parameters of conditioned eyeblink responses. Anodal tDCS of the cerebellum applied during initial acquisition seemed to reduce the increase in size of CRs in repeated conditioning sessions across four weeks, indicating a long-term detrimental effect. An augmentation of CR amplitudes during eyeblink conditioning is well known, and has already been reported in early studies investigating healthy human participants28,29. Accordingly, in the sham group, we found that the increase of CR incidences was accompanied by an increase in CR area. Tran et al.30 also found an increased CR area in healthy children parallel to an increased rate of conditioned responses, which was not observed in pre- term children. In preterm children cerebellar development is impeded31,32 and reduced size of CRs was related to disordered cerebellar function. Furthermore, animal studies show that amplitudes of conditioned responses are diminished following cerebellar lesions33. It has been argued that the cerebellum is primarily engaged in the performance of conditioned responses34–36. Anodal tDCS is thought to increase the excitability of the cerebellar cortex37,38, and may lead to an increased inhibition of the cerebellar nuclei and therefore decreased cerebellar amplitudes.hfi p Timing of CRs appeared also to be impeded by anodal tDCS. The difference was most prominent in the first 10 paired acquisition trials of day 1. To a lesser degree, this difference was also found on subsequent days, with CRs tending to occur later in the group which received anodal stimulation on the first day. In accordance with the present findings, Mitroi et al.39 reported significantly longer CR peak and onset latencies following anodal cerebellar stimulation compared to sham. Discussionh The present data confirm that anodal cerebellar tDCS has a modulatory effect on eyeblink conditioning. Dif- ferent to previous results, however, we did not observe significant beneficial effects on acquisition learning. Rather, long-term detrimental effects of cerebellar tDCS were most prominent. Anodal tDCS impeded the long- term retention of these learned motor responses. Furthermore, timing and magnitude of conditioned responses seemed to be impaired.iii We were unable to confirm our initial findings of significantly enhanced CR acquisition as a consequence of cerebellar anodal stimulation24. Similar to Beyer et al.25 CR incidences tended to be higher in the anodal group compared to sham stimulation, but this difference did not reach significance. The present and the two previous studies were performed using the same eyeblink conditioning set-up. The delay conditioning paradigm differed between the Beyer et al.25 and the Zuchowski et al.24 studies, but were the same in the present and the Zuchowski et al.24 studies. Thus, differences in findings cannot be explained by differences in tDCS onset or reinforce- ment rate. However, Zuchowski et al.24 stimulated throughout the acquisition phase of 100 paired CS–US trials, whereas in the present study tDCS stimulation stopped after the first 50 paired CS–US trials sticking to a more conventional time of stimulation of about 20 minutes26. This is unlikely to account for the lack of cerebellar tDCS effects during the first 50 acquisition trials, but may have had an impact on the second half of acquisition trials and long-term effects across the following weeks. In the present study, CR incidence was numerically higher in the anodal compared to the sham group in the last 50 paired trials. Numerically, however, there was no difference between the two groups in the first 50 acquisition trials. Of note, cerebellar tDCS effects were most prominent as early as the first 15 acquisition trials in our previous study (see Fig. 2B in Zuchowski et al.24). In that study, cathodal tDCS had also been applied and showed reduced conditioning rates. Polarity dependent effects of cerebellar tDCS on CR acquisition, however, were not confirmed in our follow-up study25. Zuchowski et al.24 used a one day paradigm and long-term effects of cerebellar tDCS were not assessed. Results U di fi Considering days 2, 8 and 29, mean CR peak time latencies were − 98.4 ± 6.7 ms in the anodal group and − 112.9 ± 6.7 ms in the sham group. Mixed model analysis did not reveal significant effects of day [F(2,76) = 1.5; p = 0.23], stimulation by day [F(2,76) = 0.33; p = 0.72] or stimulation [F(1,38) = 2.3; p = 0.13]. p y y p p In extinction trials mean CR peak time latencies were − 115.5 ± 10.3 ms in the anodal group and − 121.1 ± 9.7 ms in the sham group. Mixed model analysis did not reveal significant block [F(2,61.25) = 1.1; p = 0.35], stimulation by block [F(2,61.25) = 0.7; p = 0.50] or stimulation effects [F(1,37.97) = 0.16; p = 0.69]. In extinction trials mean CR peak time latencies were − 115.5 ± 10.3 ms in the anodal group and − 121.1 ± 9.7 ms in the sham group. Mixed model analysis did not reveal significant block [F(2,61.25) = 1.1; p = 0.35], stimulation by block [F(2,61.25) = 0.7; p = 0.50] or stimulation effects [F(1,37.97) = 0.16; p = 0.69]. Alpha responses. The mean alpha-response count was 1.1 ± 1.5 in the anodal group and 1.2 ± 1.5 in the sham group. The group difference was not significant (p = 0.67; unpaired t test). Scientific Reports \| (2020) 10:22434 \| https://doi.org/10.1038/s41598-020-80023-8 www.nature.com/scientificreports/ Spontaneous blink rate. Spontaneous blinks were assessed within one minute both at the beginning and at the end of each day (Table 1). Comparison of spontaneous blink rates did not reveal significant differences between stimulation groups on each day (all p values > 0.5; unpaired t tests). Spontaneous blink rate. Spontaneous blinks were assessed within one minute both at the beginning and at the end of each day (Table 1). Comparison of spontaneous blink rates did not reveal significant differences between stimulation groups on each day (all p values > 0.5; unpaired t tests). www.nature.com/scientificreports/ www.nature.com/scientificreports/ Most importantly, anodal cerebellar tDCS during early acquisition let to impeded retention of conditioned eyeblink responses. CR incidences were significantly less in the first and second block of the extinction phase on day 29 in the anodal stimulated group compared to sham. Decrease of CR incidences across extinction trials, however, was not different between groups, that is extinction learning did not appear to be affected by anodal tDCS during acquisition learning. In the Zuchowski et al.24 study no detrimental effects on retention in the extinction phase was observed. Extinction, however, was tested within the same session as acquisition, and there was no time for consolidation. Similar to the present study, no effects on extinction learning were found. Likewise, we did not observe that anodal cerebellar tDCS applied during the extinction phase led to changes of extinction learning25,49. This, however, does not exclude that the cerebellum contributes to extinction. There is good evidence in the rodent literature that the cerebellum contributes to extinction20,50,51. Consistent with the present findings, Jongkees et al.52 reported not only performance impairment during anodal cerebellar tDCS, but also long-term effects of stimulation in a serial reaction time task. The authors investigated effects of cerebellar tDCS on motor response selection and sequence acquisition, and found an increase in overall reaction time during stimulation. This group difference was not only present as an immediate effect during tDCS, but reappeared at 24-h follow-up when participants performed the task without stimulation. Results point to a detrimental effect of anodal cerebellar tDCS on sequence consolidation and retention. For motor sequence learning, the primary motor cortex is known to be relevant in the acquisition stage, as excitability enhancement of this area with anodal tDCS improves learning3,4. Since motor cortex excitability can be reduced by anodal cerebellar tDCS, this might explain performance-reducing effects for this task. Regarding eyeblink condition- ing, retention is thought to take place at least in part within the cerebellar nuclei, with initial learning taking place primarily in the cerebellar cortex53–55. The excitability alteration of the cerebellar cortex evoked by anodal stimulation may impede the transfer of learning from the cerebellar cortex to the cerebellar nuclei. Learning related plasticity is likely not confined to the cerebellum. www.nature.com/scientificreports/ Thus, changes in activity in cerebello-cerebral networks involved in eyeblink conditoning may also play a role56–61.hi y g y p y The present data confirm that cerebellar anodal tDCS modulates cerebellar-dependent motor performance and motor learning processes. Findings, however, provide further evidence that cerebellar tDCS effects lack robustness and are difficult to predict19,25. As outlined above, likely the most important factor is the highly con- voluted cerebellar cortex which, because of the direction dependency of tDCS effects, likely results in opposing tDCS effects across stimulated cerebellar lobules in an individual participant, which makes the net effect hard to predict62. Interindividual differences due to anatomical variability have been shown to influence the direction of the electric field and current flow in relation to the orientation of the neuronal tissue63–65. The involvement of different cerebellar cell populations as well as the anatomical complexity of the cerebellar cortex may also play a role, and may explain why different tDCS protocols result in different behavioural outcomes38. Furthermore, heterogeneous tDCS effects have been related to the BDNF polymorphism (brain-derived neutrotropic factor), a factor relevant for synaptic plasticity66–68. A specific role of the BDNF polymorphism in eyeblink conditioning has been related to specific firing patterns of Purkinje cells69. tDCS effects have also been reported to depend on individual sensitivity to non-invasive brain stimulation70. Labruna et al.70 found that sensitivity of M1 to tran- scranial magnetic stimulation (TMS) pulses correlated with tDCS effects on M1 on visuomotor adaptation. It will be of interest for future studies to investigate whether the sensitivity to effects on cerebellar-brain-inhibition (CBI) correlates with cerebellar tDCS effects on motor learning. Finally, differential tDCS effects on distinct cell populations in the cerebellar cortex have to be taken into account, which may lead to different tDCS effects depending on stimulation parameters38.hh p g p The present study has some limitations. The study may be underpowered. Although 20 participants per stimulation group were included, stimulation effects in the acquisition phase may only occur in larger study populations. Furthermore, reduced retention effects were present 4 weeks after stimulation. It would be of interest to show that similar effects can be observed on the day following tDCS, that is after one night of consolidation. Moreover, tDCS was restricted to anodal stimulation compared to sham, without a cathodal stimulated group, and the time of stimulation was shorter compared to our initial study. www.nature.com/scientificreports/ Finally, possible individual factors like anatomical variability at the site of stimulation have not been considered. Conclusions Findings of the present study suggest a detrimental effect of anodal cerebellar tDCS on the performance and timing of learned motor responses. In addition, retention was reduced as assessed four weeks after stimulation, indicating long-term detrimental effects. Enhanced acquisition of conditioned motor responses by anodal tDCS as previously reported was not confirmed. Future studies are needed to understand the factors predicting the outcome of cerebellar tDCS effects on motor performance and learning in individual participants. Discussionh This is in contrast to findings of our previous study that found that CRs occurred significantly earlier during anodal tDCS and throughout the acquisition phase24. Beyer et al.25, on the other hand, did not find any significant effects of cerebellar anodal tDCS on CR timing parameters. Thus, anodal cerebellar tDCS effects led to opposing effects on timing of conditioned responses. ff Animal studies show that conditioned responses in delay eyeblink conditioning are dependent on pauses of Purkinje cell activity which results in less inhibition of the neural activity of the cerebellar nuclei just before the US onset and induces the generation of a well-timed CR40,41. In animal models large cortical lesions that involve the anterior lobe caused short-latency responses42,43 which were also reported in transgenic mice with impaired long-term depression (LTD) at the parallel fiber-Purkinje cell synapse44. Although less shifted forward, also in patients with cortical lesions of superior parts of the cerebellar hemisphere, mean CR onset occurred too early, on average 20 ms earlier than in controls45. tDCS effects are critically dependent on orientation of the dendrites and other neuronal structures to the electric field46–48 and the highly convoluted cerebellar cortex may explain opposing effects in different participant populations: inhibition of the cerebellar nuclei may be either ramped up or down (or not changed at all) depending on the total net effects of cerebellar tDCS stimulation, and may therefore result on opposing effects on CR timing (or none at all). Furthermore, cerebellar tDCS may affect the cell populations in the different cerebellar layers to various extents which can result in a heterogeneous com- pound effect38. Scientific Reports \| (2020) 10:22434 \| https://doi.org/10.1038/s41598-020-80023-8 Participants. On days 2, 8 and 29, 50 paired CS–US trials were applied. This was followed by 30 CS-only extinction trials on day 29. Eyeblink conditioning. Participants sat comfortably in a chair with both arms resting on armrests. During eyeblink conditioning a silent movie was shown on a screen positioned in front of the participants to maintain vigilance. Conditioned responses (CRs) were recorded from orbicularis oculi muscles bilaterally via surface electrodes which were fixed to the lower eyelid and to the nasion. Signals were fed to EMG amplifiers (sampling rate 1000 Hz, band pass filter frequency between 100 Hz and 2 kHz), full wave-rectified and further low pass- filtered offline (100 Hz). A standard delay eyeblink conditioning protocol was used according to Gormezano and Kehoe73. The CS, a neutral tone (1 kHz, 70 dB SPL, duration 540 ms), was provided via headphones and superimposed on a continuous white noise (60 dB SPL) to mask environmental noise. An air-puff (duration 100 ms, intensity 400 kPa at source, 110 kPa at nozzle) was used as US. The US was directed laterally to the outer canthus of the right eye through a nozzle mounted on a helmet worn by the participants. The CS started 310 ms after onset of each trial, preceded the US onset by a fixed time interval of 440 ms and coterminated with the US. The intertrial interval varied randomly between 20 and 35 s.t h y Conditioned eyeblink responses were semiautomatically analyzed using a custom made software74. CRs were identified within the CS–US window. Responses occurring within the 150 ms interval after CS onset were considered as reflexive responses to the tone (alpha-responses) and not rated as CRs75. Trials with spontane- ous blinks occurring prior CS onset were excluded from the analysis76. Rectified EMG recordings were filtered using a series of non-linear Gaussian filters. CRs were identified when EMG activity reached 7.5% of the EMG maximum in each recording with a minimum duration of 20 ms and a minimum integral of 1 mV*ms. All trials were visually inspected and implausible identification of CRs was manually corrected. The total number of paired (and unpaired extinction) trials was subdivided into blocks of 10 trials each. The number of CRs was expressed as the percentage of trials containing responses with respect to each block of 10 trials (percentage CR incidence) and the total number of trials (total percentage CR incidence). Participants. Participants. A total of 40 young, healthy and right handed participants took part in the study. They were randomly assigned to two stimulation groups. One group (10 males, 10 females, mean age 21.7 ± SD 2.5 years received anodal tDCS, the second group (10 males, 10 females, mean age 22.8 ± SD 3.5 years) received sham tDCS. None of the participants had a history of neurological disease. None were taking centrally acting medica- tion. Neurological examination, including ataxia rating scales71,72, was performed on the first day of the experi- ment, and were unremarkable. All participants were naive to eyeblink conditioning and tDCS. In each partici- pant, hearing thresholds (dB SPL) were determined using a tone of 1 kHz. Values corresponded to normal age limits in all participants. The study was approved by the ethics committee of the Essen University Hospital and all methods and experiments were performed in accordance with relevant guidelines and regulations. Oral and written informed consent was obtained from all participants. https://doi.org/10.1038/s41598-020-80023-8 Scientific Reports \| (2020) 10:22434 \| www.nature.com/scientificreports/ Figure 5. Experimental protocol. At the beginning of day 1, 10 CS-only trials and 10 US-only trials were presented in a pseudorandom sequence (pseudo-conditioning), followed by 100 paired CS–US trials. tDCS was started after the pseudo-conditioning phase and lasted throughout the first 50 paired CS–US trials. On days 2, 8 (1 week) and 29 (1 month), 50 paired CS–US trials were given. On day 29 (1 month) the 50 paired CS–US trials were followed by 30 CS-only extinction trials. Figure 5. Experimental protocol. At the beginning of day 1, 10 CS-only trials and 10 US-only trials were presented in a pseudorandom sequence (pseudo-conditioning), followed by 100 paired CS–US trials. tDCS was started after the pseudo-conditioning phase and lasted throughout the first 50 paired CS–US trials. On days 2, 8 (1 week) and 29 (1 month), 50 paired CS–US trials were given. On day 29 (1 month) the 50 paired CS–US trials were followed by 30 CS-only extinction trials. Experimental procedure. Delay eyeblink conditioning was performed on four days: day 1, day 2, after one week (day 8), and after one month (day 29) (Fig. 5). On day 1, following a pseudoconditioning phase of 10 CS-only and 10 US-only trials presented in pseudorandom order, 100 paired CS–US trials were applied. During the first 50 paired CS–US trials, 20 participants received anodal cerebellar tDCS, and 20 participants received sham stimulation. Participants. In addition, CR onset, peak time and area were analyzed. CR onset and peak time were expressed as negative values prior US onset set as 0 ms. CR peak time was defined at the time of maximum amplitude before US onset in paired trials. Mean baseline area was assessed in an interval of 100 ms prior US onset in each trial and subtracted from CR area. CR area was expressed as percentage of mean CR area across all CRs on day 1 in each participant, set as 100%. CR area was normalized in order to allow comparisons of changes across time. The frequency of spontaneous blinks was measured on each day within 1 min at the beginning and the end of the experiment. The number of alpha-blinks was assessed. Cerebellar transcranial direct current stimulation. Cerebellar tDCS was applied using a neuroConn DC Stimulator Plus (serial number 0371; neuroConn GmbH). Two conductive carbon–rubber electrodes (5 cm × 7 cm, surface area 35 cm2) and conductive electrode paste (Weaver ten20) were used. The cerebellar electrode was centered 3 cm lateral to the inion in a vertical position over the right cerebellar hemisphere24. The return electrode was placed on the ipsilateral buccinator muscle37. The current of anodal tDCS was set to 2 mA77 with a ramp-like fade-in and fade-out stimulation of 30 s (current density 0.057 mA/cm2). Stimulation started with the acquisition phase on day one and was performed throughout 50 of the 100 paired CS–US trials (Fig. 5). The overall duration of stimulation was 24 min and 12 s including the fade-in and fade-out time. In the sham condition the same fade-in of 30 s was used followed by 48.4 s. of tDCS and a fade-out time of 30 s. The modality of stimulation was unknown to the participants as well as to the investigator. Cerebellar tDCS was well tolerated. Some participants reported a mild tingling at the beginning of the stimulation. tDCS protocols were identical to Zuchowski et al.24 with two exceptions: (1) stimulation time was reduced from 42.9 min in the previous study to a more conventional stimulation time of about 20 min in the present study, (2) conductive electrode paste was used instead of sponge electrodes soaked in saline solution. https://doi.org/10.1038/s41598-020-80023-8 Scientific Reports \| (2020) 10:22434 \| www.nature.com/scientificreports/ Data analysis. Statistical analyses were performed in SPSS (SPSS Statistics 25.0; IBM). Participants. First, timing parameters of unconditioned eyeblink responses were analyzed using unpaired t tests. Next, linear mixed model analyses were performed. To assess immediate tDCS effects on CR acquisition learning on day 1, CR incidence was used as dependent variable, block (1–10; 10 blocks of 10 paired trials) as within subject factor and stimu- lation group (anodal vs. sham) as between subject factor. To assess long-term tDCS effects on CR incidence across days, CR incidence was used as dependent variable, day (day 2, day 8, day 29) as within subject factor and stimulation group (anodal vs. sham) as between subject factor. To analyze tDCS effects on extinction, CR incidence was used as dependent variable, extinction block (1–3; 10 blocks of 10 extinction CS-only trials) as within subject factor and stimulation group (anodal vs. sham) as between subject factor. Similar mixed model analyses were performed considering CR onset, peak time and area as dependent variable. Level of significance was set at p < 0.05. Received: 19 August 2020; Accepted: 14 December 2020 Received: 19 August 2020; Accepted: 14 December 2020 References Cerebellum 18, 1064–1097 (2019) 1. Galea, J. M., Vazquez, A., Pasricha, N., de Xivry, J. J. & Celnik, P. Dissociating the roles of the cerebellum and motor cortex during adaptive learning: The motor cortex retains what the cerebellum learns. Cereb. Cortex 21, 1761–1770 (2011). h 2. Hardwick, R. M. & Celnik, P. A. Cerebellar direct current stimulation enhances motor learning in older adults. Neurobiol. Aging 35, 2217–2221 (2014). 13. Herzfeld, D. J. et al. Contributions of the cerebellum and the motor cortex to acquisition and retention of motor memories. Neu- roimage 98, 147–158 (2014).f 14. Das, S. et al. Impairment of long-term plasticity of cerebellar purkinje cells eliminates the effect of anodal direct current stimula- tion on vestibulo-ocular reflex habituation. Front. Neurosci. 11, 444 (2017). l 5. Benussi, A. et al. Cerebello-spinal tDCS in ataxia: A randomized, double-blind, sham-controlled, crossover trial. Neurology 91 e1090–e1101 (2018).f 6. Benussi, A. et al. Long term clinical and neurophysiological effects of cerebellar transcranial direct current stimulation in patients with neurodegenerative ataxia. Brain Stimul. 10, 242–250 (2017). g 17. Benussi, A., Koch, G., Cotelli, M., Padovani, A. & Borroni, B. Cerebellar transcranial direct current stimulation in patients with ataxia: A double-blind, randomized, sham-controlled study. Mov. Disord. 30, 1701–1705 (2015).ii 8. Hulst, T. et al. Cerebellar patients do not benefit from cerebellar or M1 transcranial direct current stimulation during force-field reaching adaptation. J. Neurophysiol. 118, 732–748 (2017).f i reaching adaptation. J. Neurophysiol. 118, 732–748 (2017).f g p p y 9. Jalali, R., Miall, R. C. & Galea, J. M. No consistent effect of cerebellar transcranial direct current stimulation on visuomotor adapta- tion. J. Neurophysiol. 118, 655–665 (2017).i p y 0. Medina, J. F., Nores, W. L. & Mauk, M. D. Inhibition of climbing fibres is a signal for the extinction of conditioned eyelid responses Nature 416, 330–333 (2002).h 1. Gerwig, M., Kolb, F. P. & Timmann, D. The involvement of the human cerebellum in eyeblink conditioning. Cerebellum 6, 38–57 (2007).h 22. Timmann, D. et al. The human cerebellum contributes to motor, emotional and cognitive associative learning. A review. Cortex 46, 845–857 (2010). H l G J h d D A R A & J h F Cl l d f Wh h l 3. Hesslow, G., Jirenhed, D. A., Rasmussen, A. & Johansson, F. Classical conditioning of motor responses: What is the learning mechanism?. Neural Netw. 47, 81–87 (2013). 4. Zuchowski, M. L., Timmann, D. References 1. Nitsche, M. A. & Paulus, W. Excitability changes induced in the human motor cortex by weak transcranial direct current stimula- tion. J. Physiol. 527(Pt 3), 633–639 (2000). y 2. Nitsche, M. A. & Paulus, W. Sustained excitability elevations induced by transcranial DC motor cortex stimulation in humans Neurology 57, 1899–1901 (2001). gy 3. Nitsche, M. A. et al. Facilitation of implicit motor learning by weak transcranial direct current stimulation of the primary motor cortex in the human. J. Cogn. Neurosci. 15, 619–626 (2003).f g 4. Reis, J. et al. Noninvasive cortical stimulation enhances motor skill acquisition over multiple days through an effect on consolida- tion. Proc. Natl. Acad. Sci. USA 106, 1590–1595 (2009). 4. Reis, J. et al. Noninvasive cortical stimulation enhances motor skill acquisition over multiple tion. Proc. Natl. Acad. Sci. USA 106, 1590–1595 (2009). tion. Proc. Natl. Acad. Sci. USA 106, 1590–1595 (2009). , ( ) 5. Liew, S. L., Santarnecchi, E., Buch, E. R. & Cohen, L. G. Non-invasive brain stimulation in neurorehabilitation: Local and di effects for motor recovery Front Hum Neurosci 8 378 (2014) 5. Liew, S. L., Santarnecchi, E., Buch, E. R. & Cohen, L. G. Non-invasive brain stimulation in neurorehabilitation: Local and distant effects for motor recovery. Front Hum. Neurosci. 8, 378 (2014). 5. Liew, S. L., Santarnecchi, E., Buch, E. R. & Cohen, L. G. Non-i effects for motor recovery. Front Hum. Neurosci. 8, 378 (2014) f y 6. Grimaldi, G. et al. Cerebellar transcranial direct current stimulation (ctDCS): A novel approach to understanding cerebellar func- tion in health and disease. Neuroscientist 22, 83–97 (2016).f f 6. Grimaldi, G. et al. Cerebellar transcranial direct current stimu tion in health and disease. Neuroscientist 22, 83–97 (2016).f 7. Buch, E. R. et al. Effects of tDCS on motor learning and memory formation: A consensus and critical position paper. Clin. Neu- rophysiol. 128, 589–603 (2017).hf p y ( ) 8. Kumari, N., Taylor, D. & Signal, N. The effect of cerebellar transcranial direct current stimulation on motor learning: A systematic review of randomized controlled trials. Front. Hum. Neurosci. 13, 328 (2019). 9. Jayaram, G. et al. Modulating locomotor adaptation with cerebellar stimulation. J. Neurophysiol. 107, 2950–2957 (2012). . Jayaram, G. et al. Modulating locomotor adaptation with cerebe . et al. Modulating locomotor adaptation with cerebellar stimulati y g p p y 10. Miterko, L. N. et al. Consensus paper: Experimental neurostimulation of the cerebellum. www.nature.com/scientificreports/ , y g y p g p y y , ( ) 2. Woods, A. J. et al. A technical guide to tDCS, and related non-invasive brain stimulation tools. Clin. Neurophysiol. 127, 1031–1048 (2016).i 3. Parazzini, M. et al. Modelling the electric field and the current density generated by cerebellar transcranial DC stimulation in humans. Clin. Neurophysiol. 125, 577–584 (2014). p y 64. Priori, A., Ciocca, M., Parazzini, M., Vergari, M. & Ferrucci, R. Transcranial cerebellar direct current stimulation and transcutane- ous spinal cord direct current stimulation as innovative tools for neuroscientists. J. Physiol. 592, 3345–3369 (2014). 6 F i R C F & P i i A C b ll DCS H d i C b ll ( ) p y 65. Ferrucci, R., Cortese, F. & Priori, A. Cerebellar tDCS: How to do it. Cerebellum 14, 27–30 (2015). 66. Cheeran, B. et al. A common polymorphism in the brain-derived neurotrophic factor gene (BDNF) modulates human cortical plasticity and the response to rTMS. J. Physiol. 586, 5717–5725 (2008). y y 7. Fritsch, B. et al. Direct current stimulation promotes BDNF-dependent synaptic plasticity: Potential implications for motor learn ing. Neuron 66, 198–204 (2010). g 8. Antal, A. et al. Brain-derived neurotrophic factor (BDNF) gene polymorphisms shape cortical plasticity in humans. Brain Stimul 3, 230–237 (2010). 69. van der Vliet, R. et al. Cerebellar transcranial direct current stimulation interacts with BDNF Val66Met in motor learning. Brain Stimul. 11, 759–771 (2018).fi 70. Labruna, L. et al. Efficacy of anodal transcranial direct current stimulation is related to sensitivity to transcranial magnetic stimula- tion. Brain Stimul. 9, 8–15 (2016).h 1. Trouillas, P. et al. International Cooperative Ataxia Rating Scale for pharmacological assessment of the cerebellar syndrome. The Ataxia Neuropharmacology Committee of the World Federation of Neurology. J. Neurol. Sci. 145, 205–211 (1997). 72. Schmitz-Hubsch, T. et al. Scale for the assessment and rating of ataxia: Development of a new clinical scale. Neurology 66, 1717– 1720 (2006). 73. Gormezano, I. & Kehoe, E. J. Classical conditioning: Some methodicalconceptual issues. In Handbook of Learning and Cognitive Processes Vol 2 (ed. Estes, W. K.) 143–179 (Lawrence Erlbaum, Hillsdale, 1975). 4. Gerwig, M. et al. Evaluation of multiple-session delay eyeblink conditioning comparing patients with focal cerebellar lesions and cerebellar degeneration. Behav. Brain Res. 212, 143–151 (2010).f g 75. Woodruff-Pak, D. S., Papka, M. & Ivry, R. B. Cerebellar involvement in eyeblink classical conditioning in humans. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 33. Welsh, J. P. & Harvey, J. A. Cerebellar lesions and the nictitating membrane reflex: Performance deficits of the conditioned and unconditioned response. J. Neurosci. 9, 299–311 (1989). p 34. Gruart, A., Guillazo-Blanch, G., Fernandez-Mas, R., Jimenez-Diaz, L. & Delgado-Garcia, J. M. Cerebellar posterior interpo nucleus as an enhancer of classically conditioned eyelid responses in alert cats. J. Neurophysiol. 84, 2680–2690 (2000).h y y p p y 35. Delgado-Garcia, J. M. & Gruart, A. The role of interpositus nucleus in eyelid conditioned responses. Cerebellum 1, 289–308 (2 35. Delgado-Garcia, J. M. & Gruart, A. The role of interpositus nucleus in eyelid conditioned responses. Cerebellum 1, 289–308 (2002). 36 Jimenez Diaz L Navarro Lopez Jde D Gruart A & Delgado Garcia J M Role of cerebellar interpositus nucleus in the genesis 35. Delgado-Garcia, J. M. & Gruart, A. The role of interpositus nucleus in eyelid conditioned responses. Cerebellum 1, 289–308 (2002). 36. Jimenez-Diaz, L., Navarro-Lopez Jde, D., Gruart, A. & Delgado-Garcia, J. M. Role of cerebellar interpositus nucleus in the genesis l 35. Delgado Garcia, J. M. & Gruart, A. The role of interpositus nucleus in eyelid conditioned responses. Cerebellum 1, 289 308 (2002). 36. Jimenez-Diaz, L., Navarro-Lopez Jde, D., Gruart, A. & Delgado-Garcia, J. M. Role of cerebellar interpositus nucleus in the genesis and control of reflex and conditioned eyelid responses J Neurosci 24 9138–9145 (2004) gh p y p 36. Jimenez-Diaz, L., Navarro-Lopez Jde, D., Gruart, A. & Delgado-Garcia, J. M. Role of cerebellar interpositus nucleus in the ge and control of reflex and conditioned eyelid responses. J. Neurosci. 24, 9138–9145 (2004).i l y p 7. Galea, J. M., Jayaram, G., Ajagbe, L. & Celnik, P. Modulation of cerebellar excitability by polarity-specific noninvasive direct cur- rent stimulation. J. Neurosci. 29, 9115–9122 (2009).f 8. Batsikadze, G. et al. Effects of cerebellar transcranial direct current stimulation on cerebellar-brain inhibition in humans: A sys- tematic evaluation. Brain Stimul. 12, 1177–1186 (2019). f tematic evaluation. Brain Stimul. 12, 1177–1186 (201 39. Mitroi, J. et al. Polarity- and intensity-independent modulation of timing during delay eyeblink conditioning using cerebellar transcranial direct current stimulation. Cerebellum 19, 383–391 (2020). 0. Hesslow, G. & Ivarsson, M. Suppression of cerebellar Purkinje cells during conditioned responses in ferrets. NeuroReport 5, 649–652 (1994). 1. Jirenhed, D. A., Bengtsson, F. & Hesslow, G. Acquisition, extinction, and reacquisition of a cerebellar cortical memory trace. J Neurosci. 27, 2493–2502 (2007). ( ) 42. www.nature.com/scientificreports/ & Mauk, M. D. A mechanism for savings in the cerebellum. J. Neurosci. 21, 4081–4089 (2001). hf 53. Medina, J. F., Garcia, K. S. & Mauk, M. D. A mechanism for sa 54. Mauk, M. D., Li, W., Khilkevich, A. & Halverson, H. Cerebellar mechanisms of learning and plasticity revealed by delay e conditioning. Int. Rev. Neurobiol. 117, 21–37 (2014). g 5. Herzfeld, D. J., Hall, N. J., Tringides, M. & Lisberger, S. G. Principles of operation of a cerebellar learning circuit. Elife 9, 20 (2020) g 55. Herzfeld, D. J., Hall, N. J., Tringides, M. & Lisberger, S. G. Principles of operation of a cerebellar learning circuit. Elife 9, 20 (2020). 56 Molchan S E Sunderland T McIntosh A R Herscovitch P & Schreurs B G A functional anatomical study of associative g 55. Herzfeld, D. J., Hall, N. J., Tringides, M. & Lisberger, S. G. Principles of operation of a cerebellar learning circuit. Elife 9, 20 (2020). 56. Molchan, S. E., Sunderland, T., McIntosh, A. R., Herscovitch, P. & Schreurs, B. G. A functional anatomical study of associative learning in humans Proc Natl Acad Sci USA 91 8122–8126 (1994) 55. Herzfeld, D. J., Hall, N. J., Tringides, M. & Lisberger, S. G. P g ( ) 57. Logan, C. G. & Grafton, S. T. Functional anatomy of human eyeblink conditioning determined with regional cerebral glucose metabolism and positron-emission tomography. Proc. Natl. Acad. Sci. USA 92, 7500–7504 (1995). 8. Blaxton, T. A. et al. Functional mapping of human learning: A positron emission tomography activation study of eyeblink condi- tioning. J. Neurosci. 16, 4032–4040 (1996).l 9. Schreurs, B. G. et al. Lateralization and behavioral correlation of changes in regional cerebral blood flow with classical conditioning of the human eyeblink response. J. Neurophysiol. 77, 2153–2163 (1997). y p p y 0. Knuttinen, M. G. et al. Electromyography as a recording system for eyeblink conditioning with functional magnetic resonance imaging. Neuroimage 17, 977–987 (2002). k l bl k d h l h d l h d b ll ( ) 61. Parker, K. L. et al. Eyeblink conditioning in healthy adults: A p 61. Parker, K. L. et al. Eyeblink conditioning in healthy adults: A positron emission tomography study. Cerebellum 11, 946–956 (2012). 62. Woods, A. J. et al. A technical guide to tDCS, and related non-invasive brain stimulation tools. Clin. Neurophysiol. 127, 1031–1048 (2016). www.nature.com/scientificreports/ Perrett, S. P., Ruiz, B. P. & Mauk, M. D. Cerebellar cortex lesions disrupt learning-dependent timing of conditioned eyelid responses. J. Neurosci. 13, 1708–1718 (1993). 3. Garcia, K. S. & Mauk, M. D. Pharmacological analysis of cerebellar contributions to the timing and expression of conditioned eyelid responses. Neuropharmacology 37, 471–480 (1998). y p p gy 4. Koekkoek, S. K. et al. Cerebellar LTD and learning-dependent timing of conditioned eyelid responses. Science 301, 1736–1739 (2003). ( ) 45. Gerwig, M. et al. Timing of conditioned eyeblink responses is impaired in cerebellar patients. J. Neurosci. 25, 3919–3931 (2005). 45. Gerwig, M. et al. Timing of conditioned eyeblink responses is impaired in cerebellar patients. J. Neurosci. 25, 3919–3931 (2005). 46. Kabakov, A. Y., Muller, P. A., Pascual-Leone, A., Jensen, F. E. & Rotenberg, A. Contribution of axonal orientation to pathway- d d d l f b d l l d h h l 45. Gerwig, M. et al. Timing of conditioned eyeblink responses is 45. Gerwig, M. et al. Timing of conditioned eyeblink responses is impaired in cerebellar patients. J. Neurosci. 25, 3919–3931 (2005). 46. Kabakov, A. Y., Muller, P. A., Pascual-Leone, A., Jensen, F. E. & Rotenberg, A. Contribution of axonal orientation to pathway- dependent modulation of excitatory transmission by direct current stimulation in isolated rat hippocampus. J. Neurophysiol. 107, 1881–1889 (2012).ff ( ) 7. Rahman, A. et al. Cellular effects of acute direct current stimulation: Somatic and synaptic terminal effects. J. Physiol. 591 2563–2578 (2013). 8. Rahman, A., Toshev, P. K. & Bikson, M. Polarizing cerebellar neurons with transcranial direct current stimulation. Clin. Neuro physiol. 125, 435–438 (2014). 9. Lipp, J. et al. Prefrontal but not cerebellar tDCS attenuates renewal of extinguished conditioned eyeblink responses. Neurobiol Learn. Mem. 170, 107137 (2020).h 0. Robleto, K., Poulos, A. M. & Thompson, R. F. Brain mechanisms of extinction of the classically conditioned eyeblink response Learn. Mem. 11, 517–524 (2004). 0. Robleto, K., Poulos, A. M. & Thompson, R. F. Brain mechanisms of extinction of the classically conditioned eyeblink response Learn. Mem. 11, 517–524 (2004). 51. Hu, C., Zhang, L. B., Chen, H., Xiong, Y. & Hu, B. Neurosubstrates and mechanisms underlying the extinction of associative m memory. Neurobiol. Learn. Mem. 126, 78–86 (2015).hf y 52. Jongkees, B. J. et al. The effect of cerebellar tDCS on sequential motor response selection. Cerebellum 18, 738–749 (2019). hf 53. Medina, J. F., Garcia, K. S. References & Gerwig, M. Acquisition of conditioned eyeblink responses is modulated by cerebellar tDCS Brain Stimul. 7, 525–531 (2014).ff 5. Beyer, L., Batsikadze, G., Timmann, D. & Gerwig, M. Cerebellar tDCS effects on conditioned eyeblinks using different electrode placements and stimulation protocols. Front Hum. Neurosci. 11, 23 (2017). p p 6. van Dun, K., Bodranghien, F. C., Marien, P. & Manto, M. U. tDCS of the cerebellum: Where do we stand in 2016? Technical issues and critical review of the literature. Front. Hum. Neurosci. 10, 199 (2016).f 26. van Dun, K., Bodranghien, F. C., Marien, P. & Manto, M. U. tDCS of the cerebellum: Wh and critical review of the literature. Front. Hum. Neurosci. 10, 199 (2016).f 27. Mamlins, A., Hulst, T., Donchin, O., Timmann, D. & Claassen, J. No effects of cerebellar transcranial direct current stimulation on force field and visuomotor reach adaptation in young and healthy subjects. J. Neurophysiol. 121, 2112–2125 (2019).h 28. Boneau, C. A. The interstimulus interval and the latency of the conditioned eyelid response. J. Exp. Psychol. 56, 464–471 (1958). 29. Prokasy, W. F., Ebel, H. C. & Thompson, D. D. Response shaping at long interstimulus intervals in classical eyelid conditioning. J. Exp. Psychol. 66, 138–141 (1963). h y y p p y 29. Prokasy, W. F., Ebel, H. C. & Thompson, D. D. Response shaping at long interstimulus intervals in classical eyelid conditioning. J. Exp. Psychol. 66, 138–141 (1963). p y 30. Tran, L. et al. Cerebellar-dependent associative learning is impaired in very preterm born children and young adults. Sci. Rep. 7, 18028 (2017). 31. Limperopoulos, C. et al. Late gestation cerebellar growth is rapid and impeded by premature birth. Pediatrics 115, 688–695 (2005). 32. Messerschmidt, A. et al. Disruption of cerebellar development: Potential complication of extreme prematurity. Am. J. Neuroradiol. 26, 1659–1667 (2005). p p g g p p y p 32. Messerschmidt, A. et al. Disruption of cerebellar development: Potential complication of extreme prematurity. Am. J. Neuroradiol. 26, 1659–1667 (2005). Scientific Reports \| (2020) 10:22434 \| https://doi.org/10.1038/s41598-020-80023-8 www.nature.com/scientificreports/ www.nature.com/scientificreports/ 76. Bracha, V., Zhao, L., Irwin, K. B. & Bloedel, J. R. The human cerebellum and associative learning: Dissociation between the acquisi- tion, retention and extinction of conditioned eyeblinks. Brain Res. 860, 87–94 (2000).f y ( ) 7. Iyer, M. B. et al. Safety and cognitive effect of frontal DC brain polarization in healthy individuals. Neurology 64, 872–875 (2005) Funding O A g Open Access funding enabled and organized by Projekt DEAL. Acknowledgementsh g The study was funded by Deutsche Forschungsgemeinschaft 316803389-SFB1280, projects A05 and A0 Competing interests h p g O. Kimpel, Th. Hulst, G. Batsikadze and Th. Ernst declare no competing interests. M.A. Nitsche is at the scien- tific advidory boards of Neuroelectrics, and Neurodevice, and is funded by Deutsche Forschungsgemeinschaft (DFG)—Projektnummer 316803389—SFB 1280 “Funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—Projektnummer 316803389—SFB 1280”. D. Timmann received funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation (SFB 1280 Project A05), Mercur Research Center Ruhr and the German Heredoataxia Foundation (DHAG). M. Gerwig received speaker hono- raria and/or travel reimbursement from Novartis, Pfizer and Ipsen Pharma and research support from MSD. Author contributions O.K.: data curation; formal analysis; visualization; roles/writing—original draft. T.H.: formal analysis; writing— review and editing. G.B.: formal analysis; writing—review and editing. T.E.: data curation; writing—review and editing. M.N.: funding acquisition; writing—review and editing. D.T.: conceptualization; funding acquisition; resources; supervision; roles/writing—original draft; writing—review and editing. M.G.: conceptualization; data curation; formal analysis; project administration; supervision; visualization; roles/writing—original draft; writ- ing—review and editing. www.nature.com/scientificreports/ Neuropsychology 10, 443–458 (1996). https://doi.org/10.1038/s41598-020-80023-8 Scientific Reports \| (2020) 10:22434 \| Additional information Correspondence and requests for materials should be addressed to M.G. © The Author(s) 2020 Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. 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https://openalex.org/W4320853813	https://zenodo.org/records/7533611/files/1-1-150-155.pdf	Russian	null	ОБРАЗОВАТЕЛЬНАЯ РОБОТОТЕХНИКА В УСЛОВИЯХ ДОПОЛНИТЕЛЬНОГО ОБРАЗОВАНИЯ КАК СРЕДСТВО РАЗВИТИЯ ТВОРЧЕСКОЙ АКТИВНОСТИ ШКОЛЬНИКОВ	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	2,515	Наукосфера. №1 (1), 2023 Педагогические науки УДК 372.862 DOI 10.5281/zenodo.7533611 ОБРАЗОВАТЕЛЬНАЯ РОБОТОТЕХНИКА В УСЛОВИЯХ ДОПОЛНИТЕЛЬНОГО ОБРАЗОВАНИЯ КАК СРЕДСТВО РАЗВИТИЯ ТВОРЧЕСКОЙ АКТИВНОСТИ ШКОЛЬНИКОВ EDUCATIONAL ROBOTICS IN THE CONDITIONS OF ADDITIONAL EDUCATION AS A MEANS OF DEVELOPING CREATIVE ACTIVITY OF SCHOOLCHILDREN ОНДАР ЭДУАРД ЭМИРОВИЧ, магистрант, Тувинский государственный университет. КУУЛАР ДОЛААНА ОРЛАН-ООЛОВНА, кандидат педагогических наук, доцент, Тувинский государственный университет. ТЮЛЮШ МАРТА КАН-ООЛОВНА, кандидат педагогических наук, доцент, Тувинский государственный университет. ONDAR EDUARD EMIROVICH, master's student, Tuva State University. KUULAR DOLAANA ORLAN-OOLOVNA Candidate of Pedagogical Sciences, Associate Professor, Tuva State University. TYULUSH MARTA KAN-OOLOVNA, Candidate of Pedagogical Sciences, Associate Professor, Tuva State University. В данной статье рассматривается проблема внедрения образовательной робототехники в Наукосфера. №1 (1), 2023 Педагогические науки УДК 372.862 DOI 10.5281/zenodo.7533611 ONDAR EDUARD EMIROVICH, master's student, Tuva State University. KUULAR DOLAANA ORLAN-OOLOVNA Candidate of Pedagogical Sciences, Associate Professor, Tuva State University. TYULUSH MARTA KAN-OOLOVNA, Candidate of Pedagogical Sciences, Associate Professor, Tuva State University. ONDAR EDUARD EMIROVICH, master's student, Tuva State University. KUULAR DOLAANA ORLAN-OOLOVNA Candidate of Pedagogical Sciences, Associate Professor, Tuva State University. TYULUSH MARTA KAN-OOLOVNA, Candidate of Pedagogical Sciences, Associate Professor, Tuva State University. В данной статье рассматривается проблема внедрения образовательной робототехники в систему дополнительного образования учащихся, как средство развития творческой активности у школьников. Представлено определение детского технического творчества и его основные харак- терные черты. Выяснено, что робототехника в системе дополнительного образования учащихся. Робототехника в системе дополнительного образования служит целям овладения навыками началь- ного технического конструирования, развития мелкой моторики, изучения основных технических понятий и терминов, знакомства с программированием, инструментами и материалами. Сделан вывод, что работа с образовательными конструкторами Arduino даёт возможность школьникам узнавать новое из мира техники и технологии и формировать необходимые в жизни навыки. This article discusses the problem of introducing educational robotics into the system of additional education of students as a means of developing creative activity among schoolchildren. The definition of children's technical creativity and its main characteristic features are presented. It was found out that robot- ics in the system of additional education of students. Robotics in the system of additional education serves the purposes of mastering the skills of initial technical design, the development of fine motor skills, the study of basic technical concepts and terms, familiarity with programming, tools and materials. It is concluded http://nauko-sfera.ru/ ISSN 2542-0402 150 Наукосфера. №1 (1), 2023 Педагогические науки that working with Arduino educational designers gives students the opportunity to learn new things from the world of technology and technology and form the necessary skills in life. that working with Arduino educational designers gives students the opportunity to learn new things from the world of technology and technology and form the necessary skills in life. Ключевые слова: робототехника, техническое творчество, конструирование, дополнительное образование, программирование, цифровые, конструктор ARDUINO. Key words: robotics, technical creativity, design, additional education, programming, digital, ARDUINO designer. Н а сегодняшний день важными приоритетами государственной политики в сфере об- разования становится поддержка и развитие детского технического творчества, при- влечение школьников в научно-техническую сферу профессиональной деятельности и повышение престижа научно-технических профессий. Развитие современного общества неразрывно связано с научно-техническим прогрессом. Информационно-коммуникационные и инженерные технологии становятся частью образовательной деятельности, значительно повышающей ее эффективность и способствующей развитию интеллектуальной, эмоцио- нальной и личностной сфер обучающихся. Формируется благоприятная среда для развития инновационного направления технического творчества – робототехники. Идея развития творческих способностей и совершенствование технической подготовки подрастающего по- коления приобретают государственное значение. ONDAR EDUARD EMIROVICH, master's student, Tuva State University. KUULAR DOLAANA ORLAN-OOLOVNA Candidate of Pedagogical Sciences, Associate Professor, Tuva State University. TYULUSH MARTA KAN-OOLOVNA, Candidate of Pedagogical Sciences, Associate Professor, Tuva State University. В настоящее время, когда осуществляется образовательные проекты на техническое творчество обучающихся, перед образовательными организациями стоит задача модернизации и расширения деятельности по развитию научно- технического творчества школьников. Н Необходимость передачи технических знаний из поколения в поколение привела людей к мысли об обучении детей и молодежи техническому творчеству и изобретательству. Одним из приоритетных направлений развития технического творчества является внедрение образо- вательной робототехники в систему дополнительного образования учащихся как средства формирования комплексных знаний, способствующих развитию системности мышления де- тей, возрождению научно-технического творчества, повышению интереса к инженерному образованию. Рабочий курс по робототехнике в школе должен быть нацелен на формирова- ние научного мировоззрения, освоение методов научного познания мира, развитие исследо- вательских, прикладных, конструкторских способностей обучающихся, с наклонностями в области точных наук и технического творчества. у р Изучение литературы по теме диссертационного исследования привело нас к нахожде- нию полного и единого определения понятия «детское техническое творчество». Каждый из авторов в содержание этого понятия вкладывает свой смысл. Их взгляд на термин «детское техническое творчество» представляет огромную ценность для исследования. Исходя из это- го, мы сочли необходимым рассмотреть определения для детского технического творчества, выдвинутые педагогами и психологами как отечественными, так и зарубежными (табл. 1). Таким образом, творчество является такой деятельностью, посредством которой чело- век изменяет обычную действительность по своей воле и желанию в соответствии со своими потребностями и запросами. Сама творческая деятельность может начинаться с небольших элементов, которые вызываются, прежде всего, необходимостью преодолеть затруднения, возникшие в процессе труда, когда у человека появляется желание или потребность «сделать по-своему». Это и есть первоначальный элемент творческой деятельности. Через творческую деятельность, удовлетворяя запросы общества, человек утверждается как неповторимая це- лостная личность и индивидуальность. http://nauko-sfera.ru/ ISSN 2542-0402 151 Наукосфера. №1 (1), 2023 Наукосфера. №1 (1), 2023 Педагогические науки Таблица 1. Определение детского технического творчества или основные характерные черты Фамилия ученого Определение детского технического творчества или основные характерные черты П.Н. Андрианов и М.А. Галагузова «Деятельность учащихся в области техники, когда они в процес- се работы что-то изменяют, дополняют, комбинируют, вносят «йоту нового», представляет не что иное как техническое твор- чество младших школьников» [1, c.31]. А.А. Бытев Понимал под техническим творчеством деятельность детей, при которой они самостоятельно конструируют различные модели, приборы и установки, вносят новое в содержание выполняемых заданий, облегчая изготовление и улучшая работу моделей [2]. П.Н.Андрианов, И.И.Бака, В.А.Горский, И.Г.Розанов В качестве основных критериев детского технического творче- ства при создании технических объектов они выделяют элемен- ты полезной новизны [1, 3]. В.П. Брагин Настаивал на необходимости внеклассной работы по развитию умений юных техников. ONDAR EDUARD EMIROVICH, master's student, Tuva State University. KUULAR DOLAANA ORLAN-OOLOVNA Candidate of Pedagogical Sciences, Associate Professor, Tuva State University. TYULUSH MARTA KAN-OOLOVNA, Candidate of Pedagogical Sciences, Associate Professor, Tuva State University. Утверждал, что практическая состав- ляющая процесса является неотъемлемой частью развития дет- ского изобретательства и политехнизации обучения [4]. От года к году всё чаще и шире мы встречаемся на работе и вне её с роботами и робо- тизированными комплексами. Неслучайно во внеурочную деятельность образовательных уч- реждений введён такой предмет, как робототехника. Что же такое робототехника? Робото- техника – это одно из передовых современных направлений науки и техники, прикладная наука, предметом которой является разработка автоматизированных технических систем и являющаяся одной из важнейших технических основ развития производства и всех сторон жизни общества. Робототехника базируется на таких дисциплинах, как электроника, механи- ка, кибернетика, мехатроника, информатика, радиотехника и электротехника. А робототех- ника в образовании робототехника в образовании – это новое направление обучения подрас- тающего поколения физике, технологии, математике, информатике, вовлекающее его в про- цесс технического творчества. В современном мире ускоряющимися темпами идёт активное внедрение роботов в повседневную жизнь людей, во многих профессиях людей заменяют роботы. Специалисты в области робототехники достаточно востребованы экономикой, и сте- пень этой востребованности будет только возрастать. В ежегодном послании Федеральному Собранию в декабре 2012 года президент Рос- сийской Федерации Владимир Владимирович Путин поставил задачу, что «нужно развивать систему технического и художественного творчества, открывать кружки, секции для детей» [1]. На решение этих задач была направлена программы по созданию центров научно- технической направленности, которая стартовала в России и реализуется по настоящее вре- мя. Важным фактором развития творческих способностей личности выступает современная система дополнительного образования детей, основным компонентом которой является дет- ское техническое творчество. В образовательных учреждениях система дополнительного об- разования нашла свое воплощение в активном развитии кружков, внеклассных занятий тех- нической направленности, в частности, кружков робототехники. Робототехника служит для проявления креативных способностей учащихся. Творче- ский вид робототехники, характеризуется стремлением к созданию новых роботов, которые могут найти практическое применение в нашей жизни. Таким образом, можно сделать вы- вод, что «творческая робототехника – качественно новый уровень деятельности ребенка, ISSN 2542-0402 http://nauko-sfera.ru/ ISSN 2542-0402 152 Наукосфера. №1 (1), 2023 Педагогические науки предполагающий наличие базовых и продвинутых знаний в этой области». [2] Образователь- ная робототехника предназначена для формирования базовых знаний и умений в области конструирования и программирования роботов. Это позволяет объединить вместе препода- вание черчения, физики, информатики, математики, других естественных наук и развивать инженерно-техническое мышление. р Для успешного овладения робототехникой необходимо знать современные конструк- ционные материалы, уметь их обрабатывать различными инструментами, владеть основами программирования. ONDAR EDUARD EMIROVICH, master's student, Tuva State University. KUULAR DOLAANA ORLAN-OOLOVNA Candidate of Pedagogical Sciences, Associate Professor, Tuva State University. TYULUSH MARTA KAN-OOLOVNA, Candidate of Pedagogical Sciences, Associate Professor, Tuva State University. №1 (1), 2023 Педагогические науки - познакомить учащихся с основами проектирования и конструирования в среде Arduino; - познакомить учащихся с основами проектирования и конструирования в среде Arduino; накомить учащихся с основами программирования в компьютерных средах, пр ных Arduino и на языках программирования роботов; - познакомить учащихся с основами программирования в компьютерн доставленных Arduino и на языках программирования роботов; - сформировать умения творчески подходить к решению технических задач ф р р у р р - сформировать стремление доводить конструкцию до работающей модели; - развить умение излагать свои идеи в чёткой логической последовательности, отстаи- вать свою точку зрения, анализировать складывающуюся ситуацию и самостоятельно нахо- дить правильные решения путём логических рассуждений. Формы занятий, которые должны использоваться в процессе обучения робототехнике в школе: лекции, самостоятельная работа, проектная деятельность, соревнования. По завершении изучения курса робототехники учащийся должен: р у ур р у • знать основы механики, автоматики и программирования в компьютерных средах и на языках программирования роботов; • уметь собирать модели как используя готовую схему сборки, так и по эскизу; • уметь собирать модели как используя готовую схему сборки, так и по эскизу; б б б й • уметь разрабатывать собственные проекты по созданию роботов для раз • уметь разрабатывать собственные проекты по созданию роботов для различных целей. Виды и формы контроля результатов изучения дополнительной развивающей програм- мы по робототехнике в учреждении дополнительного образования: Виды и формы контроля результатов изучения дополнительной развивающей програм- мы по робототехнике в учреждении дополнительного образования: • индивидуальные задания; • контрольные задания; • личные творческие проекты; • участие в соревнованиях и состязательных мероприятиях различного уровня. • участие в соревнованиях и состяз • участие в соревнованиях и состязательных мероприятиях различного ур Текущий контроль целесообразно проводить в форме внутренних соревнований в са- мом учреждении или выставки роботов, оцениваемых компетентным независимым жюри по технологическим картам. Итоговый контроль по окончании учебного года целесообразно проводить в виде со- ревнований роботов на последнем занятии. Соревнования должны включать в себя всю це- почку создания робота: проектирование, конструирование, создание и программирование робота, способного выполнить определённые задания. Использование Arduino конструирования в системе дополнительного образования слу- жит целями овладения навыками начального технического конструирования, развития мел- кой моторики, изучения основных технических понятий и терминов, а также знакомство с программированием. Работа с образовательными конструкторами Arduino даёт возможность школьникам уз- навать новое из мира техники и технологии и формировать необходимые в жизни навыки. ONDAR EDUARD EMIROVICH, master's student, Tuva State University. KUULAR DOLAANA ORLAN-OOLOVNA Candidate of Pedagogical Sciences, Associate Professor, Tuva State University. TYULUSH MARTA KAN-OOLOVNA, Candidate of Pedagogical Sciences, Associate Professor, Tuva State University. Конструкторы данной фирмы вырабатывают у обучающихся целостное представление о ми- ре техники, устройстве конструкций, механизмов и машин и месте их в современной жизни, позволяют расширить и углубить технические знания и навыки обучающихся, активизиро- вать интерес к техническому творчеству, выработать умение исследовать стоящую задачу, анализировать имеющиеся ресурсы и выдвигать идеи. ONDAR EDUARD EMIROVICH, master's student, Tuva State University. KUULAR DOLAANA ORLAN-OOLOVNA Candidate of Pedagogical Sciences, Associate Professor, Tuva State University. TYULUSH MARTA KAN-OOLOVNA, Candidate of Pedagogical Sciences, Associate Professor, Tuva State University. Курс робототехники в системе дополнительного образования может стать одним из способов изучения не только техники, производственных технологий, компьютерных техно- логий и программирования, но и всего окружающего мира с точки зрения сферы применения роботов. В последнее время любительская робототехника переживает настоящий интерес. Поэтому рынок насыщен всякого рода готовыми конструкторами. Они включают в себя множество датчиков, приборов и устройств, необходимых для создания робота. Но главный минус данных наборов в том, что они дорогие и не каждому доступны. При преподавании данного курса педагоги дополнительного образования сталкиваются с двумя основными проблемами: недостатком методических материалов и высокой ценой одной единицы робототехнического конструктора и дополнительных комплектующих к не- му. На данный момент в образовании, как в основном, так и дополнительном, чаще всего применяют различные готовые комплексы для робототехники, например, Arduino. Платфор- ма Arduino представляет собой комбинацию среды быстрой разработки Arduino IDE и моду- лей для прототипирования на базе микроконтроллеров. Фактически, Arduino это – простей- ший электронный конструктор для создания готовых устройств из отдельных модулей. Arduino пользуется огромной популярностью во всем мире из-за контроллеров, модулей и шилдов. [5] Arduino – это не просто универсальный микроконтроллер, который можно адаптиро- вать под любой проект за минимальное время. Это простейшая и доступная для изучения среда разработки Arduino IDE, которая служит для вовлечения в программирование и робо- тотехнику, а также для быстрой разработки проектов. [5] Базовый стартовый набор Arduino - комплект RFID с ЖК-дисплеем 1602, шаговый дви- гатель, Обучающий набор для начинающих с розничной коробкой, набор для электронных компонентов для Arduino UNO R3. Образовательный комплект для учеников школ в возрасте от 10 лет, который ознакомит с роботостроением, а также даёт понимание таких наук как фи- зика, математика, информатика и основы конструирования. Наличие большого количества деталей и элементов позволяет создавать базовые конст- рукции (примерно 21 проектов), а также дает возможность создать свою модель. Тем самым ученик станет с большим интересом заниматься конструированием, развивать нужные тех- нические навыки. Таким образом, учащиеся работают над проектом из готовых Arduino компонентов и модулей, программируют его для поставленной задачи. Стоимость одного Arduino набора держится в пределе 2000-4000 рублей. Более сложные наборы для старшей школы ещё до- роже, примерно 8000-10000 рублей. На занятиях в форме познавательного проекта работа с конструкторами Arduino позво- ляет научить генерировать интересные идеи и развивает необходимые в дальнейшей жизни практические навыки. При реализации программ по робототехнике в дополнительном образовании решаются следующие педагогические задачи: http://nauko-sfera.ru/ ISSN 2542-0402 153 Наукосфера. 1. Андрианов П.Н. Развитие технического творчества младших школьников // Андрианов П.Н., Галагузова М.А., Каюкова Л.А., Нестерова Н.А., Фетцер В.В. Москва: Просвещение, 1990. 110 с. 2. Бытев А.А. Фотопроекционный кружок в школе. Минск: Народная асвета, 1968. 112 с. 3. Бака И.И. Теоретические основы подготовки школьников к творческому труду в сфере ма- териального производства. М: МГПИ им. В.И. Ленина,1985. 133 с. 4. Брагин В.П. Техническое творчество: Пособие для руководителей технических кружков / В.П. Брагин, Н.П. Булатов, В.Г. Гаршенин, П.С. Павлов, Б.М. Сметанин, Н.Е. Цейтлин, В.П. Шафе- ров. Москва «Молодая Гвардия» 1956. 526 с. р р 5. Наборы и конструкторы Ардуино для начинающих // Российское Ардуино – сообщество. 2022. URL: https://arduinomaster.ru/arduino-kit/rus-konstruktory-arduino-dlya-detej (дата обращения 07.01.2023). http://nauko-sfera.ru/ 4. Брагин В.П. Техническое творчество: Пособие для руководителей технических кружков / В.П. Брагин, Н.П. Булатов, В.Г. Гаршенин, П.С. Павлов, Б.М. Сметанин, Н.Е. Цейтлин, В.П. Шафе- ров. Москва «Молодая Гвардия» 1956. 526 с. 5. Наборы и конструкторы Ардуино для начинающих // Российское Ардуино – сообщество. 2022. URL: https://arduinomaster.ru/arduino-kit/rus-konstruktory-arduino-dlya-detej (дата обращения 07.01.2023). СПИСОК ЛИТЕРАТУРЫ у , , р , р р , 2. Бытев А.А. Фотопроекционный кружок в школе. Минск: Народная асвета, 1968 http://nauko-sfera.ru/ ISSN 2542-0402 154 Наукосфера. №1 (1), 2023 Педагогические науки р р 5. Наборы и конструкторы Ардуино для начинающих // Российское Ардуино – сообщество. 2022. URL: https://arduinomaster.ru/arduino-kit/rus-konstruktory-arduino-dlya-detej (дата обращения 07.01.2023). © Ондар Э.Э., Куулар Д.О., Тюлюш М.К., 2023. http://nauko-sfera.ru/ ISSN 2542-0402 155
https://openalex.org/W2899408454	https://link.springer.com/content/pdf/10.1007%2F978-3-319-99948-7_5.pdf	English	null	Ebb and Flow: Breath-Writing from Ancient Rhetoric to Jack Kerouac and Allen Ginsberg	Palgrave studies in literature, science and medicine	2,018	cc-by	10,256	CHAPTER 5 Ebb and Flow: Breath-Writing from Ancient Rhetoric to Jack Kerouac and Allen Ginsberg Stefanie Heine © The Author(s) 2019 A. Rose et al., Reading Breath in Literature, Palgrave Studies in Literature, Science and Medicine, https://doi.org/10.1007/978-3-319-99948-7_5 91 © The Author(s) 2019 A. Rose et al., Reading Breath in Literature, Palgrave Studies in Literature, Science and Medicine, https://doi.org/10.1007/978-3-319-99948-7_5 Stefanie Heine Abstract  Following the path of Charles Olson, Jack Kerouac and Allen Ginsberg negotiate breath as a compositional principle for a new particu- larly American literature. Such a poetics of breathing turns out to be a revival of classical thought. For ancient rhetoricians, especially Aristotle, Cicero and Quintilian, the breath-pause is constitutive for structuring speech. Already in the ancient approaches, a dilemma emerges: breathing is supposed to cut speech into well-measured units while physical respira- tion tends to be irregular. Even though the Beat poets seem to elude this problem in their attempt to adapt composition to the writer’s individual rhythms, breath, as they theorise it, is a point where bodily processes and cultural techniques intersect. The natural, organic body as Kerouac and Ginsberg celebrate it invokes a cultural memory, and thus, the idea of a purely embodied writing is upset. Keywords  Breath · Embodied poetics · Jack Kerouac · Allen Ginsberg · Ancient rhetoric Verse now, 1950, if it is to go ahead, if it is to be of essential use, must, I take it, catch up and put into itself certain laws and possibilities of the breath, of the breathing of the man who writes as well as of his listenings.1 91 © The Author(s) 2019 A. Rose et al., Reading Breath in Literature, Palgrave Studies in Literature, Science and Medicine, https://doi.org/10.1007/978-3-319-99948-7_5 91 92 S. HEINE The opening claim of Charles Olson’s influential essay “Projective Verse,” already touched upon in the introduction of this book, responds to a set of questions that would occupy two circles of avant-garde writers in the 1950s and 1960s, the Black Mountain poets and the Beat movement: How can a new literature that radically breaks with tradition be inaugu- rated? What basis can it have, if not tradition? “The laws and possibilities of the breath,” a recourse to “natural” bodily processes, promises freer expression and an emancipation of American poetry from old, formal conventions. Liberating language from the shackles of fossilised, dusty rules of metre and rhyme will vivify and renew it, while transferring the author’s breathing rhythm to that of the words written will produce an organic, embodied literature that reconciles art and life. In his discus- sion of breath, Olson refers to the “revolution of the ear,”2 pointing to a revival of orality in American poetry starting from Walt Whitman and extending to Ezra Pound and William Carlos Williams. Stefanie Heine His claims that “breath allows all the speech-force of language back in” and “speech is … the secret of a poem’s energy”3 could be read as a call for spoken liter- ature, for words carried by physical breath, which are more lively than those “which print bred.”4 For a number of writers of both the Beat and Black Mountain con- text, “speech-force” was not only to be realised in oral performances, but should also affect the words in the composition process, in which breath would function as a measure that is “arriv[ed] at … organically.”5 Olson, like Allen Ginsberg,6 establishes a simple compositional principle: break the line when you run out of breath: And the line comes (I swear it) from the breath, from the breathing of the man who writes, at the moment that he writes, … for only he, the man who writes, can declare, at every moment, the line its metric and its ending—where its breathing, shall come to, termination.7 Similarly, Jack Kerouac proposes that a dash shall indicate the moment between inhalation and exhalation, when breath is drawn, replacing the commas and colons that more commonly separate grammatical and semantic units.8 In these approaches, “preconceived metrical pattern[s]” are counteracted with more irregular, variable and individual structures derived from “a source deeper than the mind … the breathing and the belly and the lungs.”9 Similarly, Jack Kerouac proposes that a dash shall indicate the moment between inhalation and exhalation, when breath is drawn, replacing the commas and colons that more commonly separate grammatical and semantic units.8 In these approaches, “preconceived metrical pattern[s]” are counteracted with more irregular, variable and individual structures derived from “a source deeper than the mind … the breathing and the belly and the lungs.”9 5 EBB AND FLOW: BREATH-WRITING FROM ANCIENT RHETORIC … 93 Ancient Origins of the Breath-Stop What was advocated as a fresh principle for a new literature in the essays, writing manuals and oral comments of the Beat and Black Mountain writers was actually a tacit renascence of classical thought. In ancient rhetoric, the importance of breathing as a bodily prerequisite for oral delivery and as a structuring element of speech was stressed by Aristotle, Cicero and Quintilian. Breath had a pivotal role in the creation of prose rhythm, which the rhetoricians considered as more loosely meas- ured than poetry. Prose should be structured in sequences, for example “periods,” which Aristotle defines as “sentence[s] that [have] a begin- ning and an end in [themselves].”10 In line with the compositional ideas of the Beat and Black Mountain writers, for the rhetoricians breathing marks the intervals between structural sequences. Aristotle mentions that a period should be delivered “in a breath … taken as a whole”11 and Cicero asserts that “there should be in speeches closes [of periods] where we may take breath.”12i The period in ancient rhetoric is a clearly defined unit: a segment that represents a thought with a beginning and an end. This idea is taken up by Ginsberg and Kerouac. Ginsberg claims that the “[b]reath-stop and the thought-division could be the same,”13 and Kerouac observes that a jazz musician blows “a phrase on his saxophone till he runs out of breath, and when he does, his sentence, his statement’s been made … . That’s how I therefore separate my sentences, as breath separations of the mind.”14 With the assumption that a unit of breath coincides with a unit of thought or a completed statement, Kerouac and Ginsberg con- sciously or unconsciously follow the rhetoricians.15 What Kerouac and Ginsberg designate as a poetics of the body meets an old matter of con- troversy around the sound execution of artistic composition and some- times unpredictable physical needs. The question arising for the ancient rhetoricians, Kerouac and Ginsberg, is: How does the necessity of draw- ing a breath while speaking undercut claims to a synchronicity of breath- ing and thinking?16l The period in ancient rhetoric is a clearly defined unit: a segment that represents a thought with a beginning and an end. This idea is taken up by Ginsberg and Kerouac. Ancient Origins of the Breath-Stop Ginsberg claims that the “[b]reath-stop and the thought-division could be the same,”13 and Kerouac observes that a jazz musician blows “a phrase on his saxophone till he runs out of breath, and when he does, his sentence, his statement’s been made … . , , , That’s how I therefore separate my sentences, as breath separations of the mind.”14 With the assumption that a unit of breath coincides with a unit of thought or a completed statement, Kerouac and Ginsberg con- sciously or unconsciously follow the rhetoricians.15 What Kerouac and Ginsberg designate as a poetics of the body meets an old matter of con- troversy around the sound execution of artistic composition and some- times unpredictable physical needs. The question arising for the ancient rhetoricians, Kerouac and Ginsberg, is: How does the necessity of draw- ing a breath while speaking undercut claims to a synchronicity of breath- ing and thinking?16l The reflections of the rhetoricians indicate that a seamless coincidence of sense and breath units cannot be taken for granted.17 In Quintilian’s detailed account of how a speech should be delivered orally, it becomes obvious that an exact concurrence of breathing pause and the comple- tion of a period are only an aspirational ideal.18 The rhetoricians gener- ally argue that the completion of a period should determine the moment 94 S. HEINE when a breath is drawn, and not the other way round. Cicero stresses that only the “unskilful and ignorant speaker … measures out the peri- ods of his speech, not with art, but with the power of his breath.”19 He argues that the breathing pause should be motivated by coherent seg- ments of speech rather than the bodily need to inhale: “there should be in speeches closes [of periods] where we may take breath not when we are exhausted, … but by the rhythm of language and thoughts.”20 Quintilian notes that the orators can train their breath through physical exercise in order to make it more amenable to the need to mark a period: “we ought to exercise it [the breath, or breathing], that it may hold out as long as possible.”21 h d b d ll In this respect, Kerouac’s and Ginsberg’s position is diametrically opposite: the physical need to draw a breath shall determine the interval between thoughts and constitute the structural unit. Ancient Origins of the Breath-Stop What the ancient rhetoricians have in mind is a scenario of oral composition: the orator composes his sentences as he speaks. In contrast, Ginsberg and Kerouac primarily composed in writing: by hand or with a typewriter. When the writer “pronounces” the words in his head while writing, a need to inhale does not necessarily coincide with the moment where a breathing pause would have occurred if the same sentence were spoken. In fact, we may place many more words in the span of one breath if we pronounce them in our head than if we pronounce them orally.26 In contrast to oral composition, in writing, composition is not inevitably affected by the necessity to draw a breath: while writing, one can inhale without this effecting a pause in the sentence put on paper. When breath-measure is applied to written composition, its organic foundations disappear. Concerning Kerouac’s and Ginsberg’s texts, one observation is obvious: the pause markers almost always seamlessly coin- cide with grammatical units—so either the “laws … of the breath” were ignored in the actual writing process, or they do not structure speech differently to standard grammatical units. Moreover, if a healthy body also “unconsciously” follows the control of the mind to such a degree that breathing adjusts itself to anticipated syntactic breaks, the “laws of the breath” may actually (and unintentionally) be the “laws of the mind” rather than “a source deeper than the mind.”27 segments, the “mind-breaks” or “thought-divisions” in Ginsberg’s case, and the “phrases,” “sentences” or “statements” in Kerouac’s case actu- ally consist in.24 Whether the two writers actually did break up their lines or sentences when they had to inhale is impossible to verify in written documents. While one can check drafts and manuscripts for where line- breaks are made and where dashes or other pause markers are inserted, this textual geneticism does not demonstrate Kerouac’s and Ginsberg’s actual breathing patterns.25 Moreover, their poetics of breath rests on collapsing a fundamental difference between oral and written compo- sition. What the ancient rhetoricians have in mind is a scenario of oral composition: the orator composes his sentences as he speaks. In contrast, Ginsberg and Kerouac primarily composed in writing: by hand or with a typewriter. Ancient Origins of the Breath-Stop To repeat, Ginsberg claims that the measure of the breath-stop is “arriv[ed] at … organically” and rhythmical structures come from “a source deeper than the mind … the breathing and the belly and the lungs.” Kerouac stresses that he separates his phrases when he “draw[s] a breath”22 like the saxophon- ist does when “he runs out of breath.”23 However, their commitment to what Cicero designates as rude oratory does not resolve the tension between the physical necessity to inhale and the breathing pause as a structuring principle of speech already present in antiquity. The units of thoughts and statements addressed by Kerouac and Ginsberg under- mine their claim of a compositional principle solely generated from the body. In the reference to the coincidence of breathing and structural units, the “nature” of their compositional theories as a cultural inher- itance becomes obvious; the unaddressed yet distinctly audible reso- nances with ancient rhetoric alone unsettle the idea of an art that comes to be in a fully organic manner. In the context of their writings, breath does not only refer to the body “of the man who writes,” but also back to a rhetorike techne in which they are engaged. What is proposed as a means to approach a reconciliation of art and life in fact turns out to be a discursive vitalism pointing to an older discourse and cultural tech- nique in which a seamless coincidence of body and artistic composition has already been challenged. Against the background of this incongruity, this chapter traces the contradictions of Ginsberg’s and Kerouac’s notions of a vital, bod- ily breath-writing. In the comments about their writing process, nei- ther Ginsberg nor Kerouac give a clear definition of what the proposed 5 EBB AND FLOW: BREATH-WRITING FROM ANCIENT RHETORIC … 95 segments, the “mind-breaks” or “thought-divisions” in Ginsberg’s case, and the “phrases,” “sentences” or “statements” in Kerouac’s case actu- ally consist in.24 Whether the two writers actually did break up their lines or sentences when they had to inhale is impossible to verify in written documents. While one can check drafts and manuscripts for where line- breaks are made and where dashes or other pause markers are inserted, this textual geneticism does not demonstrate Kerouac’s and Ginsberg’s actual breathing patterns.25 Moreover, their poetics of breath rests on collapsing a fundamental difference between oral and written compo- sition. Ancient Origins of the Breath-Stop Not only does he rarely use the stop and start buttons during composition, but the pauses in the recordings do not always coincide with the line-breaks in the printed poems. In most cases, it is unlikely that the pauses mark moments where Ginsberg ran out of breath; they rather indicate points where he ran out of thought: often, he only speaks two or three words, followed by very long intervals during which numerous breaths can be taken, often punctured by interjections like “ahem.” Consequently, when Ginsberg designates the “natural” end of a line as “breath-stop” in retrospect, he uses the term as an image for the mind-break, or as a name for the line-break in the written text (note that in the lecture, he comes up with breath in the context of transcribing the spoken poem), which has little to do with his actual breathing during composition. An investigation of the tapes archived at Stanford University shows that what Ginsberg presents here is indeed a theory—a theory that does not match his compositional practice. Not only does he rarely use the stop and start buttons during composition, but the pauses in the recordings do not always coincide with the line-breaks in the printed poems. In most cases, it is unlikely that the pauses mark moments where Ginsberg ran out of breath; they rather indicate points where he ran out of thought: often, he only speaks two or three words, followed by very long intervals during which numerous breaths can be taken, often punctured by interjections like “ahem.” Consequently, when Ginsberg designates the “natural” end of a line as “breath-stop” in retrospect, he uses the term as an image for the mind-break, or as a name for the line-break in the written text (note that in the lecture, he comes up with breath in the context of transcribing the spoken poem), which has little to do with his actual breathing during composition. Following these observations, it has to be stressed that Kerouac’s and Ginsberg’s reflections of breathing and writing are poetological theo- ries rather than descriptions of actual composition processes. While it is worth considering these in their own right, it is important to be aware of the ambivalent position bodily breath thereby comes to occupy: while it is celebrated as the natural source of a literary text’s structure, its actual role in the writers’ compositional practices seems to be marginal. Ancient Origins of the Breath-Stop When the writer “pronounces” the words in his head while writing, a need to inhale does not necessarily coincide with the moment where a breathing pause would have occurred if the same sentence were spoken. In fact, we may place many more words in the span of one breath if we pronounce them in our head than if we pronounce them orally.26 In contrast to oral composition, in writing, composition is not inevitably affected by the necessity to draw a breath: while writing, one can inhale without this effecting a pause in the sentence put on paper. When breath-measure is applied to written composition, its organic foundations disappear. Concerning Kerouac’s and Ginsberg’s texts, one observation is obvious: the pause markers almost always seamlessly coin- cide with grammatical units—so either the “laws … of the breath” were ignored in the actual writing process, or they do not structure speech differently to standard grammatical units. Moreover, if a healthy body also “unconsciously” follows the control of the mind to such a degree that breathing adjusts itself to anticipated syntactic breaks, the “laws of the breath” may actually (and unintentionally) be the “laws of the mind” rather than “a source deeper than the mind.”27 The only documented cases where Ginsberg adopted an oral composi- tional technique are his so-called auto-poesy tapes. In a lecture, Ginsberg later explicates his recording compositions in terms of his theory of the mind- and breath-stop: most machines have a “stop” and a “start” button …, so if you’re actually intending to do writing, one way is to use the automatic “control” button as the margin of your line … . That is, you’re talking into the machine, you don’t have anything to say, so you click it off. Then, when something 96 S. HEINE emerges, when you notice something … – click! Then, when you’re tran- scribing on a page, … which I’ve done a lot, from ’65 to’70, with a Uher machine, you can use the “click” at the end of the tape line, the tape oper- ation, as your breath stop. … [I]t’s the natural end of the line.28 An investigation of the tapes archived at Stanford University shows that what Ginsberg presents here is indeed a theory—a theory that does not match his compositional practice. Ancient Origins of the Breath-Stop Bearing this ambivalence in mind, I will elucidate the particularities of Kerouac’s and Ginsberg’s poetics of breathing, whose fixation on vitalism turns out to be grounded more in discourse than in physiology. The trajectories of their respective poetological endeavours become explicit when counter- pointed against theories of rhetorical composition. Thus, I want to pair Ginsberg with Quintilian and Kerouac with Aristotle, focusing especially on the character and function of the caesura. Ginsberg and Quintilian Ginsberg claims that the so-called natural speech pauses, which he iden- tifies with “breath-stops,” “indicate mind-breaks.”29 The “[b]reath stop is where you stop the phrase to breathe again. Stop to think and 97 5 EBB AND FLOW: BREATH-WRITING FROM ANCIENT RHETORIC … breathe.”30 By claiming that “you’re gonna stop and take a breath” when “you run out of thought and words,”31 he recalls Quintilian, who argues that the pause is the “point, where the mind takes a breath and recovers its energy.”32 For Quintilian, the breathing pause is the moment “when the rush of words comes to a halt”33 and the mind is relieved from its work. The pause should provide a rest so that the orators can assemble their mental forces anew before the next compositional effort. When claiming that the mind takes a breath, Quintilian deploys a met- aphor invoking the intake of vital breath.34 He addresses the “rush of the words” the pause interrupts and thus recalls a common associa- tion tied to the metaphor of “taking a breath” in the sense of relaxing: slowing down, i.e. the pace of one’s breathing rhythm. To do nothing except breathe seems to suggest that one does almost nothing: “taking a breath” is “pausing.” The image of the mind taking a breath during the pause implicates that the mind stops doing what it usually does, namely thinking. By claiming that the mind takes a breath in the moment of the breathing pause, Quintilian rhetorically establishes a temporal coin- cidence of metaphorical breath and its literal, or, precisely speaking non-linguistic, bodily referent. In his remarks on the breathing pause and writing, Ginsberg also tries to reconcile metaphorical and literal dimensions of breathing. In the sen- tence “when you talk and then after a while you run out of thought and words, … then you’re going to stop and take a breath and continue,” Ginsberg synchronises the metaphor of “taking a breath”35 with phys- ical inhalation. Like Quintilian, he suggests that the breathing pause between uttered words (literally taking a breath) is a moment of rest and recovery (metaphorically taking a breath)—and that the mind needs to rest when the speaker runs “out of thought.” Ginsberg also addresses the other implications of “taking a breath,” discussed in Quintilian’s use of the metaphor: inhaling vitalising air and doing almost nothing. Ginsberg and Quintilian He states that during the pause, the writer is “waiting for the next thought to articulate itself.”36 By noting “you’re improvising and you’re relying on the moment-to-moment inspiration,”37 Ginsberg suggests that phys- ical inspiration, inhaling, coincides with inspiration in the classical sense: the generation of creative ideas. The metaphorical breath of life as a vital- ising force is thus transferred to the domain of artistic work in process. Drawing on his preoccupation with Buddhist thought and meditation practices, Ginsberg considers it relevant that ideas are generated where nothing is written or thought. The “blank spots,” or “gaps in between the thoughts,”38 Ginsberg addresses in this context overlap exactly with 98 S. HEINE the point where he locates the breath-stop. Out of the “unborn aware- ness,”39 a space of pure potentiality that opens in the moment when we do nothing but breathe, new thoughts are generated. The conflation of the physiological process of breathing, that is, the so-called natural pause or breath-stop and the mind-break, with the emergence of new ideas, that is, inspiration, becomes most noticeable in his “Notes on Howl”: “Ideally each line of Howl is a single breath unit … —that’s the Measure, one physical-mental inspiration of thought contained in the elastic of a breath.”40 Even though Ginsberg encourages his readers to take both the met- aphor of “taking a breath” and the notion of inspiration literally, his theory pushes physical respiration into the background. The claim that breath is a “source deeper than the mind” is made plausible in Ginsberg’s comments on thought-generating “unborn awareness.” However, rec- onciling breathing and inspiration in this way does not explain why the end of a thought should coincide with the need to draw a breath. The neat outline of “breath-stop = mind-break = inspiration” is an attempt to bring the body into agreement with compositional techniques, tra- ditional ideas about how creative works are generated and theories of thought processes. Such a carefully constructed model—clearly a work of a well-read mind—stands in conflict with the claim that the work of the respiratory organs, which proceeds according to its own mechanisms, is supposed to generate the rhythmical structures of the poem in process. Ginsberg and Quintilian The fact that breathing rhythms are influenced by accidental external cir- cumstances and the respective bodily condition of the breather—which, quite surprisingly for a position that supposedly foregrounds the body, is never addressed by Ginsberg—counteracts the idea that “mind-breaks” should necessarily be “identical with natural speech pauses.”41 On the one hand, it is precisely the irregularity of breathing that makes it inter- esting for Ginsberg’s polemic towards a new poetry: he stresses that, in contrast to the “automatic and mechanic,” symmetrical and “even” measure of traditional metrical forms, poetry as he envisages it, “speech as breath from the body,” is more variably structured.42 On the other hand, the irregularities of a human’s breathing rhythm run counter to the smooth symmetry Ginsberg establishes in his compositional the- ory. Ginsberg considers the work of the mind as a process which is at the same time bodily and intellectual.43 His negotiations of breath and mind-breaks thus challenge a simple binary between a rational, intellec- tual mind and an irrational, animalistic body. However, the cost of this 5 EBB AND FLOW: BREATH-WRITING FROM ANCIENT RHETORIC … 99 by all means productive questioning of a dualism that keeps haunting the Western world is an eradication of difference: Ginsberg seals the gap between mind and body that especially articulates itself when the body speaks, or to be precise, breathes. He claims that mind-breaks are the same as non-metaphorical breath-stops, that is, the pauses between inha- lation and exhalation in the physical respiration process. Kerouac and Aristotle Kerouac first and foremost links breathing to the free mind-flow and uncensored expression: PROCEDURE … sketching language is undisturbed flow from the mind of personal secret idea-words, blowing (as per jazz musician) on subject of image. SCOPING Not “selectivity” of expression but following free deviation (association) of mind into limitless blow-on-subject seas of thought, swim- ming in sea of English with no discipline other than rhythms of rhetorical exhalation and expostulated statement … —Blow as deep as you want— write as deeply, fish as far down as you want.44 CENTER OF INTEREST … blow!—now!—your way is your only way— “good”—or “bad”—always honest (“ludicrous”), spontaneous, “confes- sional” interesting, because not “crafted.”45 CENTER OF INTEREST … blow!—now!—your way is your only way— “good”—or “bad”—always honest (“ludicrous”), spontaneous, “confes- sional” interesting, because not “crafted.”45 The most obvious basis for the comparison of mind-flow and breath is a term Kerouac adopts from jazz music: “blowing.” In jazz, “blowing” refers to improvisation.46 In the case of the improvised saxophone-solo Kerouac addresses in his Paris Review interview, such an improvisation is literally blown. With respect to the breath-carried sounds produced by the saxophonist and, by analogy, by the speaker who improvises literary texts, Kerouac’s image has a physiological basis. However, the suggested continuity of the flow of the mind and breathing is as rhetorically con- structed as Ginsberg’s equation of breath-stop and mind-break. This analogy is informed by the idea that physical breathing happens uncon- sciously and thus escapes from those grammatical and syntactical rules that restrict the mind’s free expression—prohibitions the conscious mind cannot ignore. Further, the flow of exhaled air lends itself to a compari- son with the stream of consciousness. 100 S. HEINE S. HEINE 100 Kerouac extends the analogy between breath and a liberated mind to language: the free flow of the mind shall be mirrored in the free flow of language. Kerouac does not go so far as to propose a purely fluent, unsegmented speech or writing. His alternative is to replace the barri- ers of conventional punctuation mirroring grammatical units with a less restraining separator, namely breath. Kerouac and Aristotle No periods separating sentence-structures already arbitrarily riddled by false colons and timid usually needless commas—but the vigorous space dash separating rhetorical breathing (as jazz musician drawing breath between outblown phrases)—47 While the ancient rhetoricians make a considerable effort to reconcile the breathing pause and grammatical units in their arguments, Kerouac is eager to separate the two. In ancient rhetoric, the image of flowing water, which Kerouac invokes in the “flow” and the “seas” of language, is used in order to depict what is spoken between the pauses: Quintilian mentions “the unbroken flow of the voice … being carried along down the stream of oratory”48 and Cicero compares ongoing speech with “the rolling stream of a river.”49 In both cases, the breathing pause is what brings that flow to a halt. Even though he takes the caesura into account, Kerouac’s reservations against anything that disturbs the flow are apparent. In the unpublished essay “History of the Theory of Breath as a Separator of Statements in Spontaneous Writing,” Kerouac extends his comparison of breath-measure to jazz music: in a handwritten addition, the jazz musician is equated with both a runner and orator, and jazz is mentioned in the same breath with oratory and a hundred-yard dash. The imperative “write excitedly, swiftly”50 became the foundation of the most prominent Beat and Kerouac-myth,51 culminating in the repeat- edly invoked scene of Kerouac taping together sheets of paper to a long scroll in order to avoid interruptions before manically typing down On the Road in three weeks.52 In Kerouac’s discussion of running, pausing and writing, we find a striking echo of Aristotle. Aristotle argues that, in contrast to a style segmented by periods, colons and commas, the loose or continuous style is “unpleasant, because it is endless, for all wish to have the end in sight.”53 He gives the following reason for the benefits of the pause: “runners, just when they have reached the goal, lose their breath and strength, whereas before, when the end is in sight, they show 5 EBB AND FLOW: BREATH-WRITING FROM ANCIENT RHETORIC … 101 no signs of fatigue.”54 The advantage of the pause is that it prevents fatigue, the loss of breath, and that it impels the runner to go on. In his argument for pauses, Aristotle looks at them prospectively, as some- thing that lies ahead. Kerouac and Aristotle Such a prospective view opens a very attractive pos- sibility for Kerouac: the break no longer blocks the flow, but generates an impetus to speed on. In a letter to his agent Sterling Lord, Kerouac stresses that the dashes indicating the breathing pause mark something impending: “Make this clear, that my prose is a series of rhythmic expos- tulations of speech visually separated for the convenience of the reader’s eye by dashes, by vigorous definite dashes, which can be seen coming as you read.”55 Kerouac also highlights the importance of looking ahead during composition in “History of the Theory of Breath”: analogous to the writer of spontaneous prose, the jazz musician has to keep track of breath when he moves from one chorus to the next in order to create a continuity between segments. For Kerouac, the pause as such, the moment when according to Aristotle the runners “lose their breath and strength,” represents the most delicate moment in his theory of writing. Whereas Ginsberg empha- sises the meditative potentiality of the pause as a moment of calmness and rest, Kerouac is focused on the speed of the flowing words.56 The idea of resting in the sense of slackening poses a threat to his obsession with mas- tery and an intact, potent masculine body mirrored in a muscular, virile prose.57 The aspired athletic speed of writing should demonstrate vigour. Kerouac claims that he wants to write “[l]ike Proust, but on the run, a Running Proust.”58 “I decided to do just like he did—but fast. … Fast. Marcel Proust had asthma and was lying around writing and eating in bed. Once in a while he’d get up feebly, put on a coat and go down a bar in Paris.”59 Just like Proust, Kerouac wants to write a monumental cycle of novels covering his entire life—but he neither wants to spend as much time as Proust did on the Recherche,60 nor, most importantly, to fail in accomplishing the oeuvre. His comments show that in wishing to be a “running Proust,” Kerouac also wanted to ensure that he didn’t mimic Proust’s frailty. What Kerouac aspires to is an athletic writing in contrast to an asthmatic one.61 The breath Kerouac wishes to incorporate in his writing is one of a healthy, well-trained, potent body. Kerouac and Aristotle It is significant that, in his emphasis on speed, Kerouac conceals the fact that a strained body may be out of breath, or that speaking on the run could be controlled by strained breath.62 A breath that indicates signs of the body’s slackening or weakens it, a writing structured by asthma attacks and apnoea would 102 S. HEINE endanger Kerouac’s poetological pursuits. In other words, Kerouac cannot envision breathlessness in his poetic theory. A physiological foun- dation of writing is only desirable if the body in question is intact and dis- ciplined into athletic strength. Spontaneous writing as such is considered as a result of discipline, or, to follow Kerouac’s own image, the runner’s sprint provides an immediate demonstration of what rigorous training and hardening muscles give rise to. … the critics have failed to realize that spontaneous writing of narrative prose is infinitely more difficult than careful slow painstaking writing with opportunities to revise—Because spontaneous writing is an ordeal requiring immediate discipline—They seem to think there’s no discipline involved—They don’t know how horrible it is to learn immediate and swift discipline and draw your breath in pain as you do so.63 Spontaneous prose is described as the empowering accomplishment of hard work. The aching breath recalling Shakespeare’s Hamlet64 results from the exertion of a well-trained body and stands in contrast to the painful asthmatic breath exhausting a body subject to illness. The refer- ence to Proust’s asthma and his debilitated physical condition shows Kerouac’s longing for mastery over his body and writing alike: the healthy and strong body is a body under command.65 The athlete’s control over his muscles creates the illusion that he is liberated from the more random works of the body that may affect a person (i.e. illness). The imperatives of a “defective” body have no place in Kerouac’s theory of writing. Spontaneous prose is described as the empowering accomplishment of hard work. The aching breath recalling Shakespeare’s Hamlet64 results from the exertion of a well-trained body and stands in contrast to the painful asthmatic breath exhausting a body subject to illness. Kerouac and Aristotle The refer- ence to Proust’s asthma and his debilitated physical condition shows Kerouac’s longing for mastery over his body and writing alike: the healthy and strong body is a body under command.65 The athlete’s control over his muscles creates the illusion that he is liberated from the more random works of the body that may affect a person (i.e. illness). The imperatives of a “defective” body have no place in Kerouac’s theory of writing. Consequently, Kerouac invests the breathing pauses with implications forbidding any possibility that they may be a symptom of the fatigued body. In this respect, it is significant how he describes the graphical sign that should mark the breathing pause and its function: No periods separating sentence-structures already arbitrarily riddled by false colons and timid usually needless commas—but the vigorous space dash separating rhetorical breathing.66 … a sentence which after all is a rhetorical expostulation based on breath- ing and has to end, and I make it end with a vigorous release sign, i.e., the dash—67 By repeatedly describing the dash as “vigorous” (in contrast to the “timid” commas), Kerouac projects the strength of the runner into the 5 EBB AND FLOW: BREATH-WRITING FROM ANCIENT RHETORIC … 103 pause, the moment when his body is in danger of collapsing and his mus- cles are bound to go limp. The aim of associating the pause with virility motivates the choice of the dash as the sign marking it on a semantic and graphical level: “dash” designates the punctuation mark Kerouac uses, but also the runner’s sprint. Through the “dash,” the pause devel- ops a sense of speeding on. In Kerouac’s handwritten manuscripts, the dashes also evoke an impression of speed graphically: often, the lines look as if they were dashed off energetically. Visually, the dash—in this case especially the printed one—establishes a proximity between words it separates: it links them by a vertical line almost touching their respec- tive ends and beginnings, so that the eye is invited to follow this connec- tion. Whereas a blank space between words encourages the eye to pause, the dash rather incites the eye to sprint between words. Moreover, in contrast to the bent commas and colons, the erect straight line of dash, which is also bigger in size, has a phallic quality. Kerouac and Aristotle When his editor at the Grove Press, Don Allen, replaced dashes by full stops and added commas in the manuscript of The Subterraneans, Kerouac complained about this “horrible castration job.” “He has broken down the organic strength of the manuscript and it is no longer THE SUBTERRANEANS by Jack K, but some feeble something by Don Allen.”68 Such a castration anxiety also explains why Kerouac mingles images of breath and sex in the “Essentials”: … write outwards swimming in sea of language to peripheral release and exhaustion—69 … write outwards swimming in sea of language to peripheral release and exhaustion—69 … write excitedly, swiftly, with writing-or-typing-cramps, in accordance (as from center to periphery) with laws of orgasm … . Come from within, out—to relaxed and said.70 … write excitedly, swiftly, with writing-or-typing-cramps, in accordance (as from center to periphery) with laws of orgasm … . Come from within, out—to relaxed and said.70 “[E]xhaustion,” which in terms of respiration represents a threat— i.e. Proust’s asthmatic feebleness and Aristotle’s drained runner who has lost his “breath and strength”—is redirected to the domain of sexual cli- max: Kerouac links the “relaxed” moment of the pause to an explosive “release” of male (creative) potency. Kerouac repeatedly writes that the dashes “release” the sentence. Beside the sexual connotations evoked in the “Essentials,” “release” also designates “liberation,” the “action of freeing, or the fact of being freed.” Moreover, in jazz music, “release” designates a “passage of music that serves as a bridge between repetitions 104 S. HEINE of a main melody.”71 By choosing the word “release” in order to describe the function of the dash, Kerouac is able to connect all the qual- ities he wants to project into the breathing pause in order not to make it appear as slackening or escaping mastery: virility, liberation and a sense of continuity pointing forward to the point after the critical moment of the pause. The word also contains Kerouac’s most eager wish: to make his writing available to the public, to release his written products, to get published and be honoured as America’s healthy Proust. Kerouac’s com- ments on his writing processes and methods, above all the “Essentials,” were most important elements in his attempt to create a public image of himself as a writer. The potent, vigorously breathing body of the author- itative and controlling author Kerouac promotes is produced by his own words. 4. Ibid., 15. Even though Olson stresses orality in “Projective Verse,” it is not his only concern, or even a primary one. As Raphael Allison notes in his book Bodies on the Line: Performance and the Sixties Poetry Reading, “competing with Olson’s emphasis on the breath, graphic text itself was to him of equal value” (68). Prescient to the authors to be discussed in more detail in this article and their relation to orality, it has to be noted that Jack Kerouac did refer to spoken language and the tongue in his Kerouac and Aristotle Kerouac’s literary texts are constructed in a way that evokes the impression of spontaneous, bodily, athletic writing executed by a vig- orous author. The comments on the writing process and methods are designed to verify and confirm—and not least co-create—the effect pro- duced in the literary texts.72 The texts by the ancient rhetoricians, Ginsberg and Kerouac, all imagine the writing or speaking body. In their discussions of the role of breath in writing, especially concerning the breathing pause, both Kerouac and Ginsberg follow in the footsteps of the rhetoricians. Whereas their poetological reflections start from the same premises, they ultimately diverge. Ginsberg’s negotiation of the breathing pause amounts to a meditatively charged stasis, he emphasises the role of qui- escent contemplation. Contrarily, Kerouac’s poetics of breathing culmi- nates in a promotion of flow, fast movement and virile athleticism. 1. Olson (1966, 15). 2. Ibid., 15. 3. Ibid., 20. 4. Ibid., 15. Even though Olson stresses orality in “Projective Verse,” it is not his only concern, or even a primary one. As Raphael Allison notes in his book Bodies on the Line: Performance and the Sixties Poetry Reading, “competing with Olson’s emphasis on the breath, graphic text itself was to him of equal value” (68). Prescient to the authors to be discussed in more detail in this article and their relation to orality, it has to be noted that Jack Kerouac did refer to spoken language and the tongue in his 3. Ibid., 20. Notes Ibid. 11. Ibid. 12. Cicero (1990, 506). I take this short summary of the role of breath in ancient rhetoric from my article “animi velut respirant. Rhythm and Breathing Pauses in Ancient Rhetoric, Virginia Woolf and Robert Musil.” 13. Ginsberg (2001, 359). 13. Ginsberg (2001, 359). 14. Kerouac (1968). 15. Neither Ginsberg nor Kerouac explicitly refers to ancient rhetoric. It is also unclear whether they read the rhetoricians’ discussions of breath or may have been familiar with their ideas through secondary sources. 15. Neither Ginsberg nor Kerouac explicitly refers to ancient rhetoric. It is also unclear whether they read the rhetoricians’ discussions of breath or may have been familiar with their ideas through secondary sources. 16. Regarding the empirical perspective on this matter, a study conducted at Northeastern University by François Grosjean and Maryann Collins from 1979 approaching the question “What is the relationship between linguistic structure and breathing?” (100) concludes that breathing pauses “occur mainly at major constituent breaks” (110). “[T]he need to breathe (at least at slow and normal rates) is not in control of pausing but … on the contrary, breathing adjusts itself to pause patterns” (109). Only when the participants of the study were asked to speak very fast, their breathing pauses did not coincide with syntactic breaks: at faster rates “the physiological need to breathe forces the speaker to stop in order to inhale,” disregarding syntactic units (112). It has to be men- tioned that the study is based on the speaking of healthy participants who were asked to read a text in which punctuation marks indicated where 16. Regarding the empirical perspective on this matter, a study conducted at Northeastern University by François Grosjean and Maryann Collins from 1979 approaching the question “What is the relationship between linguistic structure and breathing?” (100) concludes that breathing pauses “occur mainly at major constituent breaks” (110). “[T]he need to breathe (at least at slow and normal rates) is not in control of pausing but … on the contrary, breathing adjusts itself to pause patterns” (109). Only when the participants of the study were asked to speak very fast, their breathing pauses did not coincide with syntactic breaks: at faster rates “the physiological need to breathe forces the speaker to stop in order to inhale,” disregarding syntactic units (112). Notes 4. Ibid., 15. Even though Olson stresses orality in “Projective Verse,” it is not his only concern, or even a primary one. As Raphael Allison notes in his book Bodies on the Line: Performance and the Sixties Poetry Reading, “competing with Olson’s emphasis on the breath, graphic text itself was to him of equal value” (68). Prescient to the authors to be discussed in more detail in this article and their relation to orality, it has to be noted that Jack Kerouac did refer to spoken language and the tongue in his 5 EBB AND FLOW: BREATH-WRITING FROM ANCIENT RHETORIC … 105 comments on the new literature (e.g. in the unpublished essay “History of the Theory of Breath as a Separator of Statements in Spontaneous Writing”) and did give public readings, but his overall focus has always been on writing and the written text. Allen Ginsberg’s focus on the spo- ken word is much stronger: he repeatedly stresses its importance in his interviews (e.g. 2001, 81, 158, 272), and—as a grandfather of contem- porary poetry slams—presenting his poetry orally to a live audience was a priority in his literary endeavours. The legendary reading of “Howl” at the Six Gallery is only one example. 5. Ginsberg (2001, 19). 5. Ginsberg (2001, 19). 6. “Ideally each line of Howl is a single breath unit. … My breath is long— that’s the measure, one physical-mental inspiration of thought contained in the elastic of a breath” (Ginsberg 1999, 416). “So you arrange the verse line on the page according to where you have your breath stop, and the number of words within one breath, whether it’s long or short, as this long breath has just become” (Ginsberg 1997, 23). 6. “Ideally each line of Howl is a single breath unit. … My breath is long— that’s the measure, one physical-mental inspiration of thought contained in the elastic of a breath” (Ginsberg 1999, 416). “So you arrange the verse line on the page according to where you have your breath stop, and the number of words within one breath, whether it’s long or short, as this long breath has just become” (Ginsberg 1997, 23). 7. Olson (1966, 19). 8. For example, Kerouac (1992, 57) and Kerouac (1999, 15 8. For example, Kerouac (1992, 57) and Kerouac (1999, 15). 9. Ginsberg (2001, 19). 9. Ginsberg (2001, 19). 10. Aristotle (1926, 389). 10. Aristotle (1926, 389). 11. Notes It has to be men- tioned that the study is based on the speaking of healthy participants who were asked to read a text in which punctuation marks indicated where 106 S. HEINE the syntactic units are. Along with the fact that it is a quite old study, the results cannot be transferred seamlessly to the scenario of oral composi- tion the rhetoricians and Beat and Black Mountain writers have in mind. However, it is revealing that breathing pauses and syntactic units seem to co-occur smoothly, but only as long as the body is under control, and that the physiological need to inhale tends to interrupt the syntax once the circumstances of the bodily condition for some reason changes. the syntactic units are. Along with the fact that it is a quite old study, the results cannot be transferred seamlessly to the scenario of oral composi- tion the rhetoricians and Beat and Black Mountain writers have in mind. However, it is revealing that breathing pauses and syntactic units seem to co-occur smoothly, but only as long as the body is under control, and that the physiological need to inhale tends to interrupt the syntax once the circumstances of the bodily condition for some reason changes. 17. Cicero attempts to conciliate the physical need to inhale and making a pause at the completion of a period by means of a quite constructed argu- ment that beauty in artificial works is in agreement with natural utility (1875, 244). 18. Quintilian (1856, 352–353). Also in the passages on composition, there are uncertainties about the moment when a breath is required because a thought is completed at the moment when the orator should actually take a breath: “Who, for example can doubt that there is but one thought in the following passage and that it should be pronounced without a halt of breath? Still, the groups formed by the first two words, the next three, and then again by the next two and three, have each their own special rhythm and cause a slight check in our breathing” (Quintilian 1943, 545). 19. Cicero (1875, 243). 20. Cicero (1990, 506). Notes The editor’s comment to this passage shows that the rhetoricians’ attempts to reconcile the completion of the period with the need to inhale leads to inconsistencies: “There is no real, though an apparent inconsistency: the periods must furnish opportunity for taking breath, but must not be determined solely by the need for this” (Cicero 1990, 506). 21. Quintilian (1856, 357). This overview of the breathing pause in ancient rhetoric is taken from my article “animi velut respirant. Rhythm and Breathing Pauses in Ancient Rhetoric, Virginia Woolf and Robert Musil.” 22. Kerouac (1999, 15). 23. Kerouac (1968), not paginated. 24. Even though the examples Ginsberg uses as illustrations in numerous interviews and line-breaks or sentence segmentations in Ginsberg’s and Kerouac’s literary texts give some indication of these units, a precise explication is still lacking. 24. Even though the examples Ginsberg uses as illustrations in numerous interviews and line-breaks or sentence segmentations in Ginsberg’s and Kerouac’s literary texts give some indication of these units, a precise explication is still lacking. 25. Investigating the breath-stops in their oral deliveries, in contrast, is possi- ble in the cases where recordings were made. In Ginsberg’s recordings of Howl, for example, one can observe that the moments when he inhales and pauses do not always coincide with the line breaks. Even though Ginsberg stresses that he imitates the compositional process in his read- ings (2001, 126), the readings as such do not constitute valid data for 5 EBB AND FLOW: BREATH-WRITING FROM ANCIENT RHETORIC … 107 an investigation of the composition process. The only thing one might infer from Ginsberg’s Howl readings is that the moments when he has to inhale before the line ends show that his breath may not be as long as he claims in the Notes for Howl—even though he himself addresses this fact and attributes it to his exhaustion at the moment when he was reading (2001, 416). an investigation of the composition process. The only thing one might infer from Ginsberg’s Howl readings is that the moments when he has to inhale before the line ends show that his breath may not be as long as he claims in the Notes for Howl—even though he himself addresses this fact and attributes it to his exhaustion at the moment when he was reading (2001, 416). 26. Notes This may explain Ginsberg’s long lines in Howl, which he cannot pro- nounce in one breath orally (see 25). 26. This may explain Ginsberg’s long lines in Howl, which he cannot pro- nounce in one breath orally (see 25). 27. See 16. 28. Ginsberg (1974). 28. Ginsberg (1974). 29. Ginsberg (2001, 126). 30. Ibid., 108. 31. Ibid., 359. 32. Quintilian (1943, 543). 32. Quintilian (1943, 543). 33. Ibid., 543. 34. The Latin use of “respire,” the verb used by Quintilian, already included the figurative meaning of breathing as resting: “to fetch one’s breath again, to recover breath; to recover, revive, be relieved or refreshed after any thing difficult (as labor, care, etc.)” (Lewis and Short 1879). 35. In the Oxford English Dictionary, “to take breath” is considered to be a figurative use of the “[p]ower of breathing, free or easy breathing”: “to breathe freely, to recover free breathing, as by pausing after exertion” (OED online). 36. Ginsberg (2001, 126). 37. Ibid., 411. 37. Ibid., 411. 38. Ibid., 365. 39. Ibid. 40. Ginsberg (1999, 416). 41. Ginsberg (2001, 126). In the Q&A session of lecture given in 1974, Ginsberg puts this claim into perspective and admits that his conceptions of mind units and breath units are not fully fleshed out. A student asked how Ginsberg uses his breath when he writes in a notebook: “do you read it out loud as you’re writing it down?” In reply, Ginsberg mentions “It’s an interesting thing whether it’s breath or it’s mind unit. I never fig- ured that out” (Ginsberg 1974). 42. Ginsberg (2001, 107). 42. Ginsberg (2001, 107). 43. Ibid., 145. 43. Ibid., 145. 44. Kerouac (1992, 57), my emphasis. 44. Kerouac (1992, 57), my emphasis. 45. Ibid., 58, italics in the original. 45. Ibid., 58, italics in the original. 46. Witmer (2003). 46. Witmer (2003). 47. Kerouac (1992, 57). 48. Quintilian (1943, 541). 48. Quintilian (1943, 541). 108 S. HEINE 49. Cicero (1875, 247). 50. Kerouac (1992, 58). 51. Kerouac himself spent considerable efforts to create and maintain that myth, which for him goes hand in hand with having found his own style and “voice,” most prominently expressed in the “Essentials.” Significantly, the “Essentials” constitute an instruction to imitate, circu- late and popularize the style Kerouac discovered for himself. 52. Notes It has long been known that this is not an accurate description of how On the Road came to be and that Kerouac spent years taking notes and designing drafts for the novel (cf., for example Brinkley 2004, xxv). 53. Aristotle (1926, 387). 53. Aristotle (1926, 387). 54. Ibid. 55. Kerouac (1999, 11). 56. Even though Ginsberg occasionally also refers to speed, for example by referring to the next line to be written or read as “next spurt” (2001, 125), this is never at the centre of his reflections—he rather seems to be echoing Kerouac’s ideas of “athletic speech” (Ginsberg 2001, 114) in these instances. 57. Kerouac stresses these characteristics on a small undated scrap of paper containing a list of desirable prose attributes. 57. Kerouac stresses these characteristics on a small undated scrap of paper containing a list of desirable prose attributes. 58. Kerouac (1995, 515). 59. Kerouac (2005, 192). 60. Kerouac (1995, 515). 61. See Benjamin (1968). 62. See 16. 62. See 16. 63. Kerouac (1999, 325). 64. “Draw your breath in pain” is, of course, an implicit quote. Kerouac was well aware of Hamlet’s last words: he quotes “Absent thee from felicity awhile,” the line preceding “And in this harsh world draw thy breath in pain,” in a letter to Ginsberg written in 1947 (1995, 122). Moreover, in a letter to Neal Cassady in 1950, Kerouac makes an explicit reference to Hamlet, precisely when he “discovers” the strenuousness of writing spontaneously in one’s own voice: “My important recent discovery and revelation is that the voice is all. Can you tell me Shakespeare’s voice per se?—Who speaks when Hamlet speaks? HAMLET, not Will Shakespeare …. You, man, must write exactly as everything rushes in your head, and AT ONCE. The pain of writing is just that” (1995, 233). It is impor- tant to note that these earliest thoughts on spontaneous prose, in which breath is not explicitly mentioned, are inspired by Hamlet’s last sigh. 65. In Proust’s Recherche, a notion of mastery is not absent. To the contrary, the narrator uses his illness as a means to exert control over the characters he interacts with. In particular, in The Captive, the house he cannot leave 65. In Proust’s Recherche, a notion of mastery is not absent. To the contrary, the narrator uses his illness as a means to exert control over the characters he interacts with. Notes In particular, in The Captive, the house he cannot leave 5 EBB AND FLOW: BREATH-WRITING FROM ANCIENT RHETORIC … 109 due to his physical condition becomes a setting where Marcel can subject his lover Albertine to his supervision and bend her to his will as well as a stage for the dramas he directs. The space he is limited to because of his feeble physical condition is totally under Marcel’s control, precisely because it is secluded from the contingencies of the outside world. In Le Souffle coupé. Respirer et écrire, François-Bernard Michel claims that asthma implies a closure of what is supposed to be open: the asthmatic closes his bronchia and thus conserves his air, he refuses to exhale (194). The intentionality insinuated in Michel’s formulation is problematic, but it gets to the heart of Marcel’s attempt of creating an enclosed space sealed from exposure to the outside. Thus, Proust’s asthma represents a flip side to Kerouac’s poetics of breathing. Not only are the two models of literary breathers similarly subject to mystification: the aesthetic ideali- sation of the fin de siècle decadent in Proust’s case, the phallocentric, vir- ile daredevil who lives fast and dies young in Kerouac’s case. In contrast to the asthmatic, Kerouac’s athletic writing embraces exhalation: “blow- ing” is the central respirational movement for Kerouac, and it has to be noted that in contrast, he is deeply suspicious of inhalation, of everything that enters the body from without and is not his own. Through his focus on exhalation, Kerouac stages an extension of the self to the outside world and is equally paranoid of a possible interference of the outside with the self as Proust is. The analogy of his writing and sprinting sup- ports this: as an anaerobic exercise, the sprint relies on energy resources stored in the body—it allows a momentary fantasy of not being depend- ent on an oxygen supply from without. 66. Kerouac (1992, 57), my emphasis. 67. Kerouac (1995, 324), my emphasis. 68. Kerouac (1995, 11). 68. Kerouac (1995, 11). 69. Kerouac (1992, 58). 70. Ibid. 71. OED online, my emphasis. 72. For a more detailed analysis of how the “Essentials of Spontaneous Prose” themselves represent a deliberate attempt to create an effect of spontane- ity that first had to be carefully prepared, see my article “First Thought, Best Thought. Notes Improvisation bei Jack Kerouac und Allen Ginsberg.” A look at Kerouac’s manuscripts and drafts shows that the methods and techniques he proposes in his writing manuals and comments have never been consequently applied in his actual writing processes. I investigated a large bulk of materials at the Berg Collection of English and American Literature, among them drafts for The Subterraneans, On the Road and Visions of Gerard. A detailed discussion of these findings, however, exceeds the scope of this paper. Generally, it is worth noting that Kerouac 110 S. HEINE 110 S. HEINE made extensive use of “timid commas” and hardly used the dashes in a consequential manner (to replace commas, colons or full stops); most of the times, one can find a mixture of dashes, commas and full stops. I want to give only one example that demonstrates how Kerouac retrospec- tively—and against his imperative “no revisions” (1992, 57)—aligned his texts to his own writing instructions: in order to highlight that he replaces full stops by dashes, he consequently changes lowercased words succeed- ing a dash into capitalized ones in the setting copy of Visions of Gerard. References Allison, Raphael. 2014. Bodies on the Line: Performance and the Sixties Poetry Reading. Iowa, IA: University of Iowa Press. Aristotle. 1926. The “Art” of Rhetoric, ed. and trans. John Henry Freese. London: William Heinemann. Benjamin, Walter. 1968. The Image of Proust. In Illuminations, trans. Harry Zohn, intro. Hannah Arendt, 201–215. New York: Schocken Books. “breath.” OED Online. Oxford: Oxford University Press. www.oed.com. Accessed 13 Mar 2018. Brinkley, Douglas (ed.). 2004. Windblown World: The Journals of Jack Kerouac 1947–1954. New York: Viking Penguin. Cicero. 1875. Oratory and Orators, ed. and trans. J.S. Watson. New York: Harper & Brothers. Cicero. 1990. de Oratore libri tres, ed. August S. Wilkins. Hildesheim: Georg Olms Verlag. Ginsberg, Allen. 1974. Spiritual Poetics II. Lecture at the Naropa University. https://archive.org/details/Allen_Ginsberg_class_Spiritual_Poetics_part_2_ July_1974_74P002. Accessed 12 Mar 2018. Ginsberg, Allen. 1997. Allen Ginsberg: An Interview by Gary Pacernick. The American Poetry Review 26 (4): 23–27. Ginsberg, Allen. 1999. Notes for Howl and Other Poems. In The New American Poetry 1945–1960, ed. Donald M. Allen, 414–420. Berkeley: University of California Press. Ginsberg, Allen. 2001. Spontaneous Mind: Selected Interviews 1958–1996, ed. David Carter. New York: HarperCollins. Grosjean, François, and Maryann Collins. 1979. Breathing, Pausing and Reading. Phonetica 36: 98–114. Heine, Stefanie. 2014. First Thought, Best Thought. Improvisation bei Jack Kerouac und Allen Ginsberg. In Improvisation und Invention. Momente, Modelle, Medien, ed. Sandro Zanetti, 245–259. Zürich: Diaphanes. 111 5 EBB AND FLOW: BREATH-WRITING FROM ANCIENT RHETORIC … Heine, Stefanie. 2017. animi velut respirant. Rhythm and Breathing Pauses in Ancient Rhetoric, Virginia Woolf and Robert Musil. Comparative Literature 69 (4): 355–369. Kerouac, Jack. 1968. The Art of Fiction No. 41. The Paris Review. www.thep- arisreview.org/interviews/4260/the-art-of-fiction-no-41-jack-kerouac. Accessed 12 Mar 2018. Kerouac, Jack. 1992. Essentials of Spontaneous Prose. In The Portable Beat Reader, ed. Ann Charters, 57–58. New York: Viking. Kerouac, Jack. 1995. Selected Letters, 1940–1956, ed. Ann Charters. New York: Viking Penguin. Kerouac, Jack. 1999. Selected Letters, 1957–1969, ed. Ann Charters. New York: Viking Penguin. Kerouac, Jack. 2005. Dialogues in Great Books. In Empty Phantoms. Interviews and Encounters with Jack Kerouac, ed. Paul Maher, 184–202. New York: Thunder’s Mouth Press. Michel, François-Bernard. 1984. Le Souffle coupé: Respirer et écrire. Paris: Gallimard. Olson, Charles. 1966. Projective Verse. In Selected Writings, ed. Robert Creeley, 15–26. New York: New Directions. Quintilian. 1856. Quintilian’s Institutes of Oratory: Or, Education of an Orator in Twelve Books, vol. II, trans. John Selby Watson. London: Bohn’s Classical Library. Quintilian. 1943. References Institutio Oratia, trans. H.E. Butler. Cambridge: Harvard University Press. “release.” OED Online. Oxford: Oxford University Press. www.oed.com. Accessed 13 Mar 2018. “respiro.” A Latin Dictionary. 1879. Ed. Charlton T. Lewis and Charles Short. Oxford: Clarendon Press. Online Version. www.perseus.tufts.edu/hopper/ text?doc=Perseus%3Atext%3A1999.04.0059%3Aentry%3Drespiro. Accessed 13 Mar 2018. Witmer, Robert. 2003. Blow. In The New Grove Dictionary of Jazz, ed. Barry Kernfeld. Grove Music Online, Oxford Music Online: Oxford University Press. http://www.oxfordmusiconline.com. Accessed 13 Mar 2018. 112 S. HEINE 112 S. HEINE 112 S. HEINE Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ by/4.0/), 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 license and indicate if changes were made. The images or other third party material in this chapter are included in the chapter’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter’s Creative Commons license 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.
https://openalex.org/W3002917508	https://www.frontiersin.org/articles/10.3389/fgene.2019.01379/pdf	English	null	Comprehensive Analysis of Copy Number Variations in Kidney Cancer by Single-Cell Exome Sequencing	Frontiers in genetics	2,020	cc-by	6,700	ORIGINAL RESEARCH published: 23 January 2020 doi: 10.3389/fgene.2019.01379 Reviewed by: Hao Lin Reviewed by: Hao Lin, University of Electronic Science and Technology of China, China Juan Wang, Inner Mongolia University, China Jingpu Zhang, Henan University of Urban Construction, China Correspondence: Huan Nie nh1212@hit.edu.cn Lifen Yao yaolifen_2015@sina.com Qinghua Jiang qhjiang@hit.edu.cn †These authors have contributed equally to this work Keywords: copy number variations, single-cell exome sequencing, clear-cell renal cell carcinoma, receptor protein tyrosine kinase, signaling transduction pathway Specialty section: This article was submitted to Statistical Genetics and Methodology, a section of the journal Frontiers in Genetics Specialty section: This article was submitted to Statistical Genetics and Methodology, a section of the journal Frontiers in Genetics Received: 23 September 2019 Accepted: 17 December 2019 Published: 23 January 2020 INTRODUCTION Renal cell carcinoma (RCC) is one kind of kidney cancer, accounting for nearly 300,000 new cancer cases per year worldwide (Hakimi et al., 2013). RCC includes several histological subtypes, among which clear cell renal cell carcinoma (ccRCC) is the most common and lethal one (Hakimi et al., 2016). Increasing studies have shown that the development of ccRCC seems to be shaped by chromosomal lesions and a number of somatic mutations (Sato et al., 2013). VHL and PBRM1, located within the chromosome 3p25 and 3p21 segments, are the top two signiﬁcantly mutated genes in ccRCC (Sato et al., 2013). Nearly 90% of ccRCCs undertake the deletion on chromosome 3p, leading to a very high frequency of VHL inactivation (Gnarra et al., 1994). Moreover, VHL and PBRM1 are mutated in about 50 and 41% of sporadic ccRCC, respectively (Kaelin, 2004; Varela et al., 2011). However, little is known about the genetic mechanisms in VHL/PBRM1- negative ccRCC. Received: 23 September 2019 Accepted: 17 December 2019 Published: 23 January 2020 Comprehensive Analysis of Copy Number Variations in Kidney Cancer by Single-Cell Exome Sequencing Wenyang Zhou 1†, Fan Yang 2†, Zhaochun Xu 1†, Meng Luo 1, Pingping Wang 1, Yu Guo 1, Huan Nie 1, Lifen Yao 2* and Qinghua Jiang 1* 1 School of Life Science and Technology, Harbin Institute of Technology, Harbin, China, 2 Department of Neurology, The First Afﬁliated Hospital of Harbin Medical University, Harbin, China Keywords: copy number variations, single-cell exome sequencing, clear-cell renal cell carcinoma, receptor protein tyrosine kinase, signaling transduction pathway Edited by: Lei Deng, Central South University, China Clear-cell renal cell carcinoma (ccRCC) is the most common and lethal subtype of kidney cancer. VHL and PBRM1 are the top two signiﬁcantly mutated genes in ccRCC specimens, while the genetic mechanism of the VHL/PBRM1-negative ccRCC remains to be elucidated. Here we carried out a comprehensive analysis of single-cell genomic copy number variations (CNVs) in VHL/PBRM1-negative ccRCC. Genomic CNVs were identiﬁed at the single-cell level, and the tumor cells showed widespread ampliﬁcation and deletion across the whole genome. Functional enrichment analysis indicated that the ampliﬁed genes are signiﬁcantly enriched in cancer-related signaling transduction pathways. Besides, receptor protein tyrosine kinase (RTK) genes also showed widespread copy number variations in cancer cells. Our studies indicated that the genomic CNVs in RTK genes and downstream signaling transduction pathways may be involved in VHL/PBRM1-negative ccRCC pathogenesis and progression, and highlighted the role of the comprehensive investigation of genomic CNVs at the single- cell level in both clarifying pathogenic mechanism and identifying potential therapeutic targets in cancers. Copy Number Variations Calling Copy Number Variations Calling In each cell, germline and somatic copy number variations were called by Control-FREEC version 11.5 (Boeva et al., 2012). Considering the exome enrichment during library construction, read counts were calculated by exome region. The target region ﬁle of exome capture was downloaded from the Agilent website (https://earray.chem.agilent.com/suredesign/ index.htm). The germline CNVs were detected in each cell and bulk normal tissue, respectively. Somatic CNVs were detected only in single cells. Gene annotations were performed with Annovar software (Wang et al., 2010) and OAHG database (Cheng et al., 2016). Genomic copy number variations (CNVs) play an important role in cancer progression, and emerging studies indicate that genomic CNVs are associated with the ccRCC (Gerlinger et al., 2014; Nouhaud et al., 2018) and other cancers (Waddell et al., 2015; Secrier et al., 2016; Hong et al., 2019). Xu et al. (Xu et al., 2012) performed a single-cell exome sequencing to elucidate the genetic mechanisms of the ccRCC by identifying the single nucleotide variants (SNVs). However, the authors did not examine whether the genomic copy number variations play a crucial role in ccRCC. Dimensionality Reduction of Cells T-distributed stochastic neighbor embedding (t-SNE) was performed based on the germline CNVs of target regions. Both 25 single cells and bulk normal tissue were projected to 2D space using the R package named “Rtsne.” To further investigate the potential roles of CNVs in VHL/ PBRM1-negative ccRCC, we performed a comprehensive single- cell CNV analysis based on a dataset provided by Xu et al., (2012). We delineated the genomic copy number variation landscape at the single-cell level and reclassiﬁed all single cells based on the single-cell genomic CNVs. We also identiﬁed several signiﬁcantly ampliﬁed/deleted loci and genes in cancer cells. Finally, we further investigated the biological pathways which may be involved in the ccRCC pathogenesis. Datasets The sample data and information used in our article came from a previous study, and the original sequencing data were downloaded from NCBI (http://www.ncbi.nlm.nih.gov/sra) under the accession number SRA050201. py g To examine the landscape of copy number variations in RTK genes, we derived GISTIC-equivalent scores by dividing the germline copy numbers and classifying genes as ampliﬁed if score ≥0.9, deleted if score ≤−1.3, gained if score > 0.1, and loss if the score < −0.1. Signiﬁcantly Somatic Copy Number Variation Loci Analysis Signiﬁcantly ampliﬁed/deleted loci in tumor cells were identiﬁed using GISTIC2.0 (Mermel et al., 2011). GISTIC2.0 was run on an input deﬁned as the log2()-1 of somatic copy number values, with conﬁdence (-conf) threshold of 0.9. Considering for downstream analysis, thresholds suggested by GISTIC2.0 for copy number variation were as follows: if GISTIC score ≥0.9, it means ampliﬁcation; 0.1 < GISTIC score <0.9, corresponding to gain; −1.3 < GISTIC score < −0.1, loss; GISTIC score ≤−1.3, deletion. Function Analysis The signiﬁcantly ampliﬁed and deleted genes were identiﬁed according to signiﬁcantly somatic CNV loci (q-value < 0.0001) in GISTIC2.0. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway function enrichment analysis was performed using the Carcinogenic Potency Database (CPDB) (Kamburov et al., 2013). In this study, the p-value threshold for KEGG enrichment analysis is 0.05. Quality Control Quality control of the sequencing data was performed using FastQC. The adapter and low-quality ends were trimmed from reads using Trim-Galore version 0.5.0 (http://www. bioinformatics.babraham.ac.uk/projects/trim_galore/). Trimmed reads shorter than 20 bp were discarded. Citation: Zhou W, Yang F, Xu Z, Luo M, Wang P, Guo Y, Nie H, Yao L and Jiang Q (2020) Comprehensive Analysis of Copy Number Variations in Kidney Cancer by Single-Cell Exome Sequencing. Front. Genet. 10:1379. doi: 10.3389/fgene.2019.01379 January 2020 \| Volume 10 \| Article 1379 Frontiers in Genetics \| www.frontiersin.org Single-Cell CNVs Analysis in ccRCC Zhou et al. bigZips/). Short read pairs were mapped to the reference genome using Burrows-Wheeler Aligner (BWA) version 0.7.12-r1039 (Li and Durbin, 2009). In this process, we adopted the BWA-MEM algorithm and adjusted the main parameters, setting the minimum seed length to 19, the penalty for a mismatch to 4, and shorter split hits were marked as secondary. Then, Samtools was used to convert SAM ﬁles to compressed BAM ﬁles, sort the BAM ﬁles by chromosomal coordinates, and remove the PCR duplicates from BAM ﬁles. Based on the next-generation sequencing technology, previous studies identiﬁed many driver mutations in ccRCC (Gnarra et al., 1994; Kaelin, 2004; Sato et al., 2013; Cheng et al., 2019). However, limited insights are available on the genomic diversity within tumor tissues (Wang et al., 2014). Generally, tumor tissues may contain cancer cells from multiple clones and noncancerous cells, which make it difﬁcult to identify the mutations in each clone and detect the driver genes during the cancer progression (Xu et al., 2012; Casasent et al., 2018). Fortunately, single-cell DNA sequencing has been developed to meet this challenge, because it can provide unique insights into intratumor heterogeneity, development, and diversity of cancers at the single-cell level (Casasent et al., 2018). For example, Xu et al. (2012) carried out the single-cell exome sequencing on a ccRCC tumor and its adjacent normal tissue. They identiﬁed four genes (i.e., AHNAK, SRGAP3, LRRK2, and USP6) as potential driving factors for VHL/PBRM1-negative ccRCC development, which provided new insights into the pathogenesis of the ccRCC. Identiﬁcation of Single-Cell Genomic Copy Number Variations in Kidney Cancer and Normal Cells To identify genomic CNVs in ccRCC, we analyzed the sequencing data from a ccRCC patient, which includes 20 single-cell exome sequencing data from the tumor tissue, 5 single-cell exome sequencing data from the adjacent normal tissue, and a bulk exome sequencing data from the adjacent normal tissue. Trim-Galore was used to remove the low-quality and adapter segments and analyze the quality of sequencing reads. The cleaned reads were mapped to the reference genome with BWA software (Li and Durbin, 2009). The sequencing depth was more than 20X (29.68 ± 5.68) in all single cells. The genomic CNVs were called by using Control-FREEC (Boeva et al., 2012). RESULTS To remove the background mutations caused by germline or technology ﬂaws, somatic CNVs were identiﬁed in all cells using bulk normal tissue as control. The somatic CNVs showed much more ampliﬁcation than germline CNVs in the cancer cells remarkably (Figure 1B). Large-scale of somatic CNVs were found in the ccRCC single cells, which was consistent with the previous studies based on the bulk sequencing (Cancer Genome Atlas Research, N, 2013; Gerlinger et al., 2014; Nouhaud et al., 2018). What’s more, single-cell sequencing data revealed the ampliﬁcation of copy number showed a high degree of consistency, which suggests the ampliﬁcation may play an important role in the progression of ccRCC. On the contrary, the deletion showed higher intratumor heterogeneity in the cancer cells. Reads Mapping The human reference genome sequence (Hg19) was used for mapping (http://hgdownload.soe.ucsc.edu/goldenPath/hg19/ January 2020 \| Volume 10 \| Article 1379 Frontiers in Genetics \| www.frontiersin.org Single-Cell CNVs Analysis in ccRCC Zhou et al. Re-Classiﬁcation of Kidney Cancer and Normal Cells Based on Single-Cell Copy Number Variations Germline CNVs were identiﬁed in all the samples. The comparison between cancer and normal cells revealed widespread ampliﬁcation and deletion across the whole genome in tumor cells (Figure 1A). At the same time, some deleted loci were found both in normal and cancer cells, which may be caused by multiple displacement ampliﬁcation (MDA) ampliﬁcation (Yilmaz and Singh, 2012) or exome capture during DNA library preparation. Generally, surgically removed cancer tumors may contain both cancer and normal cells (Xu et al., 2012). To reclassify all the single cells accurately, the t-distributed stochastic neighbor embedding (t-SNE) was performed based on the cell copy number in exome target regions. The results of dimensionality reduction (Figure 2, Supplementary Table S1) showed that three cancer cells (CC-15, CC-17, and CC-20) clustered tightly FIGURE 1 \| The genomic copy number variations (CNVs) identiﬁed across all cells. (A) The germline CNVs in single cells and normal tissue. Genomic CNVs within the whole genome are shown, the color scale ranges from blue (deletion) to red (ampliﬁcation) with estimated copy numbers shown. The cell names are marked by different cell types. (B) The somatic CNVs in single cells. FIGURE 1 \| The genomic copy number variations (CNVs) identiﬁed across all cells. (A) The germline CNVs in single cells and normal tissue. Genomic CNVs within the whole genome are shown, the color scale ranges from blue (deletion) to red (ampliﬁcation) with estimated copy numbers shown. The cell names are marked by different cell types. (B) The somatic CNVs in single cells. FIGURE 1 \| The genomic copy number variations (CNVs) identiﬁed across all cells. (A) The germline CNVs in single cells and normal tissue. Genomic CNVs within the whole genome are shown, the color scale ranges from blue (deletion) to red (ampliﬁcation) with estimated copy numbers shown. The cell names are marked by different cell types. (B) The somatic CNVs in single cells. January 2020 \| Volume 10 \| Article 1379 3 Frontiers in Genetics \| www.frontiersin.org Single-Cell CNVs Analysis in ccRCC Zhou et al. According to the single-cell genomic CNVs, all the single cells can be reclassiﬁed into three groups, namely cancer cell (CC), normal cell (NC), and normal cell in cancer tissue (NCinCT). To address whether the genomic CNVs were signiﬁcantly different between the three groups, we calculated the proportion of whole genome that covered with ampliﬁcation (copy number ≥4) and loss (copy number = 0), respectively. Loci Distribution of Signiﬁcant Genomic Copy Number Variations in Kidney Cancer Loci Distribution of Signiﬁcant Genomic Copy Number Variations in Kidney Cancer To investigate the loci distribution of the signiﬁcant genomic CNVs across all tumor single cells, GISTIC2.0 (Mermel et al., 2011) was used to identify the signiﬁcant genomic CNVs loci based on the somatic CNVs in 17 cancer cells, but not including germline CNVs which are not involved in cancer development generally. The results indicated that copy numbers in the signiﬁcant CNV loci have a high degree of consistency across all the cancer cells. Although lots of lost loci (more slight than deletion, −1.3 < GISTIC score < −0.1) were identiﬁed, there was no signiﬁcantly deleted locus (GISTIC score ≤−1.3) found in cancer cells, which was consistent with high heterogeneity of deletion region in our cancer cells. Signiﬁcantly ampliﬁed loci (Figure 4, Supplementary Table S2A) according to GISTIC2.0 (12q13.3, 12p13.31, 5q35.3, etc.; q- value < 0.05) comprised genes such as IGFBP4, ERBB2, ERBB3, FGFR4, CDK2, FLT4, and so on. The IGFBP4 gene had been reported to be associated with several types of cancer (Hallberg FIGURE 2 \| Population analysis based on the germline copy number variations (CNVs). T-distributed stochastic neighbor embedding (T-SNE) analysis of cancer cell (red), normal cell (blue), and normal cell in cancer tissue (green) based on the germline CNVs. FIGURE 2 \| Population analysis based on the germline copy number variations (CNVs). T-distributed stochastic neighbor embedding (T-SNE) analysis of cancer cell (red), normal cell (blue), and normal cell in cancer tissue (green) based on the germline CNVs. FIGURE 3 \| The coverage of genomic copy number variations (CNV) regions in three cell types. (A) The percentage of ampliﬁcation region (copy number ≥4) across the whole genome in different cell types. (B) The percentage of loss region (copy number = 0) across the whole genome in different cell types. In the two sub-graphs (A) and (B), p-values between two groups (Wilcoxon signed-rank test) and all groups (Kruskal-Wallis test) were calculated. FIGURE 3 \| The coverage of genomic copy number variations (CNV) regions in three cell types. (A) The percentage of ampliﬁcation region (copy number ≥4) across the whole genome in different cell types. (B) The percentage of loss region (copy number = 0) across the whole genome in different cell types. In the two sub-graphs (A) and (B), p-values between two groups (Wilcoxon signed-rank test) and all groups (Kruskal-Wallis test) were calculated. Re-Classiﬁcation of Kidney Cancer and Normal Cells Based on Single-Cell Copy Number Variations The results (Figure 3) showed that there were more ampliﬁed loci in CC group than NC group (P = 3×10−4) and NCinCT group (P = 1.8×10−3). Besides, there was no signiﬁcant difference between NC and NCinCT groups (P = 0.79). The lost loci also showed a similar result. Single-cell genomic CNVs indicated that the genome of cancer cells was in an extremely unstable state. with the normal cells and tissue, suggesting that they probably were normal cells in the tumor tissue. These results were consistent with the previous ﬁndings which based on the single-cell SNVs (Xu et al., 2012). These three cells (CC-15, CC-17, and CC-20) were excluded from the cancer cell group in the downstream analysis. Focusing on the remaining cancer cells, we found no subpopulation of cancer cells within the cancer tissue. with the normal cells and tissue, suggesting that they probably were normal cells in the tumor tissue. These results were consistent with the previous ﬁndings which based on the single-cell SNVs (Xu et al., 2012). These three cells (CC-15, CC-17, and CC-20) were excluded from the cancer cell group in the downstream analysis. Focusing on the remaining cancer cells, we found no subpopulation of cancer cells within the cancer tissue. FIGURE 2 \| Population analysis based on the germline copy number variations (CNVs). T-distributed stochastic neighbor embedding (T-SNE) analysis of cancer cell (red), normal cell (blue), and normal cell in cancer tissue (green) based on the germline CNVs. Functional Analysis of Signiﬁcant Genomic Copy Number Variations in Kidney Cancer Copy Number Variations in Kidney Cancer To better understand the potential biological and functional characteristics of the signiﬁcantly ampliﬁed and deleted genes in cancer cells, biological function pathways in ccRCC had been further investigated. The KEGG functional enrichment analysis was performed using the CPDB Database based on the ampliﬁed and deleted genes, respectively. The ampliﬁed genes showed signiﬁcant enrichment (p-value < 0.05) for signal transduction, metabolism, cell cycle, immunity, and other cancer-related pathways (Figure 5, Supplementary Table S3). In contrast to ampliﬁed genes, deleted genes only showed signiﬁcant enrichment for the fatty acid elongation pathway (p-value = 7.6×10−3). While the top signiﬁcantly deleted loci (Figure 4, Supplementary Table S2B) (1q21.3, 1p35.2, 16q24.3, 3p14.1, etc.; q-value < 0.05) showed loss of Chmp1A, CADM2, PRAP1, and ULK1 genes. Chmp1A and CADM2, belonging to cell adhesion molecules family, had been found to be a tumor suppressor gene in RCC. The overexpression of Chmp1A and CADM2 signiﬁcantly suppressed cancer growth and invasion (You et al., 2012; He et al., 2013). PARP1 gene played an important role in DNA repair and cell apoptosis (Tulin, 2011), the cell with PARP1 deﬁciency show resistance to DNA damage- induced programmed cell death and increased cancer risk (Schiewer and Knudsen, 2014). ULK1 was an initiate autophagy gene, and the down-regulation of ULK1 had been The most notable result is that a large portion of enrichment pathways belong to the signaling transduction pathway. Both of the HIF-1 (Posadas et al., 2013), ErbB (Liu et al., 2015), PI3K-Akt (Linehan et al., 2010; Sato et al., 2013; Guo et al., 2015), Ras (de Araujo Junior et al., 2015; Chen et al., 2018), Rap1 (Chen et al., 2018), and MAPK signaling pathway (Liu et al., 2015) had been FIGURE 5 \| Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis for signiﬁcantly ampliﬁed genes. The size of the point means the gene number both in our ampliﬁed gene set and KEGG pathway terms. The color of point means enrichment signiﬁcance (−log10P). The pathways were sorted by rich factor (the ratio of signiﬁcantly ampliﬁed gene number in this pathway term to gene number in this pathway term). FIGURE 4 \| The signiﬁcant genomic copy number variation (CNV) loci in cancer cells. All CNV types in each cancer cell were counted for the top frequency histogram, and q-value for each signiﬁcant genomic CNV loci was shown on the right. Loci Distribution of Signiﬁcant Genomic Copy Number Variations in Kidney Cancer January 2020 \| Volume 10 \| Article 1379 4 Frontiers in Genetics \| www.frontiersin.org Single-Cell CNVs Analysis in ccRCC Zhou et al. found in cancer (Zhang et al., 2017). ULK1 may play a pivotal role in cancer by promoting cell death (Chen et al., 2014). The genes in signiﬁcantly ampliﬁed loci include a number of known driver genes, which may promote the cancer progression by the up-regulation of cell growth and cell cycle. Signiﬁcantly deleted loci include some tumor suppressor genes and autophagy genes. The inactivation of these genes leads to uncontrolled tumor growth, which may contribute to the VHL/PBRM1- negative ccRCC pathogenesis and progression found in cancer (Zhang et al., 2017). ULK1 may play a pivotal role in cancer by promoting cell death (Chen et al., 2014). et al., 2000; Romero et al., 2011; Yang et al., 2017), it can promote the RCC cell metastasis and activate Wnt/beta-catenin signaling pathway in humans (Ueno et al., 2011). ERBB2 and ERBB3 genes belong to the epidermal growth factor receptor (EGFR) family, and they had been identiﬁed as common driver genes of multiple cancer types by promoting solid tumor growth (Yarden, 2001; Henson et al., 2017; Oldrini et al., 2017). The ampliﬁcation of EGFR also was found in other cancers, which contributed to the EGFR excessive activation (Sigismund et al., 2018). FGFR4 gene belongs to the ﬁbroblast growth factor receptor family, and the activation of FGFR4 can promote cell growth and angiogenesis in cancer (Bai et al., 2015). CDK2 gene is commonly excessive activation in human cancers, and dysfunction of CDK2 can lead to uncontrolled cell growth (Mihara et al., 2001). FLT4 gene, belonging to the vascular endothelial growth factor family, had been reported to regulate cancer cell survival and proliferation (Varney and Singh, 2015). The genes in signiﬁcantly ampliﬁed loci include a number of known driver genes, which may promote the cancer progression by the up-regulation of cell growth and cell cycle. Signiﬁcantly deleted loci include some tumor suppressor genes and autophagy genes. The inactivation of these genes leads to uncontrolled tumor growth, which may contribute to the VHL/PBRM1- negative ccRCC pathogenesis and progression Functional Analysis of Signiﬁcant Genomic Copy Number Variations in Kidney Cancer Only the loci with q-value < 0.0001 were shown. FIGURE 5 \| Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis for signiﬁcantly ampliﬁed genes. The size of the point means the gene number both in our ampliﬁed gene set and KEGG pathway terms. The color of point means enrichment signiﬁcance (−log10P). The pathways were sorted by rich factor (the ratio of signiﬁcantly ampliﬁed gene number in this pathway term to gene number in this pathway term). FIGURE 5 \| Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis for signiﬁcantly ampliﬁed genes. The size of the point means the gene number both in our ampliﬁed gene set and KEGG pathway terms. The color of point means enrichment signiﬁcance (−log10P). The pathways were sorted by rich factor (the ratio of signiﬁcantly ampliﬁed gene number in this pathway term to gene number in this pathway term). FIGURE 5 \| Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis for signiﬁcantly ampliﬁed genes. The size of the point means the gene number both in our ampliﬁed gene set and KEGG pathway terms. The color of point means enrichment signiﬁcance (−log10P). The pathways were sorted by rich factor (the ratio of signiﬁcantly ampliﬁed gene number in this pathway term to gene number in this pathway term). FIGURE 4 \| The signiﬁcant genomic copy number variation (CNV) loci in cancer cells. All CNV types in each cancer cell were counted for the top frequency histogram, and q-value for each signiﬁcant genomic CNV loci was shown on the right. Only the loci with q-value < 0.0001 were shown. January 2020 \| Volume 10 \| Article 1379 Frontiers in Genetics \| www.frontiersin.org 5 Single-Cell CNVs Analysis in ccRCC Zhou et al. found involved in the pathogenesis of RCC. What’s more, the results also showed that Th17 cells (Li et al., 2015) and microRNAs (Gowrishankar et al., 2014) seem to have a connection with the ccRCC pathogenesis. Interestingly, the fatty acid elongation pathway was signiﬁcantly deleted in ccRCC, which may account for the fact that ccRCC tumors are lipid-laden (Hakimi et al., 2016). examined the copy number variations in their upstream RTK genes (Robinson et al., 2000; Secrier et al., 2016) to investigate possible reasons for the negative results that tumor did not appear known driver mutations in VHL and PBRM1. Functional Analysis of Signiﬁcant Genomic Copy Number Variations in Kidney Cancer The single cancer cells show widespread ampliﬁcation and deletion on multiple RTKs compared with the normal cells, the NC and NCinCT groups show similar RTK gene proﬁle. There were some RTK genes (EPHB6, EPHA1, EPHB3, FGFR4, PDGFRB, and FLT4) showing ampliﬁcation in cancer cells. On the contrary, EPHB2, ERBB4, FGFR1, PDGFRA, KDR, and FLT1 genes showed deletion in cancer cells (Figure 6). Genomic copy number is varied across these RTKs and downstream pathways, indicating that the genomic CNVs in RTKs and downstream Receptor Protein Tyrosine Kinase Genes Show Widespread Copy Number Variations in Cancer Cells Since lots of cancer-related signaling transduction pathways showed signiﬁcantly ampliﬁed in cancer cells, we then FIGURE 6 \| The copy number of receptor protein tyrosine kinase (RTK) genes in all single cells. The copy number variations (CNVs) on RTK genes in both tumor and normal cells were shown. The RTKs family and cell types were shown at the left and bottom of the plot. The mutation types in each cell and gene were counted for the top and right frequency histograms respectively FIGURE 6 \| The copy number of receptor protein tyrosine kinase (RTK) genes in all single cells. The copy number variations (CNVs) on RTK genes in both tumor and normal cells were shown. The RTKs family and cell types were shown at the left and bottom of the plot. The mutation types in each cell and gene were counted for the top and right frequency histograms, respectively. FIGURE 6 \| The copy number of receptor protein tyrosine kinase (RTK) genes in all single cells. The copy number variations (CNVs) on RTK genes in both tumor and normal cells were shown. The RTKs family and cell types were shown at the left and bottom of the plot. The mutation types in each cell and gene were counted for the top and right frequency histograms, respectively. FIGURE 6 \| The copy number of receptor protein tyrosine kinase (RTK) genes in all single cells. The copy number variations (CNVs) on RTK genes in both tumor and normal cells were shown. The RTKs family and cell types were shown at the left and bottom of the plot. The mutation types in each cell and gene were counted for the top and right frequency histograms, respectively. January 2020 \| Volume 10 \| Article 1379 6 Frontiers in Genetics \| www.frontiersin.org Single-Cell CNVs Analysis in ccRCC Zhou et al. FUNDING This work is supported by the National Natural Science Foundation of China [61571152, 61822108], the National Science and Technology Major Project of China [2016YFC1202302] and the Natural Science Foundation of Heilongjiang Province [F2015006]. This work is supported by the National Natural Science Foundation of China [61571152, 61822108], the National Science and Technology Major Project of China [2016YFC1202302] and the Natural Science Foundation of Heilongjiang Province [F2015006]. This work is supported by the National Natural Science Foundation of China [61571152, 61822108], the National Science and Technology Major Project of China [2016YFC1202302] and the Natural Science Foundation of Heilongjiang Province [F2015006]. Pathway analysis of these signiﬁcantly ampliﬁed and deleted genes identiﬁed several signaling transduction pathways, including HIF-1, ErbB, PI3K-Akt, Ras, Rap1, and MAPK signaling pathways, were affected by genomic ampliﬁcation. At the same time, RTK genes showed widespread copy number variations in cancer cells speciﬁcally. Mutations on RTKs may take part in the overactivity of downstream signaling transduction pathways, leading to the uncontrolled growth of ccRCC cells. DISCUSSION Previous studies have shown that VHL and PBRM1 are the top two signiﬁcantly mutated genes in ccRCC. However, the pathogenesis in VHL/PBRM1-negative ccRCC is still unclear. Our comprehensive analysis of CNVs in 25 single cells from a ccRCC patient provided new insights into the pathogenesis of the ccRCC. We reclassiﬁed all the single cells and identiﬁed pathological mutations in VHL/PBRM1-negative ccRCC cells. Similar to the genomic CNVs in other cancers, the pathogenesis in VHL/PBRM1-negative ccRCC seems to be shaped by the accumulation of ampliﬁcation in driver genes (IGFBP4, ERBB2, ERBB3, FGFR4, CDK2, and FLT4), the loss of function in tumor suppressor genes (Chmp1A, CADM2) and autophagy genes (PRAP1, ULK1). DATA AVAILABILITY STATEMENT signaling transduction pathways may have important roles in the pathogenesis and progression of the VHL/PBRM1- negative ccRCC. Publicly available datasets were analyzed in this study. The original sequencing data can be downloaded from NCBI (http://www.ncbi.nlm.nih.gov/sra) under the accession number SRA050201. AUTHOR CONTRIBUTIONS HN, LY, and QJ designed the experiments. PW obtained data from NCBI. WZ and ML analyzed the data. FY, ZX, and YG wrote the manuscript. All authors read and approved the manuscript. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fgene.2019. 01379/full#supplementary-material Overall, our single-cell analysis of the copy number in VHL/ PBRM1-negative ccRCC revealed that the genomic CNVs in RTKs may cooperate with downstream signaling transduction pathways to take part in VHL/PBRM1-negative ccRCC pathogenesis and progression. Clinically, our ﬁndings may provide more effective targeted therapeutic approaches for patients with VHL/PBRM1-negative ccRCC. However, because of the small number of cells and the high intratumor heterogeneity, our ﬁndings need to be veriﬁed in larger cohorts. TABLE S1 \| The results of dimensionality reduction based on the germline CNVs. The name and coordinate in 2D space of all single cells were shown in this table. TABLE S2 \| The results of signiﬁcantly ampliﬁed (Table S2A) and deleted (Table S2B) loci according to GISTIC2.0. The cytoband name, q-value and gene names of each ampliﬁcation/deletion loci were shown in this table. TABLE S3 \| The results of KEGG enrichment analysis based on signiﬁcantly ampliﬁed (Table S3A) and deleted (Table S3B) genes according to the CPDB database. The pathway name, p-value and gene sets of each pathway were shown in this table. TABLE S3 \| The results of KEGG enrichment analysis based on signiﬁcantly ampliﬁed (Table S3A) and deleted (Table S3B) genes according to the CPDB database. The pathway name, p-value and gene sets of each pathway were shown in this table. REFERENCES doi: 10.1093/ bioinformatics/btp324 Xu, X., Hou, Y., Yin, X., Bao, L., Tang, A., Song, L., et al. (2012). Single-cell exome sequencing reveals single-nucleotide mutation characteristics of a kidney tumor. Cell 148 (5), 886–895. doi: 10.1016/j.cell.2012.02.025 Li, L., Yang, C., Zhao, Z., Xu, B., Zheng, M., Zhang, C., et al. (2015). Skewed T- helper (Th)1/2- and Th17/T regulatorycell balances in patients with renal cell carcinoma. Mol. Med. Rep. 11 (2), 947–953. doi: 10.3892/mmr. 2014.2778 Yang, B., Zhang, L., Cao, Y., Chen, S., Cao, J., Wu, D., et al. (2017). Overexpression of lncRNA IGFBP4-1 reprograms energy metabolism to promote lung cancer progression. Mol. Cancer 16 (1), 154. doi: 10.1186/s12943-017-0722-8 Linehan, W. M., Srinivasan, R., and Schmidt, L. S. (2010). The genetic basis of kidney cancer: a metabolic disease. Nat. Rev. Urol. 7 (5), 277–285. doi: 10.1038/ nrurol.2010.47 Yarden, Y. (2001). The EGFR family and its ligands in human cancer. signalling mechanisms and therapeutic opportunities. Eur. J. Cancer 37 Suppl 4, S3–S8. doi: 10.1016/s0959-8049(01)00230-1 Liu, X., Wang, J., and Sun, G. (2015). Identiﬁcation of key genes and pathways in renal cell carcinoma through expression proﬁling data. Kidney Blood Press Res. 40 (3), 288–297. doi: 10.1159/000368504 Yilmaz, S., and Singh, A. K. (2012). Single cell genome sequencing. Curr. Opin. Biotechnol. 23 (3), 437–443. doi: 10.1016/j.copbio.2011.11.018 Mermel, C. H., Schumacher, S. E., Hill, B., Meyerson, M. L., Beroukhim, R., and Getz, G. (2011). GISTIC2.0 facilitates sensitive and conﬁdent localization of the targets of focal somatic copy-number alteration in human cancers. Genome Biol. 12 (4), R41. doi: 10.1186/gb-2011-12-4-r41 You, Z., Xin, Y., Liu, Y., Sun, J., Zhou, G., Gao, H., et al. (2012). Chmp1A acts as a tumor suppressor gene that inhibits proliferation of renal cell carcinoma. Cancer Lett. 319 (2), 190–196. doi: 10.1016/j.canlet.2012.01.010 Zhang, L., Fu, L., Zhang, S., Zhang, J., Zhao, Y., Zheng, Y., et al. (2017). Discovery of a small molecule targeting ULK1-modulated cell death of triple negative breast cancer in vitro and in vivo. Chem. Sci. 8 (4), 2687–2701. doi: 10.1039/ c6sc05368h Mihara, M., Shintani, S., Nakahara, Y., Kiyota, A., Ueyama, Y., Matsumura, T., et al. (2001). Overexpression of CDK2 is a prognostic indicator of oral cancer progression. Jpn. J. Cancer Res. 92 (3), 352–360. doi: 10.1111/j.1349- 7006.2001.tb01102.x 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. Nouhaud, F. REFERENCES 29 (12), 1078–1079. doi: 10.1038/nbt.2058 Ueno, K., Hirata, H., Majid, S., Tabatabai, Z. L., Hinoda, Y., and Dahiya, R. (2011). IGFBP-4 activates the Wnt/beta-catenin signaling pathway and induces M- CAM expression in human renal cell carcinoma. Int. J. Cancer 129 (10), 2360– 2369. doi: 10.1002/ijc.25899 He, W., Li, X., Xu, S., Ai, J., Gong, Y., Gregg, J. L., et al. (2013). Aberrant methylation and loss of CADM2 tumor suppressor expression is associated with human renal cell carcinoma tumor progression. Biochem. Biophys. Res. Commun. 435 (4), 526–532. doi: 10.1016/j.bbrc.2013.04.074 Varela, I., Tarpey, P., Raine, K., Huang, D., Ong, C. K., Stephens, P., et al. (2011). Exome sequencing identiﬁes frequent mutation of the SWI/SNF complex gene PBRM1 in renal carcinoma. Nature 469 (7331), 539–542. doi: 10.1038/ nature09639 Henson, E., Chen, Y., and Gibson, S. (2017). EGFR family members’ regulation of autophagy is at a crossroads of cell survival and death in cancer. Cancers (Basel) 9 (4), 27–40. doi: 10.3390/cancers9040027 Hong, X., Qiao, S., Li, F., Wang, W., Jiang, R., Wu, H., et al. (2019). Whole-genome sequencing reveals distinct genetic bases for insulinomas and non-functional pancreatic neuroendocrine tumours: leading to a new classiﬁcation system. Gut 0, 1–11. doi: 10.1136/gutjnl-2018-317233 Varney, M. L., and Singh, R. K. (2015). VEGF-C-VEGFR3/Flt4 axis regulates mammary tumor growth and metastasis in an autocrine manner. Am. J. Cancer Res. 5 (2), 616–628. Waddell, N., Pajic, M., Patch, A. M., Chang, D. K., Kassahn, K. S., Bailey, P., et al. (2015). Whole genomes redeﬁne the mutational landscape of pancreatic cancer. Nature 518 (7540), 495–501. doi: 10.1038/nature14169 Kaelin, W. G. Jr. (2004). The von Hippel-Lindau tumor suppressor gene and kidney cancer. Clin. Cancer Res. 10 (18 Pt 2), 6290S–6295S. doi: 10.1158/1078- 0432.CCR-sup-040025 Wang, K., Li, M., and Hakonarson, H. (2010). ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 38 (16), e164. doi: 10.1093/nar/gkq603 Kamburov, A., Stelzl, U., Lehrach, H., and Herwig, R. (2013). The ConsensusPathDB interaction database: 2013 update. Nucleic Acids Res. 41 (Database issue), D793–D800. doi: 10.1093/nar/gks1055 Wang, Y., Waters, J., Leung, M. L., Unruh, A., Roh, W., Shi, X., et al. (2014). Clonal evolution in breast cancer revealed by single nucleus genome sequencing. Nature 512 (7513), 155–160. doi: 10.1038/nature13600 Li, H., and Durbin, R. (2009). Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25 (14), 1754–1760. REFERENCES Chen, Y. L., Ge, G. J., Qi, C., Wang, H., Wang, H. L., Li, L. Y., et al. (2018). A ﬁve-gene signature may predict sunitinib sensitivity and serve as prognostic biomarkers for renal cell carcinoma. J. Cell Physiol. 233 (10), 6649–6660. doi: 10.1002/jcp.26441 Bai, Y. P., Shang, K., Chen, H., Ding, F., Wang, Z., Liang, C., et al. (2015). FGF-1/- 3/FGFR4 signaling in cancer-associated ﬁbroblasts promotes tumor progression in colon cancer through Erk and MMP-7. Cancer Sci. 106 (10), 1278–1287. doi: 10.1111/cas.12745 Cheng, L., Sun, J., Xu, W., Dong, L., Hu, Y., and Zhou, M. (2016). OAHG: an integrated resource for annotating human genes with multi-level ontologies. Sci. Rep. 6, 34820. doi: 10.1038/srep34820 Boeva, V., Popova, T., Bleakley, K., Chiche, P., Cappo, J., Schleiermacher, G., et al. (2012). Control-FREEC: a tool for assessing copy number and allelic content using next-generation sequencing data. Bioinformatics 28 (3), 423–425. doi: 10.1093/bioinformatics/btr670 Cheng, L., Wang, P., Tian, R., Wang, S., Guo, Q., Luo, M., et al. (2019). LncRNA2Target v2.0: a comprehensive database for target genes of lncRNAs in human and mouse. Nucleic Acids Res. 47 (D1), D140–D144. doi: 10.1093/ nar/gky1051 Cancer Genome Atlas Research, N. (2013). Comprehensive molecular characterization of clear cell renal cell carcinoma. Nature 499 (7456), 43–49. doi: 10.1038/nature12222 de Araujo Junior, R. F., Leitao Oliveira, A. L., de Melo Silveira, R. F., de Oliveira Rocha, H. A., de Franca Cavalcanti, P., and de Araujo, A. A. (2015). Telmisartan induces apoptosis and regulates Bcl-2 in human renal cancer cells. Exp. Biol. Med. (Maywood) 240 (1), 34–44. doi: 10.1177/ 1535370214546267 Casasent, A. K., Schalck, A., Gao, R., Sei, E., Long, A., Pangburn, W., et al. (2018). Multiclonal invasion in breast tumors identiﬁed by topographic single cell sequencing. Cell 172 (1-2), 205–217 e212. doi: 10.1016/j.cell.2017.12.007 Gerlinger, M., Horswell, S., Larkin, J., Rowan, A. J., Salm, M. P., Varela, I., et al. (2014). Genomic architecture and evolution of clear cell renal cell carcinomas deﬁned by multiregion sequencing. Nat. Genet. 46 (3), 225–233. doi: 10.1038/ ng.2891 Chen, Y., He, J., Tian, M., Zhang, S. Y., Guo, M. R., Kasimu, R., et al. (2014). UNC51-like kinase 1, autophagic regulator and cancer therapeutic target. Cell Prolif. 47 (6), 494–505. doi: 10.1111/cpr.12145 January 2020 \| Volume 10 \| Article 1379 Frontiers in Genetics \| www.frontiersin.org 7 Single-Cell CNVs Analysis in ccRCC Zhou et al. Gnarra, J. R., Tory, K., Weng, Y., Schmidt, L., Wei, M. REFERENCES H., Li, H., et al. (1994). Mutations of the VHL tumour suppressor gene in renal carcinoma. Nat. Genet. 7 (1), 85–90. doi: 10.1038/ng0594-85 Robinson, D. R., Wu, Y. M., and Lin, S. F. (2000). The protein tyrosine kinase family of the human genome. Oncogene 19 (49), 5548–5557. doi: 10.1038/sj.onc.1203957 Romero, D., O’Neill, C., Terzic, A., Contois, L., Young, K., Conley, B. A., et al. (2011). Endoglin regulates cancer-stromal cell interactions in prostate tumors. Cancer Res. 71 (10), 3482–3493. doi: 10.1158/0008-5472.CAN-10-2665 Gowrishankar, B., Ibragimova, I., Zhou, Y., Slifker, M. J., Devarajan, K., Al-Saleem, T., et al. (2014). MicroRNA expression signatures of stage, grade, and progression in clear cell RCC. Cancer Biol. Ther. 15 (3), 329–341. doi: 10.4161/cbt.27314 Sato, Y., Yoshizato, T., Shiraishi, Y., Maekawa, S., Okuno, Y., Kamura, T., et al. Sato, Y., Yoshizato, T., Shiraishi, Y., Maekawa, S., Okuno, Y., Kamura, T., et al. (2013). Integrated molecular analysis of clear-cell renal cell carcinoma. Nat. Genet. 45 (8), 860–867. doi: 10.1038/ng.2699 (2013). Integrated molecular analysis of clear-cell renal cell carcinoma. Nat. Genet. 45 (8), 860–867. doi: 10.1038/ng.2699 Guo, H., German, P., Bai, S., Barnes, S., Guo, W., Qi, X., et al. (2015). The PI3K/ AKT pathway and renal cell carcinoma. J. Genet. Genomics 42 (7), 343–353. doi: 10.1016/j.jgg.2015.03.003 Schiewer, M. J., and Knudsen, K. E. (2014). Transcriptional roles of PARP1 in cancer. Mol. Cancer Res. 12 (8), 1069–1080. doi: 10.1158/1541-7786.MCR-13-0672 Hakimi, A. A., Pham, C. G., and Hsieh, J. J. (2013). A clear picture of renal cell carcinoma. Nat. Genet. 45 (8), 849–850. doi: 10.1038/ng.2708 Secrier, M., Li, X., de Silva, N., Eldridge, M. D., Contino, G., Bornschein, J., et al. (2016). Mutational signatures in esophageal adenocarcinoma deﬁne etiologically distinct subgroups with therapeutic relevance. Nat. Genet. 48 (10), 1131–1141. doi: 10.1038/ng.3659 Hakimi, A. A., Reznik, E., Lee, C. H., Creighton, C. J., Brannon, A. R., Luna, A., et al. (2016). An integrated metabolic atlas of clear cell renal cell carcinoma. Cancer Cell 29 (1), 104–116. doi: 10.1016/j.ccell.2015.12.004 Sigismund, S., Avanzato, D., and Lanzetti, L. (2018). Emerging functions of the EGFR in cancer. Mol. Oncol. 12 (1), 3–20. doi: 10.1002/1878-0261.12155 Hallberg, L. M., Ikeno, Y., Englander, E., and Greeley, G. H.Jr. (2000). Effects of aging and caloric restriction on IGF-I, IGF-I receptor, IGFBP-3 and IGFBP-4 gene expression in the rat stomach and colon. Regul. Pept. 89 (1-3), 37–44. doi: 10.1016/s0167-0115(00)00095-1 Tulin, A. (2011). Re-evaluating PARP1 inhibitor in cancer. Nat. Biotechnol. REFERENCES X., Blanchard, F., Sesboue, R., Flaman, J. M., Sabourin, J. C., Pﬁster, C., et al. (2018). Clinical relevance of gene copy number variation in metastatic clear cell renal cell carcinoma. Clin. Genitourin Cancer 16 (4), e795–e805. doi: 10.1016/j.clgc.2018.02.013 Oldrini, B., Hsieh, W. Y., Erdjument-Bromage, H., Codega, P., Carro, M. S., Curiel- Garcia, A., et al. (2017). EGFR feedback-inhibition by Ran-binding protein 6 is disrupted in cancer. Nat. Commun. 8 (1), 2035. doi: 10.1038/s41467-017- 02185-w Copyright © 2020 Zhou, Yang, Xu, Luo, Wang, Guo, Nie, Yao and Jiang. 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. Posadas, E. M., Limvorasak, S., Sharma, S., and Figlin, R. A. (2013). Targeting angiogenesis in renal cell carcinoma. Exp. Opin. Pharmacother. 14 (16), 2221– 2236. doi: 10.1517/14656566.2013.832202 January 2020 \| Volume 10 \| Article 1379 Frontiers in Genetics \| www.frontiersin.org 8
https://openalex.org/W4391132517	https://insightsimaging.springeropen.com/counter/pdf/10.1186/s13244-023-01555-x	English	null	Assessment of lung deformation in patients with idiopathic pulmonary fibrosis with elastic registration technique on pulmonary three-dimensional ultrashort echo time MRI	Insights into imaging	2,024	cc-by	7,918	Abstract Objective To assess lung deformation in patients with idiopathic pulmonary fibrosis (IPF) using with elastic regis- tration algorithm applied to three-dimensional ultrashort echo time (3D-UTE) MRI and analyze relationship of lung deformation with the severity of IPF. Methods Seventy-six patients with IPF (mean age: 62 ± 6 years) and 62 age- and gender-matched healthy controls (mean age: 58 ± 4 years) were prospectively enrolled. End-inspiration and end-expiration images acquired with a sin- gle breath-hold 3D-UTE sequence were registered using elastic registration algorithm. Jacobian determinants were calculated from deformation fields and represented on color maps. Jac-mean (absolute value of the log means of Jacobian determinants) and the Dice similarity coefficient (Dice) were compared between different groups. Methods Seventy-six patients with IPF (mean age: 62 ± 6 years) and 62 age- and gender-matched healthy controls (mean age: 58 ± 4 years) were prospectively enrolled. End-inspiration and end-expiration images acquired with a sin- gle breath-hold 3D-UTE sequence were registered using elastic registration algorithm. Jacobian determinants were calculated from deformation fields and represented on color maps. Jac-mean (absolute value of the log means of Jacobian determinants) and the Dice similarity coefficient (Dice) were compared between different groups. Results Compared with healthy controls, the Jac-mean of IPF patients significantly decreased (0.21 ± 0.08 vs. 0.27 ± 0. 07, p < 0.001). Furthermore, the Jac-mean and Dice correlated with the metrics of pulmonary function tests and the composite physiological index. The lung deformation in IPF patients with dyspnea Medical Research Council (MRC) ≥ 3 (Jac-mean: 0.16 ± 0.03; Dice: 0.06 ± 0.02) was significantly lower than MRC1 (Jac-mean: 0. 25 ± 0.03, p < 0.001; Dice: 0.10 ± 0.01, p < 0.001) and MRC 2 (Jac-mean: 0.22 ± 0.11, p = 0.001; Dice: 0.08 ± 0.03, p = 0.006). Mean- while, Jac-mean and Dice correlated with health-related quality of life, 6 min-walk distance, and the extent of pulmo- nary fibrosis. Jac-mean correlated with pulmonary vascular-related indexes on high-resolution CT. Conclusion The decreased lung deformation in IPF patients correlated with the clinical severity of IPF patients. Elastic registration of inspiratory-to-expiratory 3D UTE MRI may be a new morphological and functional marker for non-radi- ation and noninvasive evaluation of IPF. Critical relevance statement This prospective study demonstrated that lung deformation decreased in idiopathic pulmonary fibrosis (IPF) patients and correlated with the severity of IPF. Abstract Elastic registration of inspiratory-to-expiratory three-dimensional ultrashort echo time (3D UTE) MRI may be a new morphological and functional marker for non- radiation and noninvasive evaluation of IPF. Correspondence: Min Liu mikie0763@126.com Huaping Dai daihuaping@ccmu.edu.cn Full list of author information is available at the end of the article © The Author(s) 2024. 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 Author(s) 2024. 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/. Yang et al. Insights into Imaging (2024) 15:17 https://doi.org/10.1186/s13244-023-01555-x Yang et al. Insights into Imaging (2024) 15:17 https://doi.org/10.1186/s13244-023-01555-x ORIGINAL ARTICLE Open Access Assessment of lung deformation in patients with idiopathic pulmonary fibrosis with elastic registration technique on pulmonary three‑dimensional ultrashort echo time MRI Xiaoyan Yang1,2, Pengxin Yu3, Haishuang Sun2, Mei Deng4, Anqi Liu4, Chen Li2, Wenyan Meng2, Wenxiu Xu2, Bingbing Xie2, Jing Geng2, Yanhong Ren2, Rongguo Zhang3, Min Liu4 and Huaping Dai1,2* Open Access Open Access Assessment of lung deformation in patients with idiopathic pulmonary fibrosis with elastic registration technique on pulmonary three‑dimensional ultrashort echo time MRI Xiaoyan Yang1,2, Pengxin Yu3, Haishuang Sun2, Mei Deng4, Anqi Liu4, Chen Li2, Wenyan Meng2, Wenxiu Xu2, Bingbing Xie2, Jing Geng2, Yanhong Ren2, Rongguo Zhang3, Min Liu4* and Huaping Dai1,2* Key points y • Elastic registration of inspiratory-to-expiratory three-dimensional ultrashort echo time (3D UTE) MRI could evaluate lung deformation. • Elastic registration of inspiratory-to-expiratory three-dimensional ultrashort echo time (3D UTE) MRI could evaluate lung deformation. • Lung deformation significantly decreased in idiopathic pulmonary fibrosis (IPF) patients, compared with the healthy controls. • Reduced lung deformation of IPF patients correlated with worsened pulmonary function and the composite physi- • Lung deformation significantly decreased in idiopathic pulmonary fibrosis (IPF) patients, compared with the healthy controls. • Reduced lung deformation of IPF patients correlated with worsened pulmonary function and the composite physi- ological index (CPI). Keywords Elastic registration, Idiopathic pulmonary fibrosis, Magnetic resonance imaging, Ultrashort echo time • Lung deformation significantly decreased in idiopathic pulmonary fibrosis (IPF) patients, compared with the healthy controls. • Reduced lung deformation of IPF patients correlated with worsened pulmonary function and the composite physi- ological index (CPI). Keywords Elastic registration, Idiopathic pulmonary fibrosis, Magnetic resonance imaging, Ultrashort echo time Keywords Elastic registration, Idiopathic pulmonary fibrosis, Magnetic resonance imaging, Ultrashort echo time Graphical Abstract Yang et al. Insights into Imaging (2024) 15:17 Page 2 of 13 Yang et al. Insights into Imaging Introduction transformation necessary to align a source image with a target image. This is particularly useful for images taken at different time points or stages of the same time, as the algorithm can automate and quantify the differences between the two images [4, 5]. Recently, the elastic reg- istration algorithm has been paid attention to assess- ing lung deformation in acute lung injury [6], chronic obstructive pulmonary disease [7, 8], and asthmatic [9]. Chassagnon et al. used elastic registration on HRCT images to evaluate the morphological and functional deterioration of systemic sclerosis-related interstitial lung disease (SSc-ILD) [10]. Moreover, it has been shown that performing elastic registration between baseline and follow-up HRCT can be advantageous in quantitatively Idiopathic pulmonary fibrosis (IPF) is a chronic, pro- gressive fibrotic lung disease, ultimately causing to res- piratory failure and mortality [1]. At present, pulmonary function tests (PFTs) and high-resolution computed tomography (HRCT) are the most important methods for the diagnosis and evaluation of IPF [2, 3]. However, PFTs only offer a general overview of lung function and do not provide detailed information about the regional func- tion of IPF, while visual assessments of HRCT images are subjective and depend on the expertise of the radiologist, which can limit their reliability and reproducibility. The elastic registration algorithm is a specific type of imaging registration method designed to determine the Page 3 of 13 Page 3 of 13 Yang et al. Insights into Imaging (2024) 15:17 assessing the morphological deterioration of lung shrink- age that is characteristic of IPF [11]. included. IPF patients were diagnosed by the multidisci- plinary team using the diagnostic criteria established by the American Thoracic Society, European Respiratory Society, Japanese Respiratory Society, and Latin Ameri- can Thoracic Association (ATS/ERS/JRS/ALAT) in 2018 [1]. The people with normal HRCT findings and PFTs were included in the control group. Exclusion crite- ria include (I) participants with cancer or infection; (II) participants with MRI contraindications, such as a pace- maker or claustrophobia; and (III) participants who failed to complete MRI scan or had poor image quality with the obvious artifact. All participants underwent MRI, HRCT, and PFTs within 24 h. Figure 1 showed the flowchart of how participants were enrolled. To minimize the cumulative radiation exposure associ- ated with frequent CT scans, ultrashort echo time (UTE) and zero echo time (ZTE) sequences have been intro- duced into clinical MRI protocols. Introduction These sequences can depict structural and functional alterations and can even be used in children [12, 13]. UTE is capable of generat- ing CT-like contrast in the lung parenchyma [14], and 3D-UTE MRI has the high reproducibility to identify the imaging features of IPF and evaluate the extent of pulmo- nary fibrosis [15]. i Using elastic registration analysis of inspiratory-to- expiratory UTE MRI, Chassagnon et al. demonstrated a reduction in lung base respiratory deformation in patients with systemic sclerosis-related pulmonary fibro- sis, compared to those without fibrosis [16]. Thus, we hypothesized that lung deformation with elastic regis- tration technique on 3D-UTE MRI could be used as a potential biomarker for the severity of IPF. We aimed to analyze the correlation of lung deformation with pulmo- nary function, dyspnea, exercise tolerance, health-related quality of life (HRQoL), and the extent of pulmonary fibrosis on HRCT in patients with IPF. The severity of resting dyspnea in IPF patients was assessed using the Medical Research Council (MRC) scale [17]. Health-related quality of life was evaluated based on a respiratory-specific questionnaire, the St. George’s Respiratory Questionnaire (SGRQ) [18]. This questionnaire encompasses three domains, including res- piratory symptoms, activity, and the psychosocial impact of the disease, and scores ranging from 0 to 100 indicate a worse quality of life. Additionally, the study also meas- ured the 6-min walking distance (6-MWD) of all patients. HRCT and quantitative analysis All participants underwent HRCT using multidetec- tor CT systems (Toshiba Aquilion ONE TSX-301C/320; Philips iCT/256) in full inspiration. The chest was scanned from the lung apex to the lowest hemidiaphragm in a single breath-hold in a supine position with the following acquisition parameters and reconstruction parameters: tube voltage = 100–120 kV, tube current = 100–300 mAs, section thickness = 0.625–1 mm, table speed = 39.37 mm/s, gantry rotation time = 0.8 s, and reconstruction increment = 1–1.25 mm. Segmentation of lung and fibrosis lesions was per- formed in the software InferScholar (https://www.infer vision.com/) [21]. The full lung region was automatically segmented and then manually corrected by one radi- ologist. According to Christe et al. [22], ground-glass opacities (GGO), reticulation, and honeycombing signs on HRCT were outlined (Supplementary figure 1). The extent of lung fibrosis was quantified as the percentage of the volume occupied by reticulation and honeycombing in relation to the total lung volume. Two metrics are used to evaluate the quality of elas- tic registration. The first metric is the intersection over union (IoU) [26], which measures the overlap degree between the lung area of the inspiratory image after reg- istration and that of the expiratory image. The second metric measured the distance between the landmark in the inspiratory image after registration and that in the expiratory image. Specifically, an observer manu- ally places two categories of landmarks A and B on both inspiratory and expiratory images (Supplementary fig- ure 3). After the registration is completed, the landmarks on the inspiratory image are automatically registered to the expiratory image. The landmarks coordinates were collected to calculate the mean distances of two land- marks: DA, the distance between registered landmark- A and original landmark-A in the expiratory image; and DB, the distance between two landmark-Bs. To verify the repeatability and stability of the registration method, two times of respiratory phases (inspiratory and expiratory) were acquired for each participant. IoU, DA, and DB are calculated for each respiratory phase, and the consistency between the two phases is calculated. According to previous studies [23, 24], pulmonary ves- sels on HRCT were automatically segmented using an integrated segmentation method that employs automated algorithms (the FACT-Digital Lung Workstation, Dexin) and subsequently reviewed by the radiologist and manu- ally corrected (Supplementary figure 2). HRCT and quantitative analysis The main vascu- lar parameters quantified included the volume, number, and tortuosity of pulmonary vessel branches of total pul- monary vascular (TPV), pulmonary vein vascular (PVV), and pulmonary artery vascular (PAV). Elastic registration Figure 2a shows the elastic registration of inspiratory and expiratory UTE images. First, all inspiratory and expiratory UTE images were preprocessed by isotropic sampling with 1 mm. Then, the lung regions were auto- matically segmented with the InferScholar (https://www. infervision.com/). An experienced observer checked lung segmentation results to ensure that the lungs were completely and accurately identified. The fined lung segmentation was then dilated by ten pixels to con- tain the peri-lungs. ElasticSyN algorithm was used to perform elastic registration of inspiratory (moving) to expiratory (fixed) images. This algorithm is integrated into a stable and powerful open-source software called Advanced Normalization Tools (ANTs) (https://github. com/ANTsX/ANTs) [25]. Magnetic resonance imaging All patients underwent chest MRI using a 1.5T MRI scanner (MAGNETOM Aera; Siemens Healthcare) with an 18-channel phased-array body coil. Two three-dimen- sional, ultrashort echo time, gradient echo spiral volume interpolated breath-hold examination sequences were acquired during a single respiratory phase, one follow- ing a full inspiration and the other after a full expiration. Both sequences were performed in the coronal plane and had a duration of 15 s. The key parameters as following: repetition time = 2.73 msec; echo time = 0.05 msec; flip angle = 5°; field-of-view = 500 × 500 mm; slice thick- ness = 2.5 mm; matrix = 240 × 240; in-plane resolution Materials and methods Study design and cohorth All participants underwent PFTs (MasterScreen, Vyaire Medical GmbH) according to the standards of ATS/ ERS [19]. PFT measurements included the percent- age of predicted forced vital capacity (FVC%), percent- age of forced expiratory volume in 1 s (FEV1%), FEV1/ FVC%, percentage of predicted total lung capacity This prospective single-center cohort study was approved Institutional review board of our hospital (2019-123- K85-1). All participants provided written informed con- sent. From January 2021 and June 2022, patients with IPF and age- and gender-matched healthy controls were Fig. 1 The flowchart of inclusion and exclusion criteria of idiopathic pulmonary fibrosis patients Fig. 1 The flowchart of inclusion and exclusion criteria of idiopathic pulmonary fibrosis patients Yang et al. Insights into Imaging (2024) 15:17 Page 4 of 13 = 2.08 × 2.08 mm; spiral duration = 1800 μsec. Image reconstructions used the non-uniform Fourier trans- form (NUFT) method. To promise full inspiration and expiration, each participant was trained to practice deep breathing in the supine position before the MRI scan. (TLC%), and percentage of predicted DLco corrected for measured hemoglobin (DLco%). Additionally, the com- posite physiological index (CPI) was calculated using the following formula: CPI = 91 − (0.65 × %predicted DLCO) − (0.53 × %predicted FVC) + (0.34 × %predicted FEV1) [20]. (TLC%), and percentage of predicted DLco corrected for measured hemoglobin (DLco%). Additionally, the com- posite physiological index (CPI) was calculated using the following formula: CPI = 91 − (0.65 × %predicted DLCO) − (0.53 × %predicted FVC) + (0.34 × %predicted FEV1) [20]. Analysis of lung deformation For each participant, the Jacobian determinant (JAC) was calculated through the deformation field resulting from respiratory registration. JAC is a matrix of the same size as the expiratory image, where each value indicates whether the corresponding voxel stretched (greater than 1) or shrunk (less than 1). The JAC of each participant Yang et al. Insights into Imaging (2024) 15:17 Page 5 of 13 Yang et al. Insights into Imaging Fig. 2 The flowchart of elastic registration. a The procedure of inspiratory-to-expiratory imaging registration of transversal lung MRI. The inspiratory and expiratory images of the patient were subjected to isotropic resampling and then the lung and peri-lung regions were extracted as regions of interest (ROI). Based on the ROI only, the inspiratory image is registered to the expiratory image to obtain the registered inspiratory image. b The steps of Jacobian determinant analysis. First, a common space is defined, and the expiratory images of all participants are registered to this common space, and the registration affine matrix of each participant is obtained. Then, based on each participant’s affine matrix, a quadratic transformation of the respective Jacobian determinant (JAC) is performed. Finally, participants were grouped, and various comparisons and analyses were made between groups based on the registered JAC Fig. 2 The flowchart of elastic registration. a The procedure of inspiratory-to-expiratory imaging registration of transversal lung MRI. The inspiratory and expiratory images of the patient were subjected to isotropic resampling and then the lung and peri-lung regions were extracted as regions of interest (ROI). Based on the ROI only, the inspiratory image is registered to the expiratory image to obtain the registered inspiratory image. b The steps of Jacobian determinant analysis. First, a common space is defined, and the expiratory images of all participants are registered to this common space, and the registration affine matrix of each participant is obtained. Then, based on each participant’s affine matrix, a quadratic transformation of the respective Jacobian determinant (JAC) is performed. Finally, participants were grouped, and various comparisons and analyses were made between groups based on the registered JAC project the IAJ and HAJ to different directions along the x, y, and z axis to get the visual deformation on coronal, sagittal, and axial views.i was first normalized by the ratio of their inspiratory and expiratory lung volumes, resulting in JAC-N. Analysis of lung deformation To better distinguish stretched and shrunk numerically, JAC-N is performed logarithmically to obtain JAC-NL, where a positive value implies voxel stretch, a negative value implies voxel shrinkage, and 0 implies no change. For quantitative analysis, Jac-mean, defined as the absolute value of mean JAC-NLC for each participant, was calculated for inter-group comparative analysis. In addition, followed Chassagnon et al. [16], we defined marked deformation lung areas as those with JAC-NLC values below 0.15 (cutoff value). First, healthy deforma- tion template is segmented by using the cutoff value on the HAJ, which represents the lung motion pattern when breathing in a healthy volunteer group. Then, Dice simi- larity coefficient was used to calculate the consistency of the marked deformation area with healthy deforma- tion template for each IPF patient. Specifically, the area Figure 2b shows analysis of lung deformation. A full- expiratory image of a healthy volunteer was randomly selected as a common space. The JAC-NL of each partici- pant was registered to this common space based on their lung mask, resulting in JAC-NLC. For visual analysis, JAC-NLC of all IPF patients and healthy volunteers were averaged to obtain the corre- sponding average Jacobian determinant respectively: IAJ for IPF patients and HAJ for healthy volunteers. Then, Page 6 of 13 Yang et al. Insights into Imaging (2024) 15:17 distance A, ICC = 0.83; distance B, ICC = 0.80) (Fig. 3). The mean difference and 95% limits of agreement (95% LOA) between two respiratory phrases for IoU, DA, and DB were 0.01 (− 0.04 to 0.04), 0.26 (− 4.01 to 4.59), and 0.03 (− 3.89 to 3.94), respectively. that overlaps the marked deformation area of each IPF patient and the HAJ is recorded as true positive (TP), the marked deformation area that belongs to the HAJ but not the IPF patient is recorded as false negative (FN), and the marked deformation area belonging to the IPF patient but not belonging to the HAJ is recorded as false posi- tive (FP), then the Dice similarity coefficient is calculated as 2 × TP/(2 × TP + FN + FP). For each IPF patient, the larger the Dice, the more consistent the lung motion pat- tern is with that of healthy volunteer group, and, con- versely, the lower the consistency. Statistical analysis All statistical analyses were performed with SPSS 26.0 (IBM Corp, Armonk). The unpaired t-test and Mann- Whitney U test (quantitative data) or chi-square test (categorical variables) were used for comparing different groups. The Spearman correlation coefficient was used to analyze the correlation between the lung deforma- tion and severity of IPF. The Bland-Altman analysis [27] and intraclass coefficients (ICC) were used to determine the repeatability of the method. ICCs were classified from null (= 0) to very good (> 0.80) and almost perfect (> 0.95) [28]. Statistical significance was assumed when two-tail p ≤ 0.05. Lung deformation and the severity of IPF Figure 5 (a–e) indicated that the Jac-mean positively cor- related with the FVC% predicted (r = 0.407, p < 0.01), FEV1% predicted (r = 0.379, p < 0.01), TLC% predicted (r = 0.357, p < 0.01), DLco% predicted (r = 0.486, p < 0.01), and negatively correlated with CPI (r = − 0.477, p < 0.01). Meanwhile, the Dice positively correlated with FVC% predicted (r = 0.248, p < 0.05), FEV1% predicted (r = 0.265, p < 0.05), DLco% predicted (r = 0.305, p < 0.05), and negatively correlated with CPI (r = − 0.245, p < 0.05) (Fig. 5f–i). Baseline characteristics Seventy-six patients with IPF (72 men, mean age: 62 ± 6 years) and 62 healthy controls (58 men, mean age: 58 ± 4 years) were prospectively enrolled. Table 1 showed the demographic characteristics of IPF patients and healthy controls. Gender and body mass index (BMI) were com- parable between two groups. The mean values of FVC% predicted (80.9 ± 14.5), FEV1% (82.4 ± 14.4), TLC% (67.7 ± 11.1), and DLco% (54.8 ± 15.2) predicted for IPF patients significantly decreased in comparison with the controls. CPI in IPF patients (39.1, [IQR: 32.4, 48.1]) was significantly more than healthy controls (5.6, [IQR: − 8.0, 13.7], p < 0.001). As shown in Table 2, the lung deformation in IPF patients with MRC ≥ 3 (Jac-mean: 0.16 ± 0.03; Dice: 0.06 ± 0.02) was significantly lower than MRC 1 (Jac-mean: 0.25 ± 0.03, p < 0.001; Dice: 0.10 ± 0.01, p < 0.001) and MRC 2 (Jac-mean: 0.22 ± 0.11, p = 0.001; Dice: 0.08 ± 0.03, p = 0.006). Meanwhile, the Jac-mean in patients with MRC 1 was higher than MRC 2 (p = 0.026); however, the Dice was comparable between MRC 1 and MRC 2 (p = 0.236). In the aspect of health-related quality of life, the Jac- mean (Fig. 6a–d) was negatively correlated with res- piratory symptoms (r = − 0.401, p < 0.01), activity (r = − 0.456, p < 0.01), psychosocial impact (r = − 0.349, p < 0.01), and total score (r = − 0.465, p < 0.01). Moreover, the Jac-mean also correlated with 6MWD (r = 0.504, p < 0.01) (Fig. 6e) and the extent of pulmonary fibrosis on HRCT(r = − 0. 352, p < 0.01 ) (Fig. 6f). The Dice similarity coef- ficient negatively correlated with respiratory symptoms (r = − 0.418, p < 0. 01), activity (r = − 0.414, p < 0.05), Comparison of lung deformation between IPF patients and the controlsf Figure 4 displays color maps illustrating the differ- ences in lung deformation between IPF patients and healthy controls. These deformation maps were gener- ated in the coronal, sagittal, and transversal directions. Comparing the deformation patterns between the two groups, healthy controls had a significant deforma- tion during expiration, particularly in the peripheral regions of the lungs. In contrast, IPF patients exhibited the decreased deformation in the peripheral regions of the lungs during expiration. The Jac-mean of IPF patients was found to be 0.21 ± 0.08, which was sig- nificantly lower than the Jac-mean of healthy controls (0.27 ± 0.07, p < 0.001). All the aforementioned procedures were implemented using the Python programming language (version 3.8; Python Software Foundation) within the Ubuntu operat- ing system environment (version 16.04; Canonical Ltd.). Repeatability of elastic registration algorithmh 190 Weight (kg) 72.1 ± 11.2 71.7 ± 7.4 0.203 0.839 BMI (m/kg2) 25.0 ± 2.7 24.9 ± 2.3 0.294 0.769 Pulmonary function FVC % predicted 80.9 ± 14.5 105.9 ± 14.1 − 10.229 < 0.001* FEV1% predicted 82.4 ± 14.4 100.3 ± 12.2 − 7.775 < 0.001* FEV1/FVC% predicted 81.4 ± 6.1 81.7 ± 8.8 − 0.236 0.814 TLC % predicted 67.7 ± 11.1 99.2 ± 13.9 − 14.753 < 0.001* DLCO % predicted 54.8 ± 15.2 102.3 ± 18.1 − 16.801 < 0.001* CPI 39.1 (32.4, 48.1) 5.6 (− 8.0, 13.7) − 9.845 < 0.001* 6-MWD (m) 462.7 ± 67.1 \ Resting dyspnea (MRC), n (%) 0 0 \ 1 24 (31.6%) \ 2 34 (44.7%) \ 3 12 (15.8%) \ 4 6 (7.9%) \ HRQoL Respiratory symptoms (%) 51.5 (32.1, 63.5) \ Activity (%) 42.1 (27.6, 55.6) \ Psychosocial impact (%) 16.9 (7.8, 28.4) \ Total (%) 31.1 (14.9, 40.4) \ The percentage of pulmonary fibrosis on HRCT Pulmonary fibrosis (%) 8.3 (4.8, 16.3) \ Pulmonary vascular-related indexes TPV volume (ml) 181.22 (160.61, 219.29) 194.56 (182.75, 211.33) − 1.627 0.104 TPV number 1706 (1422, 2185) 2716 (2414, 2998) − 8.408 < 0.001* TPV tortuosity 1.12 (1.09, 1.18) 1.07 (1.05, 1.08) − 8.396 < 0.001* PVV volume (ml) 94.55 (80.44, 114.72) 95.78 (86.74, 99.98) − 0.634 0.526 PVV number 930 (722, 1139) 1320 (1190, 1478) − 6.353 < 0.001* PVV tortuosity 1.11 (1.08, 1.16) 1.07 (1.05, 1.08) − 7.087 < 0.001* PAV volume (ml) 90.20 (78.32, 105.80) 99.37 (92.05, 113.25) − 2.877 0.004 PAV number 805 (550, 956) 1352 (1173, 1476) − 9.487 < 0.001* PAV tortuosity 1.12 (1.10, 1.12) 1.06 (1.05, 1.08) − 8.194 < 0.001* Table 1 The characteristics of all participants * p < 0.05, IPF, idiopathic pulmonary fibrosis; BMI, body mass index; FEV1, forced expiratory volume; FVC, forced vital capacity; TLC, total lung capacity; DLco, diffusing capacity of the lungs for carbon monoxide; CPI, composite physiological index; 6-MWD, 6 min-walk distance; HRQoL, health-related quality of life; ILD, interstitial lung disease; HRCT, high-resolution computed tomography; TPV, total pulmonary vascular; PVV, pulmonary vein vascular; PAV, pulmonary artery vascular 0.245, p < 0.01; number: r = 0.277, p < 0.01; tortuosity: r = − 0.320, p < 0.01), PVV (number: r = 0.188, p < 0.05; tortuosity: r = − 0.210, p < 0.05), and PAV (volume: r = 0.267, p < 0.01; number: r = 0.303, p < 0.01; tortuosity: r = − 0.350, p < 0.01), while the Dice similarity coefficient showed no correlation with pulmonary vascular-related indexes on HRCT. Repeatability of elastic registration algorithmh The mean value of IoU was 0.88 ± 0.03. In addition, the mean distance between the landmark as (DA) and landmark-Bs (DB) were 7.1 ± 3.8 mm and 7.0 ± 3.1 mm, respectively. The inter-observer agreement between the two respiratory phases was very good (IoU, ICC = 0.86; Yang et al. Insights into Imaging (2024) 15:17 Page 7 of 13 Table 1 The characteristics of all participants * p < 0.05, IPF, idiopathic pulmonary fibrosis; BMI, body mass index; FEV1, forced expiratory volume; FVC, forced vital capacity; TLC, total lung capacity; DLco, diffusing capacity of the lungs for carbon monoxide; CPI, composite physiological index; 6-MWD, 6 min-walk distance; HRQoL, health-related quality of life; ILD, interstitial lung disease; HRCT, high-resolution computed tomography; TPV, total pulmonary vascular; PVV, pulmonary vein vascular; PAV, pulmonary artery vascular Patients IPF (N = 76) Control (N = 62) t/χ2 p Mean age (years old) 62 ± 6 58 ± 4 1.957 0.055 Gender, male, n (%) 72 (94.7%) 58 (93.5 %) 0.088 0.767 Height (cm) 168.3 ± 7.1 169.8 ± 5.8 − 1.318 0. Repeatability of elastic registration algorithmh Insights into Imaging ( Fig. 5 Relationship between the value of elastic registration (Jac-mean and Dice similarity coefficient) and measurements from pulmonary function tests. FVC, forced vital capacity; FEV1, forced expiratory volume; TLC, total lung capacity; DLCO, diffusing capacity of the lungs for carbon monoxide; CPI, composite physiological index Fig. 5 Relationship between the value of elastic registration (Jac-mean and Dice similarity coefficient) and measurements from pulmonary function tests. FVC, forced vital capacity; FEV1, forced expiratory volume; TLC, total lung capacity; DLCO, diffusing capacity of the lungs for carbon monoxide; CPI, composite physiological index Table 2 The characteristic of lung shrinkage is based on elastic registration between dyspnea score groups in patients with IPF Table 2 The characteristic of lung shrinkage is based on elastic registration between dyspnea score groups in patients with IPF in IPF patients; (II) the decreased lung deformation in IPF patients correlated with the worsen FVC%, FEV1%, TLC%, DLco%, and CPI; and (III) the decreased lung deformation in IPF patients correlated with the deterio- rating 6-MWD, HRQoL, and the extent of lung fibrosis on HRCT. Table 2 The characteristic of lung shrinkage is based on elastic registration between dyspnea score groups in patients with IPF IPF, idiopathic pulmonary fibrosis; MRC, Medical Research Council a The difference between MRC 1 and MRC 2 b The difference between MRC 2 and MRC ≥ 3 c The difference between MRC 1 and MRC ≥ 3 * p < 0.05 Dyspnea score MRC 1 (n = 24) MRC 2 (n = 34) MRC ≥ 3 (n = 18) Jac-mean 0.25 ± 0.03 0.22 ± 0.11a* 0.16 ± 0.03bc Dice 0.10 ± 0.01 0.08 ± 0.03a 0.06 ± 0.02bc During the progressive of interstitial fibrosis, normal interstitial tissue is replaced by an altered extracellular matrix and alveolar architecture, resulting in deteriora- tion of lung compliance and decrease of lung deformation and elasticity [29, 30]. The elastic registration algorithm utilizes a dynamic linear elastic model to capture tissue deformation, which is discretized using the finite element method. In the context of follow-up for fibrotic intersti- tial lung disease, lung shrinkage has been employed as an additional CT marker [31]. In current study, the Jacobian maps from elastic registration showed the marked defor- mation areas mainly distributed in the dorsal aspect of lung bases in healthy controls and significantly decreased in the peripheral region of the lung bases in IPF patients, Repeatability of elastic registration algorithmh psychosocial impact (r = − 0.369, p < 0.05), and the total score (r = − 0.434, p < 0.05) (Fig. 6g–j) and also correlated with 6MWD (r = 0.577, p < 0.01) (Fig. 6k). The Dice simi- larity coefficient also negatively correlated with pulmo- nary fibrosis on HRCT (r = − 0. 312, p < 0. 01) (Fig. 6l). i As Table 3 shows, the Jac-mean correlated with the volume, number, and tortuosity of TPV (volume: r = Yang et al. Insights into Imaging (2024) 15:17 Yang et al. Insights into Imaging Page 8 of 13 Fig. 3 The inter-observer agreement (Intraclass coefficients and Bland-Altman plot agreement) of intersection over union (IoU), distance A (DA), and distance B (DB) between the two respiratory phases. 95% LOA, 95%limits of agreement Fig. 4 The coronal, sagittal, and transversal projection of mean of Jacobian determinants in IPF patient and control. Marked shrinkage areas (in red) are found in the peripheral region of lung bases in healthy volunteers (a–c) and study participants with idiopathic pulmonary fibrosis (IPF) (d–f) in MRI. In corresponding areas, the lungs of IPF patients show little deformation with mean Jacobian determinant Fig. 3 The inter-observer agreement (Intraclass coefficients and Bland-Altman plot agreement) of intersection over union (IoU), distance A (DA), and distance B (DB) between the two respiratory phases. 95% LOA, 95%limits of agreement Fig. 3 The inter-observer agreement (Intraclass coefficients and Bland-Altman plot agreement) of intersection over union (IoU), distance A (DA), and distance B (DB) between the two respiratory phases. 95% LOA, 95%limits of agreement Fig. 4 The coronal, sagittal, and transversal projection of mean of Jacobian determinants in IPF patient and control. Marked shrinkage areas (in red) are found in the peripheral region of lung bases in healthy volunteers (a–c) and study participants with idiopathic pulmonary fibrosis (IPF) (d–f) in MRI. In corresponding areas, the lungs of IPF patients show little deformation with mean Jacobian determinant Fig. 4 The coronal, sagittal, and transversal projection of mean of Jacobian determinants in IPF patient and control. Marked shrinkage areas (in red) are found in the peripheral region of lung bases in healthy volunteers (a–c) and study participants with idiopathic pulmonary fibrosis (IPF) (d–f) in MRI. In corresponding areas, the lungs of IPF patients show little deformation with mean Jacobian determinant Yang et al. Insights into Imaging (2024) 15:17 Page 9 of 13 Yang et al. * p < 0.01 and p < 0.05 indicated have significance Discussion Insights into Imaging (2024) 15:17 Page 11 of 13 of IPF; however, it limits to provide the subtle and regional functional alteration as well as the extent of fibrosis. The increased value of Jac-mean and Dice similarity coefficient correlated with the increased FVC%, FEV1%, TLC%, and DLco%. Furthermore, the decreased value of Jac-mean and Dice similarity coefficient correlated with the increased CPI. These results demonstrated that decreased lung deforma- tion is consistent with the deteriorated lung function. The Dice similarity coefficient is used to compare the similarity of the marked shrinkage area between IPF and healthy con- trols. Therefore, both Jac-mean (including color map) and Dice similarity coefficient can provide visual and quantita- tive analysis of regional or global lung function. research. In our patients, we had no follow-up MR and the application of elastic registration in assessing the sta- tus of IPF (stable or progressive) need further research. Second, considering that the registration method we used is derived from open-source software (ANTs), it is sufficiently general. At the same time, we compared the registration accuracy of each participant’s two breathing phases to confirm that this registration method is repro- ducible. Nonetheless, the possible impact of different reg- istration methods remains unknown. In conclusion, lung deformation decreased in patients with IPF and correlated with the severity of IPF. Elastic registration of inspiratory-to-expiratory 3D UTE MRI may be a new morphological and functional marker for non-radiation and noninvasive evaluation of IPF. With the progress of IPF, the clinical symptoms and signs of the patient gradually deteriorate. We found the value of Jac-mean and Dice similarity coefficient decreased in patients with MRC3 and 4, compared with patients with MRC1 and 2. This revealed that the more lung deforma- tion of IPF patients decreases, the more severe dyspnea they will have. Furthermore, we also found the value of Jac-mean and Dice similarity coefficient correlated with 6MWD, respiratory symptoms, activity, psychosocial impact, and the total score of IPF patients, confirming that decreased lung deformation correlated with worse exer- cise tolerance and the poor quality of life. In addition, Jac- mean had a weak correlation with the extent of pulmonary fibrosis and pulmonary vascular-related indexes on HRCT. Discussion In this study, we evaluated the lung deformation in IPF patients by elastic registration algorithm on 3D-UTE MRI and there are several findings: (I) the lung deforma- tion decreased in the peripheral region of the lung bases Yang et al. Insights into Imaging (2024) 15:17 Yang et al. Insights into Imaging (2 Page 10 of 13 Fig. 6 Relationship between Jac-mean (a–f), Dice similarity coefficient (g–l), and indicators of health-related quality of life, 6 min-walk distance (6-MWD), and the extent of pulmonary fibrosis on HRCT Fig. 6 Relationship between Jac-mean (a–f), Dice similarity coefficient (g–l), and indicators of health-related quality of life, 6 min-walk distance (6-MWD), and the extent of pulmonary fibrosis on HRCT These were consistent with Chassagnon et al. [16], and they reported that the lesser lung deformation of SSc patients compared with participants without fibrosis. which were consistent with the decreased lung deforma- tion in patient with systemic sclerosis-related ILD [16]. The Jac-mean represents the deformation of lung. The negative value indicates lung shrinkage, and the posi- tive value indicates lung stretch. Moreover, the smaller Jac-mean is, the weaker the lung deformation is. The Jac- mean of IPF patients significantly decreased, indicating that the lung deformation deteriorated in IPF patients. i In order to accurately evaluate the value of lung defor- mation in IPF, we included functional and morphological parameters including PFT, CPI, Medical Research Council scale, HRQoL, 6-MWD, and the extent of fibrosis on HRCT. PFT is vital marker for evaluating the functional severity Table 3 Relationship between the value of elastic registration (Jac-mean and Dice similarity coefficient) and pulmonary vascular- related indexes on HRCT p < 0.01 and p < 0.05 indicated have significance Jac-mean Dice r p r p Total pulmonary vascular Volume (ml) 0.245 < 0.01 − 0.146 > 0.05 Number 0.277 < 0.01* − 0.168 > 0.05 Tortuosity − 0.320 < 0.01* − 0.050 > 0.05 Pulmonary vein vascular Volume (ml) 0.162 > 0.05 − 0.088 > 0.05 Number 0.188 < 0.05* − 0.212 > 0.05 Tortuosity − 0.210 < 0.05* 0.001 > 0.05 Pulmonary artery vascular Volume (ml) 0.267 < 0.01* − 0.146 > 0.05 Number 0.303 < 0.01* − 0.113 > 0.05 Tortuosity − 0.350 < 0.01* − 0.107 > 0.05 Table 3 Relationship between the value of elastic registration (Jac-mean and Dice similarity coefficient) and pulmonary vascular- related indexes on HRCT Yang et al. Abbreviations Abbreviations 6-MWD 6 min-walk distance ALAT Latin American Thoracic Association ALI Acute lung injury ATS American Thoracic Society BMI Body mass index COPD Chronic obstructive pulmonary disease CPI Composite physiological index DLCO Diffusing capacity of the lungs for carbon monoxide ERS European respiratory society FEV1 Forced expiratory volume FVC Forced vital capacity GGO Ground-glass opacity HRCT High-resolution computed tomography HRQoL Health-related quality of life ICC Intraclass coefficients IoU Intersection over union IPF Idiopathic pulmonary fibrosis JRS Japanese Respiratory Society MRC Medical Research Council MRI Magnetic resonance imaging PFTs Pulmonary function tests ROI Regions of interest SSc-ILD Interstitial lung disease in patients with systemic sclerosis UIP Usual interstitial pneumonia UTE Ultrashort echo time ZTE Zero echo time Accuracy and reproducibility of elastic registration are critical for the analysis of lung deformation in IPF patients. Chassagnon et al. used the distances between landmarks to evaluate the registration performance, vali- dating the accuracy of the elastic registration [16]. In our study, we used the landmark distance and IoU to evaluate the accuracy of the elastic registration, where our result- ing landmark distance is good as Chassagnon et al., and our IoU reaches 0.88 [16]. Furthermore, we go beyond the limitation of Chassagnon et al. [16] and verify that the elastic registration results are reproducible by acquir- ing images of two respiratory phases for each patient. Discussion These results confirmed that lung deformation based on elastic registration of UTE MRI correlated with clinical severity of IPF patients and further proved that the elastic registration on UTE MRI can evaluate morphological and functional alterations during the follow-up. Availability of data and materials 11. Sun H, Yang X, Sun X et al (2022) Lung shrinking assessment on HRCT with elastic registration technique for monitoring idiopathic pulmo- nary fibrosis. Eur Radiol 33:2279–2288 The data used or analyzed during the current study are available from the cor- responding author on reasonable request. i 12. Bae K, Jeon KN, Hwang MJ et al (2019) Comparison of lung imaging using three-dimensional ultrashort echo time and zero echo time sequences: preliminary study. Eur Radiol 29(5):2253–2262 Declarations 13. Geiger J, Zeimpekis KG, Jung A et al (2021) Clinical application of ultrashort echo-time MRI for lung pathologies in children. Clin Radiol 76(9):708.e9–708.e17 Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s13244-023-01555-x. The online version contains supplementary material available at https://doi. org/10.1186/s13244-023-01555-x. Additional file 1: Supplementary figure 1. The segmentation model for tissue characterization of idiopathic pulmonary fibrosis on HRCT. Ground- glass opacities (red), reticulation (orange), and honeycombing(green). Supplementary figure 2. The segmentation model for pulmonary vascular of healthy controls(a-d) and idiopathic pulmonary fibrosis(e-h) on HRCT. Total lung segmentation (a, e); pulmonary vascular (b, f); pulmonary vein(blue) and artery(red) vascular (c, g); the skeleton of pulmonary vein(blue) and artery(red) vascular (d, h). Supplementary figure 3. The picture showed the position of points A and B. The location of points A and B were manually drawn and derived from the anterior edge of the thoracic vertebra intersects the chest walls on both sides at the planer of the lower margin of the manubrium sterni on multiplanar reconstructions (MPR). In this prospective study, we employed elastic registra- tion of inspiratory-to-expiratory 3D-UTE MRI to assess the severity of IPF. However, it is important to acknowl- edge several limitations in our study. First, this study may restrict generalizability of results due to its single- center design and focus on predominantly mild cohort of patients. For patients with advanced or acute exacerbation of IPF, the lung deformation with prognosis needs further Page 12 of 13 Yang et al. Insights into Imaging (2024) 15:17 Yang et al. Insights into Imaging (2024) 15:17 Yang et al. Insights into Imaging (2024) 15:17 8. Shibata H, Iwasawa T, Gotoh T et al (2012) Automatic tracking of the respiratory motion of lung parenchyma on dynamic magnetic resonance imaging: comparison with pulmonary function tests in patients with chronic obstructive pulmonary disease. J Thorac Imaging 27(6):387–92 Author details 1 Department of Pulmonary and Critical Care Medicine, General Hospital of Ningxia Medical University, Yinchuan 750004, Ningxia, China. 2 National Center for Respiratory Medicine, National Clinical Research Center for Respira- tory Diseases, Institute of Respiratory Medicine, Chinese Academy of Medical Sciences, Department of Pulmonary and Critical Care Medicine, Center of Res- piratory Medicine, China-Japan Friendship Hospital, 2 Yinghua Dong Street, Hepingli, Chao Yang District, Beijing 100029, China. 3 Institute of Advanced Research, Infervision Medical Technology Co., Ltd, Beijing 100025, China. 4 Department of Radiology, China-Japan Friendship Hospital, 2 Yinghua Dong Street, Hepingli, Chao Yang District, Beijing 100029, China. y y 17. Mahler DA, Weinberg DH, Wells CK, Feinstein AR (1984) The measure- ment of dyspnea. Contents, interobserver agreement, and physiologic correlates of two new clinical indexes. Chest 85(6):751–8 18. Zimmermann CS, Carvalho CR, Silveira KR et al (2007) Comparison of two questionnaires which measure the health-related quality of life of idiopathic pulmonary fibrosis patients. Braz J Med Biol Res 40(2):179–87 19. Laveneziana P, Albuquerque A, Aliverti A et al (2019) ERS statement on respiratory muscle testing at rest and during exercise. Eur Respir J 53(6):1801214 20. Sharp C, Adamali HI, Millar AB (2017) A comparison of published multidimensional indices to predict outcome in idiopathic pulmonary fibrosis. ERJ Open Res 3(1):00096–02016 Received: 4 July 2023 Accepted: 28 October 2023 Received: 4 July 2023 Accepted: 28 October 2023 21. Sun HLM, Kang H, Yang X, et al. (2022) Quantitative analysis of high- resolution computed tomography features of idiopathic pulmonary fibrosis: a structure-function correlation study. Quant Imaging Med Surg 12(7):3655–3665 Funding 10. Chassagnon G, Vakalopoulou M, Regent A et al (2021) Elastic registration-driven deep learning for longitudinal assessment of systemic sclerosis interstitial lung disease at CT. Radiology 298(1):189–198 10. Chassagnon G, Vakalopoulou M, Regent A et al (2021) Elastic registration-driven deep learning for longitudinal assessment of systemic sclerosis interstitial lung disease at CT. Radiology 298(1):189–198 This work was supported by National Key Technologies R & D Program Preci- sion China (No. 2021YFC2500700; 2016YFC0901101) and National Natural Science Foundation of China (No.81870056). Competing interests The authors declare no competing interests. 16. Chassagnon G, Martin C, Marini R et al (2019) Use of elastic registration in pulmonary MRI for the assessment of pulmonary fibrosis in patients with systemic sclerosis. Radiology 291(2):487–492 Authors’ contributions (1) The conception and design of the study: ML, HPD; (2) acquisition of data, analysis, and interpretation of data: XYY, ML, PXY, HSS, MD, AQL, CL, WYM, WXX, BBX, JG, YHR, RGZ; (3) drafting the article or revising it critically for important intellectual content: XYY, ML, HPD; (4) final approval of the version to be submitted: all authors. 9. Jahani N, Choi S, Choi J et al (2017) A four-dimensional computed tomography comparison of healthy and asthmatic human lungs. J Biomech 56:102–110 Ethics approval and consent to participate This study was approved by Institutional ethics committee of China-Japan Friendship Hospital (2019-123-K85-1). All participants provided written informed consent. 14. Torres L, Kammerman J, Hahn AD et al (2019) Structure-function imaging of lung disease using ultrashort echo time MRI. Acad Radiol 26(3):431–441 Consent for publication Consent for publication All authors read and approved the final manuscript. 15. Yang X, Liu M, Duan J et al (2022) Three-dimensional ultrashort echo time magnetic resonance imaging in assessment of idiopathic pulmonary fibrosis, in comparison with high-resolution computed tomography. Quant Imaging Med Surg 12(8):4176–4189 All authors read and approved the final manuscript. References Biometrics 33(1):159–74 Page 13 of 13 Yang et al. Insights into Imaging (2024) 15:17 Yang et al. Insights into Imaging (2024) 15:17 29. Haak AJ, Tan Q, Tschumperlin DJ (2018) Matrix biomechanics and dynamics in pulmonary fibrosis. Matrix Biol 73:64–76 30 Mei Q, Liu Z, Zuo H et al (2022) Idiopathic pulmonary fibrosis: an update on pathogenesis. Front Pharmacol 12:797292 31. Verschakelen JA (2021) Lung shrinkage: an additional CT marker in the follow-up of fibrotic interstitial lung disease. Radiology 298(1):199–200 29. Haak AJ, Tan Q, Tschumperlin DJ (2018) Matrix biomechanics and dynamics in pulmonary fibrosis. Matrix Biol 73:64–76 30 Mei Q, Liu Z, Zuo H et al (2022) Idiopathic pulmonary fibrosis: an update on pathogenesis. Front Pharmacol 12:797292 31. Verschakelen JA (2021) Lung shrinkage: an additional CT marker in the follow-up of fibrotic interstitial lung disease. Radiology 298(1):199–200 References 1. Raghu G, Remy-Jardin M, Myers JL et al (2018) Diagnosis of idiopathic pulmonary fibrosis. an official ATS/ERS/JRS/ALAT clinical practice guideline. Am J Respir Crit Care Med 198(5):e44–e68 22. Christe A, Peters AA, Drakopoulos D et al (2019) Computer-aided diag- nosis of pulmonary fibrosis using deep learning and CT images. Invest Radiol 54(10):627–632 guideline. Am J Respir Crit Care Med 198(5):e44–e68 g 2. Mogulkoc N, Brutsche MH, Bishop PW et al (2001) Pulmonary function in idiopathic pulmonary fibrosis and referral for lung transplantation. Am J Respir Crit Care Med 164(1):103–8 23. Sun X, Meng X, Zhang P et al (2022) Quantification of pulmonary vessel volumes on low-dose computed tomography in a healthy male Chinese population: the effects of aging and smoking. Quant Imaging Med Surg 12(1):406–416 p 3. Kwon BS, Choe J, Do KH et al (2020) Computed tomography patterns pre- dict clinical course of idiopathic pulmonary fibrosis. Respir Res 21(1):295 24. Sun H, Liu M, Kang H et al (2022) Quantitative analysis of high-resolu- tion computed tomography features of idiopathic pulmonary fibrosis: a structure-function correlation study. Quant Imaging Med Surg 12(7):3655–3665 4. Glocker B, Sotiras A, Komodakis N, Paragios N (2011) Deformable medical image registration: setting the state of the art with discrete methods. Annu Rev Biomed Eng 13:219–44 5. Marami B, Sirouspour S, Ghoul S et al (2015) Elastic registration of pros- tate MR images based on estimation of deformation states. Med Image Anal 21(1):87–103 25. Avants BB, Tustison NJ, Stauffer M et al (2014) The Insight ToolKit image registration framework. Front Neuroinform 8:44 26. Rezatofighi H, Tsoi N, Gwak J et al. (2019) Generalized intersection over union: a metric and a loss for bounding box regression. In Proceedings of the IEEE/CVF conference on computer vision and pattern recogni- tion, pp 658-666 6. Kaczka DW, Cao K, Christensen GE et al (2011) Analysis of regional mechanics in canine lung injury using forced oscillations and 3D image registration. Ann Biomed Eng 39(3):1112–24 7. Nishio M, Matsumoto S, Tsubakimoto M et al (2015) Paired inspiratory/ expiratory volumetric CT and deformable image registration for quan- titative and qualitative evaluation of airflow limitation in smokers with or without copd. Acad Radiol 22(3):330–6 27. Bland JM, Altman DG (1986) Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1(8476):307–10 28. Landis JR, Koch GG (1977) The measurement of observer agreement for categorical data. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations.
https://openalex.org/W4220908687	https://www.preprints.org/manuscript/202112.0017/v1/download	English	null	Temporal Muscle and Stroke—A Narrative Review on Current Meaning and Clinical Applications of Temporal Muscle Thickness, Area, and Volume	Nutrients	2,022	cc-by	6,964	Abstract: Background: Evaluating muscle mass and function among stroke patients is important. However, evaluating muscle volume and function is not easy due to the disturbance of consciousness and paresis. Temporal muscle thickness (TMT) has been introduced as a novel surrogate marker for muscle mass, function, and nutritional status. We herein performed a narrative literature review on temporal muscle and stroke to understand the current meaning of the TMT in the clinical stroke practice. Methods: The search was performed in PubMed, last updated in October 2021. Report on temporal muscle morphomics and stroke-related diseases or clinical entities were collected. Results: Four studies reported on TMT and subarachnoid hemorrhage, 2 intracerebral hemorrhage, 2 is- chemic stroke, 2 standard TMT values, and 2 nutritional status. TMT was reported as a prognostic factor for several diseases, surrogate markers for skeletal muscle mass, and an indicator of nutri- tional status. Computed tomography, magnetic resonance imaging, and ultrasonography were used to measure TMT. Conclusions: TMT is gradually used as a prognostic factor of stroke or surrogate marker for skeletal muscle mass and nutritional status. Establishing standard methods to measure TMT and large prospective studies to investigate the further relationship between TMT and diseases are needed. Keywords: frailty; muscle volume; nutritional status; prognostic factor; sarcopenia; skeletal muscle mass; stroke; temporal muscle thickness. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 1 December 2021 doi:10.20944/preprints202112.0017 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 1 December 2021 doi:10.20944/preprints202112.0017 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 1 December 2021 doi:10.20944/preprints202112.0017.v1 Masahito Katsuki1,2†, Yukinari Kakizawa1†, Akihiro Nishikawa1, Yasunaga Yamamoto1, Toshiya Uchiyama1, Masahiro Agata1, Naomichi Wada1, Shin Kawamura2, Akihito Koh2 1 Department of Neurosurgery, Suwa Red Cross Hospital, Suwa, Nagano, Japan; ykakizawajp@ 1 Department of Neurosurgery, Suwa Red Cross Hospital, Suwa, Nagano, Japan; ykakizawajp@yahoo.co.jp 2 Department of Neurosurgery Itoigawa General Hospital Itoigawa Niigata Japan; 1 Department of Neurosurgery, Suwa Red Cross Hospital, Suwa, Nagano, Japan; ykakizawajp@yahoo.co.jp 2 Department of Neurosurgery, Itoigawa General Hospital, Itoigawa, Niigata, Japan; ktk1122nigt@gmail.com Correspondence: Yukinari Kakizawa, Department of Neurosurgery, Suwa Red Cross Hospital, 5-11-50, Kogandori, Suwa, 392-8510, Nagano, Japan. ykakizawajp@yahoo.co.jp † E ll t ib t d fi t th * Correspondence: Yukinari Kakizawa, Department of Neurosurgery, Suwa Red Cross Hospital, 5-11-50, Kogandori, Suwa, 392-8510, Nagano, Japan. ykakizawajp@yahoo.co.jp † Equally contributed first authors Type of the Paper: Review Temporal Muscle and Stroke—A Narrative Review on Current Meaning and Clinical Applications of Temporal Muscle Thickness, Area, and Volume Masahito Katsuki1,2†, Yukinari Kakizawa1†, Akihiro Nishikawa1, Yasunaga Yamamoto1, Toshiya Uchiyama1, Masahiro Agata1, Naomichi Wada1, Shin Kawamura2, Akihito Koh2 Masahito Katsuki1,2†, Yukinari Kakizawa1†, Akihiro Nishikawa1, Yasunaga Yamamoto1, Toshiya Uchiyama1, Masahiro Agata1, Naomichi Wada1, Shin Kawamura2, Akihito Koh2 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 1 December 2021 doi:10.20944/preprints202112.0017.v1 cannot be performed because stroke patients often have a disturbance of consciousness, sedation, rest due to surgical treatment, and paresis. Therefore, an alternative method to evaluate muscle mass and function is needed. Recently, temporal muscle thickness (TMT) on the computed tomography (CT) im- age or magnetic resonance images (MRI) has been introduced as a novel surrogate marker with which to measure the muscle mass [15], function [16], and nutritional status [17,18]. CT and MRI are routinely performed for stroke patients, and TMT measurement is easy. Therefore, TMT is attractive as the alternative method to evaluate muscle mass and func- tion for stroke patients. The purpose of this narrative literature review, we herein present, is to investigate the reports on TMT and stroke and to understand the current meaning of the TMT in the clinical stroke practice. In addition to TMT [19,20], we also examined tem- poral muscle area (TMA) [19,21,22] and temporal muscle volume (TMV) [9] as the similar things of TMT. 2. Materials and Methods Studies regarding TMT and stroke were examined. The search was performed in PubMed, last updated in October 2021, using the terms “stroke” OR “intracerebral hem- orrhage (ICH)” OR “subarachnoid hemorrhage (SAH)” OR “cerebral infarction” OR “re- habilitation” OR “sarcopenia” OR “frailty” OR “nutrition” AND “temporal muscle thick- ness”. The PubMed search resulted in a total of 73 articles. We systematically read through the abstracts of all original articles available in English. We included the studies on the association between stroke and TMT with a sample size of around 50 cases and appropri- ate statistical analyses. We also checked through the lists of the references to complete our collection of studies. All the authors verified the correct transcription of the data to our manuscript. Finally, we included 8 studies related to stroke in our review. 3. Results Four studies reported the association between TMT and SAH [9,19,21,22], 2 ICH [23,24], 2 ischemic stroke [25,26]. The other 4 studies described the standard TMT values [16,27] and the relationship between TMT and nutritional status [17,18]. 1. Introduction Stroke is a widely known cause of disability [1]. Stroke also increases the risk of skel- etal muscle loss [2], sarcopenia, which contributes to further disability related to stroke [3]. Furthermore, pre-stroke sarcopenia is also associated with poor functional outcomes [4,5]. Therefore, evaluating muscle mass and function among stroke patients is important [6,7], and aggressive nutrition therapy [8–10] or rehabilitation [11] is applicable for those with stroke as well as high risks for muscle loss. Measuring skeletal muscle mass and function is an evolving parameter for the clini- cal evaluation of physiological conditions [12]. The golden standard to evaluate muscle loss, sarcopenia, is muscle function test, such as gait speed test and grip strength test, according to the European Working Group on Sarcopenia in Older People (EWGSOP), EWGSOP2, and the Asian Working Group for Sarcopenia (AGWS) [13,14]. However, measuring muscle function like grip strength and gait speed measurement sometimes Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 1 December 2021 doi:10.20944/preprints202112.0017.v1 characteristic analysis found that the threshold of TMT was 4.9 mm in women and 6.7 mm in men, and that of TMA was 193 mm2 in women and 333 mm2 in men, which were the cut-off values for the functional outcomes among SAH patients under 75 y.o. characteristic analysis found that the threshold of TMT was 4.9 mm in women and 6.7 mm in men, and that of TMA was 193 mm2 in women and 333 mm2 in men, which were the cut-off values for the functional outcomes among SAH patients under 75 y.o. g p y Onodera [9] examined TMV using volume rendering software (Ziostation 2 version 2.9.5.1, Ziosoft, Tokyo) because TMT might be less reproducible. They investigated 60 SAH patients and measured TMV on the CT images at admission and 2 weeks after an- eurysm treatment. Patients whose TMV had decreased by ≥20% were classified into the “atrophy group,” whereas those whose TMV had decreased by <20% were classified into the “maintenance group.” Their study showed that the food intake score and the func- tional outcome were significantly more positive in the TMV maintenance group than the TMV atrophy group. Therefore, they suggested that the importance of early high-protein administration to maintain TMV in the acute term (Table 1). Table 1. Previous reports on the association between temporal muscle and SAH Author Year Number of cases Abstract Katsuki [19] 2019 49 Large TMT was related to favorable outcomes among elderly SAH. Katsuki [21] 2020 298 TMT and TMA were related to Hunt and Kosnik Grade and functional outcome at 6 months after endovascular coiling regardless of age. Katsuki [22] 2021 127 The threshold of TMT was 4.9 mm in women and 6.7 mm in men, and that of TMA was 193 mm2 in women and 333 mm2 in men, which were the cut-off val- ues for the functional outcomes at 6 months among SAH patients under 75 y.o. Onodera [9] 2021 60 The food intake score and the functional outcome at discharge were significantly more positive in the TMV maintenance group than the TMV atrophy group after SAH. Abbreviations: SAH: subarachnoid hemorrhage; TMA: temporal muscle area, TMT: temporal mus- cle thickness, TMV: temporal muscle volume Abbreviations: SAH: subarachnoid hemorrhage; TMA: temporal muscle area, TMT: temporal mus- cle thickness, TMV: temporal muscle volume 3.1. Temporal muscel and SAH Katsuki first reported TMT as a prognostic factor for SAH outcome in 2019, investi- gating 49 SAH patients over 75 y.o. who were treated by clipping with craniotomy [19]. The TMT was measured on CT image on admission, using Aquilion ONE (Canon Medical Systems Corporation, Tochigi, Japan) with 0.5 × 0.5 x 1.0 mm voxels. The slice thickness was reconstructed to 5 mm. The window width was adjusted to 300, and the window level was adjusted to 20. The TMT was measured bilaterally perpendicular to the long axis of the temporal muscle at the slice 5 mm above the orbital roof using SYNAPSE V 4.1.5 im- aging software (Fujifilm Medical, Tokyo, Japan). Then, the averages of the left and right of the TMTs were used. This method to measure TMT on CT was first defined. They per- formed univariate analysis regarding TMT and functional outcome at 6 months The study was preliminary, but the study first suggested that larger TMT was related to favorable outcomes among elderly SAH. They next investigated the relationship between temporal muscle and Hunt and Kos- nik Grade on admission and functional outcome at 6 months [21]. They examined 298 all age-group patients, and all the patients were treated by endovascular coiling. They re- vealed that Hunt and Kosnik grade on admission and functional outcome were related to TMT and TMA. TMA was measured manually by tracing the outline of the temporal mus- cle on the same CT slice as that used for measuring the TMT. Notably, this study suggests that TMT and TMA are related to both the severity of SAH and functional outcome re- gardless of age, not only for the elderly. They then investigated 127 SAH patients under 75 y.o. who were treated by clipping [22]. They examined the cut-off values for the functional outcomes. Receiver operating 3.2. Temporal muscle and ICH However, TMT was not independently related to the functional outcome (Table 3). Table 3. Previous reports on the association between temporal muscle and stroke Author Year Number of cases Abstract Sakai [25] 2021 70 TMT was a significant explanator of dysphagia severity following acute ischemic stroke. Nozoe [26] 2021 289 Sarcopenia risk was independently associated with TMT in older patients with acute stroke, but TMT was not independently related to the functional outcome. Abbreviations: TMT: temporal muscle thickness 3.4 Standard values of TMT Steindl [16] investigated 624 normal collective cohort to establish standard reference values of TMT on the T1-weighted image. TMT was measured on isovoxel (1 × 1 × 1 mm3) T1-weighted MR images perpendicularly to the long axis of the temporal muscle on an axial plane, which was oriented parallel to the anterior commissure-posterior commissure line. They also examined 422 healthy volunteers and 130 cases as prospective validation cohort and found that TMT and grip strength were correlated. This was the first report to Table 2. Previous reports on the association between temporal muscle and ICH Author Year Number of cases Abstract Katsuki [23] 2019 75 Low nutritional status, indicated by low total protein level and low TMA alto- gether, seemed to be associated with the poor functional outcomes at 6 months after endoscopic hematoma removal. Gomes [24] 2021 24 TMT at the unaffected side was larger than the affected side after a hemorrhagic stroke. Abbreviations: ICH; intracerebral hemorrhage, TMA: temporal muscle area, TMT: temporal muscle thickness Abbreviations: ICH; intracerebral hemorrhage, TMA: temporal muscle area, TMT: temporal muscle thickness Sakai [25] investigated 70 acute cerebral infarction patients’ TMT on the T2-weight MR image and functional oral intake scales. They revealed that TMT was a significant explanator of dysphagia severity following acute ischemic stroke, along with age and the National Institute of Health Stroke Scale score. The measuring method of TMT using T2- weighted images was similar to the previous report from Furtner using T1-weighted im- ages [28]. They first reported the association between TMT and ischemic stroke-related dysphagia in the acute term. Nozoe [26] examined 289 acute elderly stroke patients and investigated the TMT on CT image as an indicator of sarcopenia risk and its relationship with the functional out- come at 3 months. They found that sarcopenia risk was independently associated with TMT in older patients with acute stroke. 3.2. Temporal muscle and ICH Katsuki examined 75 ICH patients treated by endoscopic hematoma removal and in- vestigated the factors related to the functional outcome [23]. They revealed that lower total protein level was related to the poor outcome at 6 months. In addition, they mentioned TMA as an indicator of nutrition, but TMA itself was not significantly related to the out- come (p = 0.08). However, they suggested that low nutritional status, indicated by lower total protein level and low TMA altogether, seemed to be associated with poor outcomes. Gomes examined 24 post-hemorrhagic stroke patients in the chronic stage and tested the bite force and TMT [24]. Maximum molar bite force was verified using a digital dyna- mometer. TMT was measured using ultrasound images obtained at rest and during max- imal voluntary contraction of the masseter and temporalis muscles. The TMT at the unaf- fected side was larger than the affected side. This study first focused on the functional and morphological changes in the stomatognathic system after a hemorrhagic stroke. The clin- ical meaning of these changes would be investigated (Table 2). doi:10.20944/preprints202112.0017.v1 Table 2. Previous reports on the association between temporal muscle and ICH Author Year Number of cases Abstract Katsuki [23] 2019 75 Low nutritional status, indicated by low total protein level and low TMA alto- gether, seemed to be associated with the poor functional outcomes at 6 months after endoscopic hematoma removal. Gomes [24] 2021 24 TMT at the unaffected side was larger than the affected side after a hemorrhagic stroke. Abbreviations: ICH; intracerebral hemorrhage, TMA: temporal muscle area, TMT: temporal muscle thickness 3.3 Temporal muscle and stroke Sakai [25] investigated 70 acute cerebral infarction patients’ TMT on the T2-weight MR image and functional oral intake scales. They revealed that TMT was a significant explanator of dysphagia severity following acute ischemic stroke, along with age and the National Institute of Health Stroke Scale score. The measuring method of TMT using T2- weighted images was similar to the previous report from Furtner using T1-weighted im- ages [28]. They first reported the association between TMT and ischemic stroke-related dysphagia in the acute term. Nozoe [26] examined 289 acute elderly stroke patients and investigated the TMT on CT image as an indicator of sarcopenia risk and its relationship with the functional out- come at 3 months. They found that sarcopenia risk was independently associated with TMT in older patients with acute stroke. 3.2. Temporal muscle and ICH However, TMT was not independently related to the functional outcome (Table 3). Table 3. Previous reports on the association between temporal muscle and stroke Table 3. Previous reports on the association between temporal muscle and stroke Author Year Number of cases Abstract Sakai [25] 2021 70 TMT was a significant explanator of dysphagia severity following acute ischemic stroke. Nozoe [26] 2021 289 Sarcopenia risk was independently associated with TMT in older patients with acute stroke, but TMT was not independently related to the functional outcome. Abbreviations: TMT: temporal muscle thickness 4. Discussion We herein reviewed the reports on TMT and stroke. TMT is useful as a prognostic marker for SAH, ICH, and dysphagia after stroke. It also indicates nutritional status and risk of sarcopenia. As the number of reports on TMT and stroke has been increasing rap- idly in recent years, we believe that TMT is one of the crucial factors in clinical practice. In addition to this review, we discuss the TMT measurement method and TMT use in other neurosurgical practices. 3.4 Standard values of TMT TMT is useful as a prognostic marker for SAH, ICH, and dysphagia after stroke. It also indicates nutritional status and risk of sarcopenia. As the number of reports on TMT and stroke has been increasing rap- idly in recent years, we believe that TMT is one of the crucial factors in clinical practice. In addition to this review, we discuss the TMT measurement method and TMT use in other neurosurgical practices. 4.1 TMT measurement method The standard TMT measurement method has not been established. The old report used volume rendering software [29,30], and Onodera also used a similar approach [9] to measure TMV, not TMT. Then, Furtner established TMT measurement using T1-weighted MR images. They measured TMT perpendicular to the long axis of the temporal muscle Table 5. TMT and nutritional status Author Year Number of cases Abstract Hasegawa [18] 2019 73 TMT was strongly correlated with calf circumference and arm muscle circumference. However, there were no strong correlations with serum protein levels nor fat mass evaluated as triceps skinfold thickness. Hasegawa [17] 2021 48 TMT changes were directly correlated with energy adequacy in bedridden older adults. 3.4 Standard values of TMT 3.4 Standard values of TMT Steindl [16] investigated 624 normal collective cohort to establish standard reference values of TMT on the T1-weighted image. TMT was measured on isovoxel (1 × 1 × 1 mm3) T1-weighted MR images perpendicularly to the long axis of the temporal muscle on an axial plane, which was oriented parallel to the anterior commissure-posterior commissure line. They also examined 422 healthy volunteers and 130 cases as prospective validation cohort and found that TMT and grip strength were correlated. This was the first report to validate the relationship between the TMT and grip strength, namely muscle function, prospectively. p p y Katsuki [27] investigated 360 Japanese individuals’ brain check-up database obtained by MRI. They measured TMT in the same way previously reported [16] to obtain the standard values of TMT among Japanese individuals. They compared their result to Steindl’s results to know the racial difference, but the background of the participants so doi:10.20944/preprints202112.0017.v1 differed. They did not perform any muscle function test, so further investigation is needed (Table 4). Table 4. TMT and nutritional status Author Year Number of cases Abstract Steindl [16] 2020 1175 Standard values of TMT were investigated, and TMT and grip strength were correlated. Katsuki [27] 2021 360 Standard values of TMT were investigated among Japanese individuals who underwent brain check-ups. differed. They did not perform any muscle function test, so further investigation is needed (Table 4). Table 4. TMT and nutritional status Author Year Number of cases Abstract Steindl [16] 2020 1175 Standard values of TMT were investigated, and TMT and grip strength were correlated. Katsuki [27] 2021 360 Standard values of TMT were investigated among Japanese individuals who underwent brain check-ups. Abbreviations: TMT: temporal muscle thickness 3.5 TMT and nutritional status Hasegawa [18] investigated 73 elderly individuals to measure their TMT using ultra- sonography and nutritional status assessed with anthropometric measurements and la- boratory tests. TMT was strongly correlated with calf circumference and arm muscle cir- cumference. However, there were no strong correlations with serum protein levels nor fat mass evaluated as triceps skinfold thickness. They also examined the reliability to meas- ure TMT using ultrasonography, and the inter-rater reliability was 0.99. They also performed a prospective study [17]. 3.4 Standard values of TMT The study aimed to examine whether a change in TMT evaluated by the ultrasonography was directly correlated with energy adequacy and to determine the cut-off value of a change in TMT to detect energy inade- quacy. They investigated 48 bedridden elderly patients and revealed that percentage change in TMT was significantly correlated with energy adequacy. They suggested that the assessment of TMT changes could be helpful for performing better nutritional therapy (Table 5). Table 5. TMT and nutritional status Author Year Number of cases Abstract Hasegawa [18] 2019 73 TMT was strongly correlated with calf circumference and arm muscle circumference. However, there were no strong correlations with serum protein levels nor fat mass evaluated as triceps skinfold thickness. Hasegawa [17] 2021 48 TMT changes were directly correlated with energy adequacy in bedridden older adults. 4. Discussion We herein reviewed the reports on TMT and stroke TMT is useful as a prognostic differed. They did not perform any muscle function test, so further investigation is needed (Table 4). Abbreviations: TMT: temporal muscle thickness 3.5 TMT and nutritional status Hasegawa [18] investigated 73 elderly individuals to measure their TMT using ultra- sonography and nutritional status assessed with anthropometric measurements and la- boratory tests. TMT was strongly correlated with calf circumference and arm muscle cir- cumference. However, there were no strong correlations with serum protein levels nor fat mass evaluated as triceps skinfold thickness. They also examined the reliability to meas- ure TMT using ultrasonography, and the inter-rater reliability was 0.99. They also performed a prospective study [17]. The study aimed to examine whether a change in TMT evaluated by the ultrasonography was directly correlated with energy adequacy and to determine the cut-off value of a change in TMT to detect energy inade- quacy. They investigated 48 bedridden elderly patients and revealed that percentage change in TMT was significantly correlated with energy adequacy. They suggested that the assessment of TMT changes could be helpful for performing better nutritional therapy (Table 5). Table 5. TMT and nutritional status Author Year Number of cases Abstract Hasegawa [18] 2019 73 TMT was strongly correlated with calf circumference and arm muscle circumference. However, there were no strong correlations with serum protein levels nor fat mass evaluated as triceps skinfold thickness. Hasegawa [17] 2021 48 TMT changes were directly correlated with energy adequacy in bedridden older adults. 4. Discussion We herein reviewed the reports on TMT and stroke. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 1 December 2021 doi:10.20944/preprints202112.0017.v1 at the level of the orbital roof [12,15,16,31,32]. This method is widely used, but low acces- sibility to MRI in routine work is a problem. Sakai [25] used T2-weight MR images, not T1-weighted images. The difference between the T1- and T2-weighted images should be discussed. Katsuki first defined TMT and TMA on CT images [19]. CT is more accessible rather than MRI, so TMT on CT seems better for routine clinical work. Hasegawa used ultrasonography (M-Turbo; SonoSite, Bothell, WA, USA) to measure TMT at 4 cm from the eyelid and 2 cm above the reference line that was the orbitomeatal line [18]. Ultraso- nography is not so reproducible, but their study reported that TMT measurement by ul- trasonography is reliable. g p y As described above, there are some ways to measure temporal muscle morphomics, including TMT, TMA, and TMV. Easiness and high reproducibility are needed to establish the standard method. Further study on the measurement method is desired. 4.3 Limitations As described above, TMT is now attractive, and many studies have been performed, but some issues should be addressed. First, most of the studies were retrospective, so fur- ther prospective study is needed. Second, the sample sizes were small, so studies with large sample sizes are desired. Third, the TMT measurement method was not established, and several ways can be used, like MRI, CT, and ultrasonography. The standard approach to measuring TMT is needed. Fourth, the direct mechanism of why large temporal muscle relates to favorable prognosis has not been clarified. The true mechanism between TMT and outcomes should be discussed from several perspectives, such as rehabilitation, nu- trition, frailty, deglutition, or basic medicine. Some of the problems may be resolved as TMT measurements will be routinely taken with tracking time-course changes. 6. Patents Not applicable. Supplementary Materials: Not applicable. 4.2 Temporal muscle in other neurosurgical practice The first report on the temporal muscle as a prognostic factor in neurosurgical prac- tice was to evaluate the operative risk in nonsyndromic craniosynostosis in 2013 [30]. The authors used volume rendering software to assess the temporal fat pad. From this report, there have been several papers on the temporalis muscle and prognosis, especially in brain tumors. There are several reports on the overall survival and temporal muscle in glioblas- toma [32–39], metastatic brain tumor [28,40,41], and primary central nervous system lym- phoma[31,42]. Like those on TMT and stroke, all of these reports have in common that the temporal muscle was used to indicate nutritional status and skeletal muscle mass volume. The larger the temporal muscle, the better the outcome, probably due to good nutritional status and more skeletal muscle mass. 5. Conclusions TMT seems a useful surrogate marker for skeletal muscle volume and function, and would be a potential prognostic factor. The research on the association between stroke and TMT is now increasing. Further research is needed to establish the usefulness of TMT. 4.1 TMT measurement method The standard TMT measurement method has not been established. The old report used volume rendering software [29,30], and Onodera also used a similar approach [9] to measure TMV, not TMT. Then, Furtner established TMT measurement using T1-weighted MR images. They measured TMT perpendicular to the long axis of the temporal muscle Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 1 December 2021 doi:10.20944/preprints202112.0017.v1 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 1 December 2021 References 1. Toyoda, K.; Sonoda, K.; Sato, S.; Yoshimura, S. [The Japan Stroke Databank -Current stroke care in Japan and ideal stroke registries-] (Japanese). Neurol. Ther. 2018, 35, 188–192, doi:https://doi.org/10.15082/jsnt.35.3_188. 2. Ryan, A.S.; Ivey, F.M.; Serra, M.C.; Hartstein, J.; Hafer-Macko, C.E. Sarcopenia and physical function in middle-aged and older stroke survivors. Arch. Phys. Med. Rehabil. 2017, 98, 495–499, doi:10.1016/j.apmr.2016.07.015. 3. Scherbakov, N.; von Haehling, S.; Anker, S.D.; Dirnagl, U.; Doehner, W. Stroke induced sarcopenia: Muscle wasting and disability after stroke. Int. J. Cardiol. 2013, 170, 89–94, doi:10.1016/j.ijcard.2013.10.031. 4. Yoshimura, Y.; Wakabayashi, H.; Bise, T.; Nagano, F.; Shimazu, S.; Shiraishi, A.; Yamaga, M.; Koga, H. Sarcopenia is associated with worse recovery of physical function and dysphagia and a lower rate of home discharge in Japanese hospitalized adults undergoing convalescent rehabilitation. Nutrition 2019, 61, 111–118, doi:10.1016/j.nut.2018.11.005. 4. Yoshimura, Y.; Wakabayashi, H.; Bise, T.; Nagano, F.; Shimazu, S.; Shiraishi, A.; Yamaga, M.; Koga, H. Sarcopenia is associated with worse recovery of physical function and dysphagia and a lower rate of home discharge in Japanese hospitalized adults undergoing convalescent rehabilitation. Nutrition 2019, 61, 111–118, doi:10.1016/j.nut.2018.11.005. 5. Nozoe, M.; Kanai, M.; Kubo, H.; Yamamoto, M.; Shimada, S.; Mase, K. Prestroke sarcopenia and functional outcomes in elderly patients who have had an acute stroke: A prospective cohort study. Nutrition 2019, 66, 44–47, doi:10.1016/j.nut.2019.04.011. 6. Nozoe, M.; Kubo, H.; Kanai, M.; Yamamoto, M. Relationships between pre-stroke SARC-F scores, disability, and risk of malnutrition and functional outcomes after stroke—A prospective cohort study. Nutrients 2021, 13, 3586, doi:10.3390/nu13103586. 7. Nakanishi, N.; Okura, K.; Okamura, M.; Nawata, K.; Shinohara, A.; Tanaka, K.; Katayama, S. Measuring and monitoring skeletal muscle mass after stroke: A review of current methods and clinical applications. J. Stroke Cerebrovasc. Dis. 2021, 30, 105736, doi:10.1016/j.jstrokecerebrovasdis.2021.105736. 8. Yoshimura, Y.; Wakabayashi, H.; Momosaki, R.; Nagano, F.; Bise, T.; Shimazu, S.; Shiraishi, A. Stored energy increases body weight and skeletal muscle mass in older, underweight patients after stroke. Nutrients 2021, 13, 3274, doi:10.3390/nu13093274. 9. Onodera, H.; Mogamiya, T.; Matsushima, S.; Sase, T.; Kawaguchi, K.; Nakamura, H.; Sakakibara, Y. High protein intake after subarachnoid hemorrhage improves oral intake and temporal muscle volume. Clin. Nutr. 2021, 40, 3147–4191, doi:10.1016/j.clnu.2021.01.040. 10. Onodera, H.; Mogamiya, T.; Mori, M.; Matsushima, S.; Sase, T.; Nakamura, H.; Sakakibara, Y. High protein intake after subarachnoid hemorrhage improves ingestion function and temporal muscle volume. Clin. Nutr. ESPEN 2020, 40, 595, doi:10.1016/j.clnesp.2020.09.566. 11. Yoshimura, Y. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 1 December 2021 doi:10.20944/preprints202112.0017.v1 Informed Consent Statement: This is a review of literatures, so informed consent statement is not applicable. Data Availability Statement: Not applicable. Acknowledgments: We are thankful for the hospital staffs. Acknowledgments: We are thankful for the hospital staffs. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Supplementary Materials: Not applicable. Author Contributions: Conceptualization, K.Y. and M.K.; Literature search, A.N., Y.Y., T.U., N.W., S.K. and A.K.; Drafting manuscript, M.K.; Supervision, Y.K.; All authors have read and agreed to the published version of the manuscript. Funding: None. Funding: None. Institutional Review Board Statement: Not applicable. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 1 December 2021 doi:10.20944/preprints202112.0017.v1 14. Cruz-Jentoft, A.J.; Bahat, G.; Bauer, J.; Boirie, Y.; Bruyère, O.; Cederholm, T.; Cooper, C.; Landi, F.; Rolland, Y.; Sayer, A.A.; et al. Sarcopenia: Revised European consensus on definition and diagnosis. Age Ageing 2019, 48, 16–31, doi:10.1093/ageing/afy169. 14. 15. Leitner, J.; Pelster, S.; Schöpf, V.; Berghoff, A.S.; Woitek, R.; Asenbaum, U.; Nenning, K.H.; Widhalm, G.; Kiesel, B.; Gatterbauer, B.; et al. High correlation of temporal muscle thickness with lumbar skeletal muscle cross-sectional area in patients with brain metastases. PLoS One 2018, 13, e0207849, doi:10.1371/journal.pone.0207849. 15. Leitner, J.; Pelster, S.; Schöpf, V.; Berghoff, A.S.; Woitek, R.; Asenbaum, U.; Nenning, K.H.; Widhalm, G.; Kiesel, B.; Gatterbauer, B.; et al. High correlation of temporal muscle thickness with lumbar skeletal muscle cross-sectional area in patients with brain metastases. PLoS One 2018, 13, e0207849, doi:10.1371/journal.pone.0207849. 16. Steindl, A.; Leitner, J.; Schwarz, M.; Nenning, K.-H.; Asenbaum, U.; Mayer, S.; Woitek, R.; Weber, M.; Schöpf, V.; Berghoff, A.S.; et al. Sarcopenia in neurological patients: Standard values for temporal muscle thickness and muscle strength evaluation. J. Clin. Med. 2020, 9, 1272, doi:10.3390/jcm9051272. 16. Steindl, A.; Leitner, J.; Schwarz, M.; Nenning, K.-H.; Asenbaum, U.; Mayer, S.; Woitek, R.; Weber, M.; Schöpf, V.; Berghoff, A.S.; et al. Sarcopenia in neurological patients: Standard values for temporal muscle thickness and muscle strength evaluation. J. Clin. Med. 2020, 9, 1272, doi:10.3390/jcm9051272. 17. Hasegawa, Y.; Yoshida, M.; Sato, A.; Fujimoto, Y.; Minematsu, T.; Sugama, J.; Sanada, H. A change in temporal muscle thickness is correlated with past energy adequacy in bedridden older adults: a prospective cohort study. BMC Geriatr. 2021, 21, 182, doi:10.1186/s12877-021-02086-0. 17. Hasegawa, Y.; Yoshida, M.; Sato, A.; Fujimoto, Y.; Minematsu, T.; Sugama, J.; Sanada, H. A change in temporal muscle thickness is correlated with past energy adequacy in bedridden older adults: a prospective cohort study. BMC Geriatr. 2021, 21, 182, doi:10.1186/s12877-021-02086-0. 18. Hasegawa, Y.; Yoshida, M.; Sato, A.; Fujimoto, Y.; Minematsu, T.; Sugama, J.; Sanada, H. Temporal muscle thickness as a new indicator of nutritional status in older individuals. Geriatr. Gerontol. Int. 2019, 19, 135–140, doi:10.1111/ggi.13570. 18. Hasegawa, Y.; Yoshida, M.; Sato, A.; Fujimoto, Y.; Minematsu, T.; Sugama, J.; Sanada, H. Temporal muscle thickness as a new indicator of nutritional status in older individuals. Geriatr. Gerontol. Int. 2019, 19, 135–140, doi:10.1111/ggi.13570. 19. Katsuki, M.; Yamamoto, Y.; Uchiyama, T.; Wada, N.; Kakizawa, Y. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 1 December 2021 Clinical characteristics of aneurysmal subarachnoid hemorrhage in the elderly over 75; would temporal muscle be a potential prognostic factor as an indicator of sarcopenia? Clin. Neurol. Neurosurg. 2019, 186, 105535, doi:10.1016/j.clineuro.2019.105535. 19. Katsuki, M.; Yamamoto, Y.; Uchiyama, T.; Wada, N.; Kakizawa, Y. Clinical characteristics of aneurysmal subarachnoid hemorrhage in the elderly over 75; would temporal muscle be a potential prognostic factor as an indicator of sarcopenia? Clin. Neurol. Neurosurg. 2019, 186, 105535, doi:10.1016/j.clineuro.2019.105535. 20. Gonçalves, R.C.G.; Rabelo, N.N.; Figueiredo, E.G.; Welling, L.C. Oral health and temporal muscle thickness. Surg. Neurol. Int. 2021, 12, 527, doi:10.25259/SNI_905_2021. 20. Gonçalves, R.C.G.; Rabelo, N.N.; Figueiredo, E.G.; Welling, L.C. Oral health and temporal muscle thickness. Surg. Neurol. Int. 2021, 12, 527, doi:10.25259/SNI_905_2021. 21. Katsuki, M.; Suzuki, Y.; Kunitoki, K.; Sato, Y.; Sasaki, K.; Mashiyama, S.; Matsuoka, R.; Allen, E.; Saimaru, H.; Sugawara, R.; et al. Temporal muscle as an indicator of sarcopenia is independently associated with Hunt and Kosnik grade on admission and the modified Rankin Scale at 6 month of patients with subarachnoid hemorrhage treated by endovascular coiling. World Neurosurg. 2020, 137, e526–e534, doi:10.1016/j.wneu.2020.02.033. 21. Katsuki, M.; Suzuki, Y.; Kunitoki, K.; Sato, Y.; Sasaki, K.; Mashiyama, S.; Matsuoka, R.; Allen, E.; Saimaru, H.; Sugawara, R.; et al. Temporal muscle as an indicator of sarcopenia is independently associated with Hunt and Kosnik grade on admission and the modified Rankin Scale at 6 month of patients with subarachnoid hemorrhage treated by endovascular coiling. World Neurosurg. 2020, 137, e526–e534, doi:10.1016/j.wneu.2020.02.033. 22. Katsuki, M.; Kakizawa, Y.; Nishikawa, A.; Yamamoto, Y.; Uchiyama, T. Temporal muscle thickness and area are an independent prognostic factors in patients aged 75 or younger with aneurysmal subarachnoid hemorrhage treated by clipping. Surg. Neurol. Int. 2021, 12, 151, doi:10.25259/SNI_814_2020. 22. Katsuki, M.; Kakizawa, Y.; Nishikawa, A.; Yamamoto, Y.; Uchiyama, T. Temporal muscle thickness and area are an independent prognostic factors in patients aged 75 or younger with aneurysmal subarachnoid hemorrhage treated by clipping. Surg. Neurol. Int. 2021, 12, 151, doi:10.25259/SNI_814_2020. 23. Katsuki, M.; Kakizawa, Y.; Nishikawa, A.; Yamamoto, Y.; Uchiyama, T. Lower total protein and absence of neuronavigation are novel poor prognostic factors of endoscopic hematoma removal for intracerebral hemorrhage. J. Stroke Cerebrovasc. Dis. 2020, 29, 105050, doi:10.1016/j.jstrokecerebrovasdis.2020.105050. 23. Katsuki, M.; Kakizawa, Y.; Nishikawa, A.; Yamamoto, Y.; Uchiyama, T. Lower total protein and absence of neuronavigation are novel poor prognostic factors of endoscopic hematoma removal for intracerebral hemorrhage. J. Stroke Cerebrovasc. Dis. References [Treating sarcopenia with a hybrid of aggressive exercise therapy and nutritional therapy: Approaches at Kumamoto Rehabilitation Hospital] (Japanese). Mon. B. Med. Rehabil. 2018, 224, 16–24. 12. Furtner, J.; Weller, M.; Weber, M.; Gorlia, T.; Nabors, B.; Reardon, D.A.; Tonn, J.-C.; Stupp, R.; Preusser, M. Temporal muscle thickness as a prognostic marker in newly diagnosed glioblastoma patients: translational imaging analysis of the CENTRIC EORTC 26071-22072 and CORE trials. Clin. Cancer Res. 2021, online ahead, doi:10.1158/1078-0432.CCR-21-1987. 13. Chen, L.K.; Liu, L.K.; Woo, J.; Assantachai, P.; Auyeung, T.W.; Bahyah, K.S.; Chou, M.Y.; Chen, L.Y.; Hsu, P.S.; Krairit, O.; et al. Sarcopenia in Asia: Consensus report of the Asian working group for sarcopenia. J. Am. Med. Dir. Assoc. 2014, 15, 95– 101, doi:10.1016/j.jamda.2013.11.025. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 1 December 2021 35 Yesil Cinkir H ; Colakoglu Er H Is temporal muscle thickness a survival predictor in newly diagnosed glioblastoma p y g g J 35. Yesil Cinkir, H.; Colakoglu Er, H. Is temporal muscle thickness a survival predictor in newly diagnosed glioblastoma multiforme? Asia. Pac. J. Clin. Oncol. 2020, 16, e223-227, doi:10.1111/ajco.13369. 35. Yesil Cinkir, H.; Colakoglu Er, H. Is temporal muscle thickness a survival predictor in newly diagnosed glioblastoma multiforme? Asia. Pac. J. Clin. Oncol. 2020, 16, e223-227, doi:10.1111/ajco.13369. 36. Muglia, R.; Simonelli, M.; Pessina, F.; Morenghi, E.; Navarria, P.; Persico, P.; Lorenzi, E.; Dipasquale, A.; Grimaldi, M.; Scorsetti, M.; et al. Prognostic relevance of temporal muscle thickness as a marker of sarcopenia in patients with glioblastoma at diagnosis. Eur. Radiol. 2021, 31, 4079–4086, doi:10.1007/s00330-020-07471-8. 36. Muglia, R.; Simonelli, M.; Pessina, F.; Morenghi, E.; Navarria, P.; Persico, P.; Lorenzi, E.; Dipasquale, A.; Grimaldi, M.; Scorsetti, M.; et al. Prognostic relevance of temporal muscle thickness as a marker of sarcopenia in patients with glioblastoma at diagnosis. Eur. Radiol. 2021, 31, 4079–4086, doi:10.1007/s00330-020-07471-8. 37. Liu, F.; Xing, D.; Zha, Y.; Wang, L.; Dong, W.; Li, L.; Gong, W.; Hu, L. Predictive value of temporal muscle thickness measurements on cranial magnetic resonance images in the prognosis of patients with primary glioblastoma. Front. Neurol. 2020, 11, 523292, doi:10.3389/fneur.2020.523292. 37. Liu, F.; Xing, D.; Zha, Y.; Wang, L.; Dong, W.; Li, L.; Gong, W.; Hu, L. Predictive value of temporal muscle thickness measurements on cranial magnetic resonance images in the prognosis of patients with primary glioblastoma. Front. Neurol. 2020, 11, 523292, doi:10.3389/fneur.2020.523292. 38. Guven, D.C.; Aksun, M.S.; Cakir, I.Y.; Kilickap, S.; Kertmen, N. The association of BMI and sarcopenia with survival in patients with glioblastoma multiforme. Futur. Oncol. 2021, 17, 4405–4413, doi:10.2217/fon-2021-0681. 38. Guven, D.C.; Aksun, M.S.; Cakir, I.Y.; Kilickap, S.; Kertmen, N. The association of BMI and sarcopenia with survival in patients with glioblastoma multiforme. Futur. Oncol. 2021, 17, 4405–4413, doi:10.2217/fon-2021-0681. 39. Huq, S.; Khalafallah, A.M.; Ruiz-Cardozo, M.A.; Botros, D.; Oliveira, L.A.P.; Dux, H.; White, T.; Jimenez, A.E.; Gujar, S.K.; Sair, H.I.; et al. A novel radiographic marker of sarcopenia with prognostic value in glioblastoma. Clin. Neurol. Neurosurg. 2021, 207, 106782, doi:10.1016/j.clineuro.2021.106782. 40. Furtner, J.; Berghoff, A.S.; Schöpf, V.; Reumann, R.; Pascher, B.; Woitek, R.; Asenbaum, U.; Pelster, S.; Leitner, J.; Widhalm, G.; et al. Temporal muscle thickness is an independent prognostic marker in melanoma patients with newly diagnosed brain metastases. J. Neurooncol. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 1 December 2021 doi:10.20944/preprints202112.0017.v1 28. Furtner, J.; Berghoff, A.S.; Albtoush, O.M.; Woitek, R.; Asenbaum, U.; Prayer, D.; Widhalm, G.; Gatterbauer, B.; Dieckmann, K.; Birner, P.; et al. Survival prediction using temporal muscle thickness measurements on cranial magnetic resonance images in patients with newly diagnosed brain metastases. Eur. Radiol. 2017, 27, 3167–3173, doi:10.1007/S00330-016-4707-6. 29. Ranganathan, K.; Terjimanian, M.; Lisiecki, J.; Rinkinen, J.; Mukkamala, A.; Brownley, C.; Buchman, S.R.; Wang, S.C.; Levi, B. Temporalis muscle morphomics: The psoas of the craniofacial skeleton. J. Surg. Res. 2014, 186, 246–252, doi:10.1016/j.jss.2013.07.059. 30. Rinkinen, J.; Zhang, P.; Wang, L.; Enchakalody, B.; Terjimanian, M.; Holcomb, S.; Wang, S.C.; Buchman, S.R.; Levi, B. Novel temporalis muscle and fat pad morphomic analyses aids preoperative risk evaluation and outcome assessment in nonsyndromic craniosynostosis. J. Craniofac. Surg. 2013, 24, 250–255, doi:10.1097/SCS.0b013e31827006f5. 31. Furtner, J.; Nenning, K.-H.; Roetzer, T.; Gesperger, J.; Seebrecht, L.; Weber, M.; Grams, A.; Leber, S.L.; Marhold, F.; Sherif, C.; et al. Evaluation of the temporal muscle thickness as an independent prognostic biomarker in patients with primary central nervous system lymphoma. Cancers (Basel). 2021, 13, 566, doi:10.3390/cancers13030566. 32. Furtner, J.; Genbrugge, E.; Gorlia, T.; Bendszus, M.; Nowosielski, M.; Golfinopoulos, V.; Weller, M.; Van Den Bent, M.J.; Wick, W.; Preusser, M. Temporal muscle thickness is an independent prognostic marker in patients with progressive glioblastoma: translational imaging analysis of the EORTC 26101 trial. Neuro. Oncol. 2019, 21, 1587–1594, doi:10.1093/neuonc/noz131. 33. Hsieh, K.; Hwang, M.; Estevez-Inoa, G.; Saraf, A.; Spina, C.S.; Smith, D.; Wu, C.C.; Wang, T.J.C. Temporalis muscle width as a measure of sarcopenia independently predicts overall survival in patients with newly diagnosed glioblastoma. Int. J. Radiat. Oncol. 2018, 102, e225, doi:10.1016/j.ijrobp.2018.07.771. 33. Hsieh, K.; Hwang, M.; Estevez-Inoa, G.; Saraf, A.; Spina, C.S.; Smith, D.; Wu, C.C.; Wang, T.J.C. Temporalis muscle width as a measure of sarcopenia independently predicts overall survival in patients with newly diagnosed glioblastoma. Int. J. Radiat. Oncol. 2018, 102, e225, doi:10.1016/j.ijrobp.2018.07.771. 34. An, G.; Ahn, S.; Park, J.-S.; Jeun, S.-S.; Hong, Y.-K. Association between temporal muscle thickness and clinical outcomes in patients with newly diagnosed glioblastoma. J. Cancer Res. Clin. Oncol. 2021, 147, 901–909, doi:10.1007/s00432-020-03386-5. 34. An, G.; Ahn, S.; Park, J.-S.; Jeun, S.-S.; Hong, Y.-K. Association between temporal muscle thickness and clinical outcomes in patients with newly diagnosed glioblastoma. J. Cancer Res. Clin. Oncol. 2021, 147, 901–909, doi:10.1007/s00432-020-03386-5. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 1 December 2021 2020, 29, 105050, doi:10.1016/j.jstrokecerebrovasdis.2020.105050. 24. Gomes, G.G.C.; Palinkas, M.; da Silva, G.P.; Gonçalves, C.R.; Lopes, R.F.T.; Verri, E.D.; Fabrin, S.C.V.; Fioco, E.M.; Siéssere, S.; Regalo, S.C.H. Bite force, thickness, and thermographic patterns of masticatory muscles post-hemorrhagic stroke. J. Stroke Cerebrovasc. Dis. 2021, 31, 106173, doi:10.1016/j.jstrokecerebrovasdis.2021.106173. 24. Gomes, G.G.C.; Palinkas, M.; da Silva, G.P.; Gonçalves, C.R.; Lopes, R.F.T.; Verri, E.D.; Fabrin, S.C.V.; Fioco, E.M.; Siéssere, S.; Regalo, S.C.H. Bite force, thickness, and thermographic patterns of masticatory muscles post-hemorrhagic stroke. J. Stroke Cerebrovasc. Dis. 2021, 31, 106173, doi:10.1016/j.jstrokecerebrovasdis.2021.106173. 25. Sakai, K.; Katayama, M.; Nakajima, J.; Inoue, S.; Koizumi, K.; Okada, S.; Suga, S.; Nomura, T.; Matsuura, N. Temporal muscle thickness is associated with the severity of dysphagia in patients with acute stroke. Arch. Gerontol. Geriatr. 2021, 96, 104439, doi:10.1016/j.archger.2021.104439. 25. Sakai, K.; Katayama, M.; Nakajima, J.; Inoue, S.; Koizumi, K.; Okada, S.; Suga, S.; Nomura, T.; Matsuura, N. Temporal muscle thickness is associated with the severity of dysphagia in patients with acute stroke. Arch. Gerontol. Geriatr. 2021, 96, 104439, doi:10.1016/j.archger.2021.104439. 26. Nozoe, M.; Kubo, H.; Kanai, M.; Yamamoto, M.; Okakita, M.; Suzuki, H.; Shimada, S.; Mase, K. Reliability and validity of measuring temporal muscle thickness as the evaluation of sarcopenia risk and the relationship with functional outcome in older patients with acute stroke. Clin. Neurol. Neurosurg. 2021, 201, 106444, doi:10.1016/j.clineuro.2020.106444. 26. Nozoe, M.; Kubo, H.; Kanai, M.; Yamamoto, M.; Okakita, M.; Suzuki, H.; Shimada, S.; Mase, K. Reliability and validity of measuring temporal muscle thickness as the evaluation of sarcopenia risk and the relationship with functional outcome in older patients with acute stroke. Clin. Neurol. Neurosurg. 2021, 201, 106444, doi:10.1016/j.clineuro.2020.106444. 27. Katsuki, M.; Narita, N.; Sasaki, K.; Sato, Y.; Suzuki, Y.; Mashiyama, S.; Tominaga, T. Standard values for temporal muscle thickness in the Japanese population who undergo brain check-up by magnetic resonance imaging. Surg. Neurol. Int. 2021, 12, 67, doi:10.25259/SNI_3_2021. 27. Katsuki, M.; Narita, N.; Sasaki, K.; Sato, Y.; Suzuki, Y.; Mashiyama, S.; Tominaga, T. Standard values for temporal muscle thickness in the Japanese population who undergo brain check-up by magnetic resonance imaging. Surg. Neurol. Int. 2021, 12, 67, doi:10.25259/SNI_3_2021. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 1 December 2021 2018, 140, 173–178, doi:10.1007/s11060-018-2948-8. 41. Ilic, I.; Faron, A.; Heimann, M.; Potthoff, A.-L.; Schäfer, N.; Bode, C.; Borger, V.; Eichhorn, L.; Giordano, F.A.; Güresir, E.; et al. Combined assessment of preoperative frailty and sarcopenia allows the prediction of overall survival in patients with lung cancer (NSCLC) and surgically treated brain metastasis. Cancers (Basel). 2021, 13, 3353, doi:10.3390/cancers13133353. 41. Ilic, I.; Faron, A.; Heimann, M.; Potthoff, A.-L.; Schäfer, N.; Bode, C.; Borger, V.; Eichhorn, L.; Giordano, F.A.; Güresir, E.; et al. Combined assessment of preoperative frailty and sarcopenia allows the prediction of overall survival in patients with lung cancer (NSCLC) and surgically treated brain metastasis. Cancers (Basel). 2021, 13, 3353, doi:10.3390/cancers13133353. 42. Leone, R.; Sferruzza, G.; Calimeri, T.; Steffanoni, S.; Conte, G.M.; De Cobelli, F.; Falini, A.; Ferreri, A.J.M.; Anzalone, N. Quantitative muscle mass biomarkers are independent prognosis factors in primary central nervous system lymphoma: 42. Leone, R.; Sferruzza, G.; Calimeri, T.; Steffanoni, S.; Conte, G.M.; De Cobelli, F.; Falini, A.; Ferreri, A.J.M.; Anzalone, N. Quantitative muscle mass biomarkers are independent prognosis factors in primary central nervous system lymphoma: doi:10.20944/preprints202112.0017.v1 The role of L3-skeletal muscle index and temporal muscle thickness. Eur. J. Radiol. 2021, 143, 109945, doi:10.1016/j.ejrad.2021.109945. The role of L3-skeletal muscle index and temporal muscle thickness. Eur. J. Radiol. 2021, 143, 109945, doi:10.1016/j.ejrad.2021.109945.
https://openalex.org/W3011664897	https://mdsoar.org/bitstream/11603/19935/1/1-s2.0-S0022169420303152-main.pdf	English	null	Pavement alters delivery of sediment and fallout radionuclides to urban streams	Journal of hydrology	2,020	public-domain	13,953	This work was written as part of one of the author's official duties as an Employee of the United States Government and is therefore a work of the United States Government. In accordance with 17 U.S.C. 105, no copyright protection is available for such works under U.S. Law. Access to this work was provided by the University of Maryland, Baltimore County (UMBC) ScholarWorks@UMBC digital repository on the Maryland Shared Open Access (MD-SOAR) platform. This work was written as part of one of the author's official duties as an Employee of the United States Government and is therefore a work of the United States Government. In accordance with 17 U.S.C. 105, no copyright protection is available for such works under U.S. Law. Access to this work was provided by the University of Maryland, Baltimore County (UMBC) ScholarWorks@UMBC digital repository on the Maryland Shared Open Access (MD-SOAR) platform. A B S T R A C T This manuscript was handled, with the assistance of Jian Luo, Associate Editor Sediment from urban impervious surfaces has the potential to be an important vector for contaminants, parti- cularly where stormwater culverts and other buried channels draining large impervious areas exit from un- derground pipes into open channels. To better understand urban sediment sources and their relation to fallout radionuclides, we collected samples of rainfall, urban sediment (pavement sediment, topsoil), streambank se- diment, and fluvial sediment (suspended sediment and bed sediment) for 7Be, 210Pbex, and 137Cs analysis. The results indicate that each rainfall event tags pavement sediment with elevated activities of 7Be and 210Pbex such that runoff from impervious surfaces in the buried channel part of the stream network contains the highest activities. Pavement sediment, because it is characteristically a thin veneer, has a small mass to rainwater ratio resulting in a greater tagging of 7Be and 210Pbex activity than does topsoil on a per gram basis. An unmixing model indicated that suspended-sediment samples collected at the culvert outlet from the buried-channel net- work are from pavement sediment sources (45 ± 25%) with a smaller component of topsoil (22 ± 19%), and a component from streambanks (32 ± 35%) that we infer to be older channel material and subsoil eroded from within the culvert system. Downstream from the culvert, suspended sediment collected from the open-channel parts of the stream had 7Be and 210Pbex activities that were substantially reduced by the contribution of sediment from streambanks (57 ± 15%), with pavement contributions decreasing to 15 (±9%) and topsoil contributing 28 (±7%). The results highlight the utility of 7Be, 210Pbex, and 137Cs as tracers of urban sediment sources, resulting in a unique radionuclide signature for urban watersheds compared to other sediment-source settings. This manuscript was handled, with the assistance of Jian Luo, Associate Editor Keywords: A B S T R A C T https://doi.org/10.1016/j.jhydrol.2020.124855 Received 20 August 2019; Received in revised form 14 January 2020; Accepted 13 March 2020 Available online 16 M arch 2020 0022-1694/ Published by Elsevier B.V. ⁎ Corresponding author. E-mail address: agellis@usgs.gov (A.C. Gellis). a U.S. Geological Survey, Baltimore, MD, United States b U.S. Geological Survey, Menlo Park, CA, United States c U.S. Geological Survey, Austin, TX, United States d Center for Urban Environmental Research and Education, University of Maryland, Baltimore County, Baltimore, MD, United States e Department of Chemical, Biochemical, and Environmental Engineering, University of Maryland, Baltimore County, Baltimore, MD, United States f Department of Geography and Environmental Systems, University of Maryland, Baltimore County, Baltimore, MD, United States g Department of Geological Sciences, University of Alabama, Tuscaloosa, AL, United States h Warner College of Natural Resources, Colorado State University, Fort Collins, CO, United States E-mail address: agellis@usgs.gov (A.C. Gellis). ⁎ Corresponding author. Please provide feedback Please support the ScholarWorks@UMBC repository by emailing scholarworks-group@umbc.edu and telling us what having access to this work means to you and why it’s important to you. Thank you. Journal of Hydrology 588 (2020) 124855 Pavement alters delivery of sediment and fallout radionuclides to urban streams Allen C. Gellisa,⁎, Christopher C. Fullerb, Peter C. Van Metrec, Barbara J. Mahlerc, Claire Weltyd,e Andrew J. Millerd,f, Lucas A. Nibertg, Zach J. Cliftona, Jeremy J. Malena, John T. Kemperh a U.S. Geological Survey, Baltimore, MD, United States b U.S. Geological Survey, Menlo Park, CA, United States c U.S. Geological Survey, Austin, TX, United States d Center for Urban Environmental Research and Education, University of Maryland, Baltimore County, Baltimore, MD, United States e Department of Chemical, Biochemical, and Environmental Engineering, University of Maryland, Baltimore County, Baltimore, MD, United States f Department of Geography and Environmental Systems, University of Maryland, Baltimore County, Baltimore, MD, United States g Department of Geological Sciences, University of Alabama, Tuscaloosa, AL, United States h Warner College of Natural Resources, Colorado State University, Fort Collins, CO, United States Keywords: Fallout radionuclides Sediment Urban Buried channels 1. Introduction been well quantified. The three major sources of urban sediment—impervious-surface sediment, upland soils, and streambanks—are associated with different types and concentrations of contaminants. Impervious surface sediment can have high concentra- tions of contaminants from atmospheric deposition (e.g., Hg, PAHs) (Callender and Rice, 2000; Van Metre and Mahler, 2003; Many common urban contaminants, such as metals and polycyclic aromatic hydrocarbons (PAHs), sorb to sediment and are transported to streams with runoff (Sartor et al., 1974; Paul and Meyer, 2001; Mahler et al., 2005), yet sources of sediment to urban streams have not Journal of Hydrology 588 (2020) 124855 A.C. Gellis, et al. Fig. 1. Map of the Dead Run study area showing pavement sweep sites (Streets A-D, Parking Lot E),runoff samples, soil samples, and bank samples and tributaries (T1,T2) entering between the sediment collection sites Alexander Culvert (#2) and the Dead Run Gage (#1). [r = runoff sample, collection, f = soil sample collection, b = bank sample collection] Fig. 1. Map of the Dead Run study area showing pavement sweep sites (Streets A-D, Parking Lot E),runoff samples, soil samples, and bank samples and tributaries (T1,T2) entering between the sediment collection sites Alexander Culvert (#2) and the Dead Run Gage (#1). [r = runoff sample, collection, f = soil sample collection, b = bank sample collection] however, stream channels are paved over or buried during urbanization and directed into culverts, pipes, and concrete-lined ditches (Elmore and Kaushal, 2008; Napieralski and Carvalhaes, 2016). In these buried- channel parts of the watershed, streambank erosion does not occur and surface-derived sediment (e.g. soil, impervious surfaces) might be ex- pected to dominate fluvial sediment (suspended sediment (SS) and bed sediment). Boonyatumanond et al., 2007; Eckley and Branfireun, 2008; Thapalia et al., 2010), and wear of solid materials (e.g. roofing materials, pa- vement sealants, and galvanized metal) (Van Metre and Mahler, 2003; Mahler et al., 2005). Upland soils can contain pesticides and other products used in home and commercial outdoor applications as well as contaminants associated with urban non-point sources, legacy in- dustrial sources, and atmospheric deposition (Cannon and Horton 2009; Hatcher and Filippelli 2010; Jorgenson and Young 2010; Watts et al. 2010, Bain et al., 2012). processes in urban settings.l Survey (USGS) stream gaging site (Dead Run near Catonsville, Mary- land, USGS station ID 01589312), about 0.7 km downstream from Alexander Culvert (“Dead Run gage”; 1.63 km2 drainage area). The drainage area at the gage, which includes the Alexander Culvert drai- nage, is 42% impervious and 58% open space (Table 1). The watershed drains the Piedmont physiographic province and is underlain by the Mount Washington Amphibolite subunit of the Baltimore Complex (Crowley, 1976) and by interbedded gravels, sands, silts, and clays of the Potomac Group in the upper reaches of the watershed. processes in urban settings.l In an analysis of fluvial sediment for wadeable streams in the Midwest U.S., Gellis et al. (2017b) reported that stream sediment in watersheds with urban areas had higher activities of 7Be and 210Pbex than stream sediment in agricultural and undeveloped settings (Supporting Information (Fig. S1). That study, however, considered only surface (agricultural) soil and streambank and channel erosion as endmember sources of stream sediment and did not differentiate sedi- ment delivered from impervious surfaces in source partitioning. Froger et al. (2018) reported high 7Be and 210Pbex activities in sediment col- lected from paved road surfaces and attributed elevated activities in suspended sediments in streams as resulting from particles rapidly transported from urban areas. In these settings, 7Be and 210Pbex activ- ities may be higher in the buried-channel portions of the watershed with substantial impervious surfaces, in contrast to the open channel parts of the watershed, where streambank erosion processes become important. Understanding sediment sources and 7Be and 210Pbex dy- namics in urban settings could provide insight into particle-associated contaminant sources and transport processes affecting urban streams. p pp Flow exits the Alexander Culvert and continues in an open channel to the Dead Run gage (Fig. 1). Two tributaries (T1 and T2, Fig. 1) enter Dead Run between Alexander Culvert and the Dead Run gage; these tributaries are buried channels in their upper reaches and exit at cul- verts to open channels. Several small storm drains also enter the channel from neighborhoods upstream of the gage. During the study, parts of Dead Run between the Alexander Culvert and tributary T1 were undergoing restoration to create a stabilized reach with an engineered riparian zone (Fig. 1; Fig. 2A). processes in urban settings.l Although some disturbance of the channel may have occurred during the restoration, measures were taken during the restoration to reduce erosion, including pinning of erosion-control nets and fabric along the stream. Our results at the Dead Run gage are unlikely to have been compromised by these activities. Between the gage and the restoration area and along T1, channels are incised (Fig. 2B, C) with steep eroding streambanks and bed material that ranges from gravel and sands to fines. f Here we examine sediment sources and transport processes and associated 7Be, 210Pbex, and 137Cs in an urban watershed. We address three key questions: How do activities of 7Be and 210Pbex vary in urban sediments by source type and in response to rain events? What do these activities tell us about the sources of sediment to urban streams? How do stream-channel dynamics affect activities of 7Be and 210Pbex as se- diment is transported from urban source areas with buried channels into open-channel downstream areas? i Dead Run is one of many urban watersheds in Baltimore City and Baltimore County in a research network operated by the University of Maryland, Baltimore County (UMBC)–Center for Urban Environmental Research and Education (CUERE) (https://cuere.umbc.edu). Among the research objectives of the UMBC-CUERE program in Dead Run are to understand flow, sediment, and contaminant dynamics in urban set- tings using high-frequency sampling with in-stream sensors and high spatial-resolution numerical models (Duncan et al., 2017; Barnes et al., 2018; Kemper et al., 2019). 2. Materials and methods 2.1. Study area 2.2. Rainfall, runoff, pavement, soil, streambank, and fluvial sediment 2.2. Rainfall, runoff, pavement, soil, streambank, and fluvial sediment Rainfall, stormwater runoff, pavement sediment (road and parking lot particulates), soil, streambank material, and fluvial sediment (bed and SS) were collected during three periods: 1) summer (14 July–17 Aug., 2017); 2) fall (20 Oct.–10 Nov., 2017), and 3) winter/spring (27 Feb.–14 May, 2018) seasons. 2.1. Study area To address the questions described above, we examined 7Be, 210Pbex, and 137Cs activities for two locations in the Dead Run wa- tershed, an urban watershed in Baltimore, Maryland (Fig. 1). One lo- cation was the upper part of the watershed where, except for two gullies in a small open space between roads, there are no open channels—all the natural stream channels were buried during urbanization. All sur- face runoff that enters the buried channel network through numerous storm drains exits to an open channel at the Alexander Avenue Culvert (herein called the “Alexander Culvert”)(drainage area = 0.369 km2) (Fig. 1). About one-half of this area is impervious (roads, rooftops, and parking lots) and the other half is non-impervious or open space (lawns, parks, and green space) (Table 1). The second sampling location was the area that drains from Alexander Culvert to the U.S. Geological 2.2.1. Sample collection Rainfall (n = 33 samples) was collected in two 9.5-L acid-washed plastic buckets placed ~ 1.5 m above the ground. Rainfall for the summer was collected at the USGS office in Catonsville, MD (6 km from the Dead Run gage), and for the other seasons was collected at a rain gage at the Dead Run gage. The rain sample was acidified with 10% hydrochloric acid (HCl) and transferred to 1-L bottles along with three 10% HCl rinses of the collection bucket prior to shipping to the USGS Sediment Radioisotope Laboratory in Menlo Park, CA, for 7Be, 210Pbex, and 137Cs analysis. Runoff samples, as defined here, are stormwater runoff that occurs on impervious surfaces and contains sediment. Two types of runoff were sampled: stormwater runoff collected at the Alexander Culvert (n = 9 samples) and road runoff collected at road storm drains and parking lot storm drains (n = 8 samples). Both types of runoff were collected by submerging an 18.9-L bucket into the runoff (flow). Table 1 Classification of area draining to Alexander Culvert (0.37 km2) and Dead Run gage (1.63 km2). Contributing Area (km2) Percentage Alexander Culvert 0.369 Roads 0.048 13 Driveways 0.010 3 Residential Rooftops 0.028 7 Commercial Rooftops 0.028 8 Parking Lots 0.071 19 Total Impervious area 0.185 50 Other (parks, lawns, green space, etc.) 0.184 50 Dead Run Gage 1.630 Roads 0.226 14 Driveways 0.044 3 Residential Rooftops 0.141 9 Commercial Rooftops 0.085 5 Parking Lots 0.191 12 Total Impervious area 0.687 42 Other (parks, lawns, green space, etc.) 0.947 58 Table 1 Classification of area draining to Alexander Culvert (0.37 km2) and Dead Run gage (1.63 km2). 1. Introduction Streambanks are assumed to have neg- ligible concentrations of anthropogenic contaminants, except perhaps in older developed parts of the United States where some streambanks contain legacy contamination from sediment deposited in the past (18th–19th centuries) (Southworth et al., 2013; Taylor and Owens, 2009). Radionuclides in atmospheric fallout—fallout radionuclides 7Be, 210Pbex (excess 210Pb, the part not supported by decay of parent radionuclides in geologic materials), and 137Cs—are delivered to the Earth’s surface primarily by rainfall and become a marker of surface- derived sediment. Eroding streambanks typically contain lower activ- ities of 7Be, 210Pbex, and 137Cs, because the vertical position of streambanks leads to little direct contact with rainfall (Matisoff et al., 2005; Hancock et al., 2014). Differences in 7Be, 210Pbex, and 137Cs ac- tivities between geomorphic features (streambanks and upland sur- faces) have been used to discriminate sediment sources and estimate the transport and transit times of sediment (Dominik et al., 1987; Evrard et al., 2010; Gellis et al., 2017b; Huon et al., 2017; Mabit et al., 2014; Matisoff et al., 2005; Olley et al., 2013a,b; Slimane et al., 2016; Froger et al., 2018; Wallbrink et al., 2002). Of these, only Froger et al. (2018) examined 7Be, 210Pbex, and 137Cs activities and sediment It is well documented that urbanization and increased impervious cover leads to more runoff and higher peak flows (Leopold 1968; Paul and Meyer, 2001 Walsh et al., 2005). These hydrologic changes in- crease streampower, leading to channel widening, streambank erosion, and channel incision (Booth, 1990, 1991; Gellis et al., 2017a; Wolman, 1967). Thus, streambanks in urban environments are expected to con- tribute a large part of the total sediment load (Devereux et al., 2010; Gellis et al., 2017a; Cashman et al., 2018). In many urban areas, 2 A.C. Gellis, et al. Journal of Hydrology 588 (2020) 124855 2.2.2. Sample analysis All samples were transported to the USGS laboratory in Baltimore, MD, and refrigerated. Sediment for radionuclide analysis—pavement sediment, soil, bank material, decanted SS samples (following a minimum of 1 week of refrigeration and settling), and bed materi- al—were wet-sieved with distilled deionized (DI) water using a 63-μm polyester sieve. The sieved slurry was collected in glass bowls and dried at 65 °C for 2 or more days. The dried fine sediment (<63 μm) was removed with a plastic utensil and sent for analysis of fallout radio- nuclides, organic carbon content, and grain size. Sediment samples were analyzed for 7Be, 210Pb, 226Ra, and 137Cs at the USGS Sediment Radioisotope Laboratory in Menlo Park, CA, using high-resolution germanium detector gamma spectrometers following methods described in Fuller et al. (1999) and Van Metre et al. (2004). Measured activities of 7Be and 137Cs were corrected for radioactive decay from the date of sample collection to the date of analysis. Excess 210Pbex was calculated as the difference between the measured total 210Pb and 226Ra, which is determined from the short-lived intermediate gamma-emitting isotopes 214Pb and 214Bi. Method detection limits (MDLs) for 7Be and 210Pbex were 0.0067 Bq/g. The sample-specific detection limit for 7Be was determined by correcting the MDL for decay and varied among samples (0.0117 to 0.0317 Bq/g) as a result of dif- ferent times between sample collection and analysis. The MDL for 137Cs was 0.0004 Bq/g. A sediment yield at each vacuum site was determined by dividing the sediment mass recovered by the area vacuumed, determined as the width of the nozzle (12.5 cm) multiplied by the road or parking lot length sampled (m). Yields are reported as a total (unsieved) sediment yield (g/m2) and a fine (<63 µm) sediment yield (g/m2). We consider both of these yields as “potential sediment yields,” as the entire mass of sediment may not be mobilized and transported from the pavement by any given rain event. Soil samples were collected at nine sites (Fig. 1) in the Dead Run watershed. At each site, three to five subsamples were taken using a plastic spatula from the top 1 cm of surficial soil within 3 m of one another and composited. Four samples of streambank material were taken from eroding streambanks between the restored area and the Dead Run gage (Fig. 1). Prior to sample collection, the exposed surface of a streambank was scraped off and discarded. Table 1i l Pavement sediment samples (n = 85 samples) were collected at the beginning of each sampling season and after rainfall events at four road sites chosen to represent the range of road types present in the wa- tershed (residential and commercial), and from one parking lot in the Alexander Culvert watershed (Fig. 1). Similar to Collins et al. (2011), pavement sediment was collected with a hand broom and dustpan from alongside the curb and upgradient of a storm grate and placed in a 3 Journal of Hydrology 588 (2020) 124855 A.C. Gellis, et al. Fig. 2. (A) Dead Run below Alexander Culvert in restored area (11 April, 2018); (B) Incision along tributary T2 showing upstream view (11 April, 2018); (C) Dead Run upstream of gage (9 November 2018); (D) Alexander Culvert samplers (29 July, 2017); (E) Dead Run samplers (29 July, 2017). [Photograph by A.C. Gellis, U.S. Geological Survey] Fig. 2. (A) Dead Run below Alexander Culvert in restored area (11 April, 2018); (B) Incision along tributary T2 showing upstream view (11 April, 2018); (C) Dead Run upstream of gage (9 November 2018); (D) Alexander Culvert samplers (29 July, 2017); (E) Dead Run samplers (29 July, 2017). [Photograph by A.C. Gellis, U.S. Geological Survey] plastic bag. In order to obtain at least 3 g of fine sediment, the area swept varied between samples; and although each area was swept clean, it is likely that some sediment remained behind. deposits using a stainless-steel spatula. Sediment from at least five de- posits was collected and composited into one sample. A bed sample was not collected at Alexander Culvert because the channel immediately below the culvert was a transport reach with a gravel bed devoid of fines for most of the collection period. To estimate the mass of material present on pavement in the area draining to Alexander Culvert, sediment also was sampled 4 times with a high efficiency vacuum (Envirometrics Model HVS3 High-Volume Surface Sampler) (n = 39 samples). The vacuum, which is designed for sampling settled house dust at particle sizes 5 µm or larger, was fitted with a 12.5-cm-long, 1-cm-wide nozzle. Prior to outdoor sampling, controlled tests with the vacuum were done in the USGS Baltimore, MD, laboratory, where known masses of fine material (<63 µm) were va- cuumed. Table 1i The HVS3 averaged 98.3% efficiency for the range of masses tested (Table S1), although wind and unevenness of the pavement surface may introduce variability when sampling outdoors. For two of the four vacuuming events, only the pavement sites used in the sweep samples (not including Street C) (Fig. 1) were vacuumed. For the other two vacuuming events, sites were selected randomly to cover the spatial extent of the Alexander Culvert drainage area. Each sample was col- lected by starting at the midpoint in the road and ending at the curb. 2.4. Sediment sourcing Pavement sediment can be from a mixture of sources, including upland soil from lawns and open space, building and construction material, leaf litter, atmospheric deposition of particles, worn tires, and pavement erosion (Taylor and Owens, 2009; Sansalone and Cristina, 2004). A two-endmember mixing model using 137Cs as a tracer was used to calculate soil and non-soil sources to pavement sediment. One endmember is soil, with an activity equal to the median of the 137Cs activity in soil samples (0.0071 Bq/g) (Clifton et al., 2019). The non- soil endmember is assigned zero activity under the assumption that the non-soil material is from sources such as construction materials and tire and asphalt wear that are not expected to have detectable 137Cs activity. The percentage of pavement residue originating from soil was de- termined as: i Runoff samples were split in the laboratory to determine fallout radionuclide activities in whole water and in isolated particulates. One split was churned and an aliquot was taken to determine the suspended- sediment concentration (SSC). The volume of the aliquot was recorded and the aliquot was pumped through a 0.45-µm glass fiber filter. The filter was dried at 105 °C and weighed. The remaining slurry from this split was processed for analysis in the same manner as SS from the passive samplers to record activity in the particulates in Bq/g. A second split was prepared and analyzed in the same way as rainwater to measure “total” whole-water 7Be and 210Pbex activities. It was assumed that 7Be and 210Pb are effectively solubilized from the SS at the low pH of the acidified sample. It also was assumed that isotopic equilibrium is attained between yield tracers (stable Pb and Be) and 7Be and 210Pb both in solution and on sediments. 210Pb activity in “total” whole-water runoff samples is considered unsupported 210Pb (210Pbex) because the supported activity defined by 226Ra was not detectable. Reported ac- tivities in the whole-water samples in units of Bq/L were normalized to SSC to obtain units of Bq/g. Additional analytical methods for sediment are shown in SI Text I. 2.2.2. Sample analysis The resulting activity was then divided by volume of rain collected to yield activities in units of Bq/L. 137Cs and 226Ra were below detection limits in rain samples, indicating negligible contributions of 210Pb and 7Be from aeolian dust that may have entered the rainfall collector. Additionally, all 210Pb activity in rainfall is considered unsupported 210Pb (210Pbex) because the supported activity defined by 226Ra was not detectable.f where RDSEDpot(t) is the potential activity of 7Be or 210Pbex in pavement sediment for sample period t as a result of precipitation (Bq/g); RAINtot is the total rainfall over 1 m2 of the ground surface (L/m2); RAINact is the measured activity of 7Be or 210Pbex in rainfall (Bq/L); SEDyld is the median fine sediment yield from vacuuming (g/m2) = 1.57 g/m2 (Clifton et al., 2019); and RDSEDpre is the pavement sediment activity for 7Be or 210Pbex (Bq/g) prior to the rainfall event and corrected for 7Be decay since the time of the last pavement sediment sweep.fii Because of the strong affinity of 7Be and 210Pbex to fine particulate matter (Benmansour et al., 2014; Taylor et al., 2012), we assume that all whole-water runoff samples have an activity per liter (Bq/L) equal to rainfall, and that some of the 7Be and 210Pbex are likely sorbed to particles and surfaces that are not transported with runoff to the cul- vert. The calculation therefore provides an upper limit for radionuclide tagging. 2.4. Sediment sourcing = Soil Cs Cs % / 100 sample med (2) (2) = Soil Cs Cs % / 100 sample med where Soil% is the percentage of the sample that is derived from soil, Cssample is the 137Cs activity (Bq/g) of the sample, and Csmed is the median of the 137Cs activities in the soil samples (0.0071 Bq/g). To determine sources of SS samples collected from Alexander Culvert and SS and bed sediment at Dead Run, an unmixing model commonly used in sediment source studies was applied: = = = Cssi CsiPs Cssi Res ( ) i n s m 1 1 2 (3) (3) where Res is the residual sum of squares, Cssi is the concentration of tracer property i in the target sediment, Csi is the mean concentration of the tracer property in the source group s, and Ps is the relative con- tribution from source group s (after Walling, 2005; after Schuller et al., 2013). 2.2.3. Collection and computation of rainfall, turbidity, suspended sediment, and flow The unmixing model equation assumes that 0 ≤Ps ≤1 and l UMBC operates a rainfall and water-quality network in the Baltimore Metropolitan region that includes Dead Run. Water-quality sensors and a rain-gage station are co-located with USGS stream gage 01589312. = = Ps 1 s m 1 (4) The Ps that yields the lowest residual sum of squares corresponds to the final source contributions (pavement, soil, banks) for a given target sample. The collection and computation methods for rainfall, turbidity, suspended sediment, and flow are described in SI Text II. 2.2.2. Sample analysis A subsample was trans- ferred and weighed into a polyethylene vial for radionuclide analysis by high resolution gamma spectrometry as described for sediment samples. Activities of 210Pb and 7Be in rainfall samples were corrected for the recovery yield of Pb and Be (ratio of measured to added mass of stable and Pb and Be) and the fraction of redissolved sample analyzed. This method assumes that the stable Pb and Be attain isotopic equilibrium with all fallout radionuclides in the sample such that the radioisotopes are recovered in the same proportion as the yield tracer. The resulting activity was then divided by volume of rain collected to yield activities in units of Bq/L. 137Cs and 226Ra were below detection limits in rain samples, indicating negligible contributions of 210Pb and 7Be from aeolian dust that may have entered the rainfall collector. Additionally, all 210Pb activity in rainfall is considered unsupported 210Pb (210Pbex) because the supported activity defined by 226Ra was not detectable.f Five mL of 10% (wt/v) solution of ferric chloride was added to facilitate concentration of radionuclides by sorption of Pb and Be to iron hy- droxide formed on raising pH. Samples were equilibrated on an orbital shaker for 48 h. Concentrated ammonium hydroxide was then added to increase pH to 10 and precipitate iron hydroxide to sorb both the stable and radioactive isotopes of Pb and Be. The iron hydroxide floc was recovered by centrifugation after settling and decanting off the over- lying solution by siphon. The concentrated iron hydroxide was then dissolved in concentrated HCl and transferred to a tared bottle. Con- centrations of the added stable Pb and Be yield tracers in rainfall samples were measured by ICP-MS. Recoveries averaged 95 ± 8% and 86 ± 6% for stable Pb and Be, respectively. A subsample was trans- ferred and weighed into a polyethylene vial for radionuclide analysis by high resolution gamma spectrometry as described for sediment samples. Activities of 210Pb and 7Be in rainfall samples were corrected for the recovery yield of Pb and Be (ratio of measured to added mass of stable and Pb and Be) and the fraction of redissolved sample analyzed. This method assumes that the stable Pb and Be attain isotopic equilibrium with all fallout radionuclides in the sample such that the radioisotopes are recovered in the same proportion as the yield tracer. = + RDSED RAIN RAIN SED ( RDSEDpre pot t tot act yld ( ) ) 2.2.2. Sample analysis Each streambank sample is a composite of subsamples from 3 to 5 eroding streambanks collected from the bottom to the top of each streambank using a plastic hand shovel. The reported 1-sigma uncertainty in the measured radionuclide activity (σ1) was calculated from the random counting error of samples and background standard spectra at the 1 standard deviation level. Uncertainty in measured activity was typically within ±10% of the measured activity for total 210Pb and 226Ra, ±18% for 7Be, and ±20% for 137Cs. Uncertainty in unsupported 210Pb was propagated from the uncertainties in total 210Pb and 226Ra activity and averaged ±20%.i Fluvial sediment consisted of samples of SS and bed material. SS was collected at Alexander Culvert and the Dead Run gage (Figs. 1 and 2D,E) using passive samplers (Phillips et al., 2000). After a rainfall event, water (if present) and sediment were removed from the passive samplers and transferred to 18.9-L buckets. Acidified rain samples were screened through 63-µm polypropylene mesh into a container along with three 10% HCl rinses of each shipping bottle and processed following the methods outlined in Conaway et al. (2013) and Nakano et al. (2008) to concentrate the radionuclides into a small volume. Briefly, 0.100 ml each of stable Pb and Be inductively coupled plasma mass spectrometry (ICP-MS) stock standard solutions (1000 µg/mL) was added to the acidified sample to determine recovery. Samples of bed material were collected at the Dead Run gage. Bed material was collected from the surface (<1cm) of fine-grained channel 4 Journal of Hydrology 588 (2020) 124855 A.C. Gellis, et al. Five mL of 10% (wt/v) solution of ferric chloride was added to facilitate concentration of radionuclides by sorption of Pb and Be to iron hy- droxide formed on raising pH. Samples were equilibrated on an orbital shaker for 48 h. Concentrated ammonium hydroxide was then added to increase pH to 10 and precipitate iron hydroxide to sorb both the stable and radioactive isotopes of Pb and Be. The iron hydroxide floc was recovered by centrifugation after settling and decanting off the over- lying solution by siphon. The concentrated iron hydroxide was then dissolved in concentrated HCl and transferred to a tared bottle. Con- centrations of the added stable Pb and Be yield tracers in rainfall samples were measured by ICP-MS. Recoveries averaged 95 ± 8% and 86 ± 6% for stable Pb and Be, respectively. 3.1. Rainfall and flow during the study We used measured activities of 210Pbex and 7Be in rainfall, potential sediment yields from pavement, and 210Pbex and 7Be activities in pa- vement sediment measured prior to the rainfall event to estimate the enrichment of pavement sediment that might occur (potential activity) in response to a rainfall event. The potential activity of 7Be and 210Pbex in the sweep samples after being tagged by rainfall was determined as: Rainfall, pavement, and fluvial samples were collected for 17 sam- pling periods over a range of flows distributed over the three sampling seasons (Fig. 3; Table 2; SI Table S2). A sample period begins after the last sample is collected from the previous sampling period (the end of a rainfall event) and ends when the last sample of the current sampling period is collected (also a rainfall event). Samples collected during each sampling period can include pavement sweeps (dry weather) and (1) 5 Journal of Hydrology 588 (2020) 124855 A.C. Gellis, et al. Fig. 3. Summary of flow events at Dead Run gage (USGS station ID 01589312) for the 17 sampling periods in this study in (A) Summer 2017, (B) Fall 2017, and (C) Winter/Spring 2018 (data available at U. S. Geological Survey, 2019). Fig. 3. Summary of flow events at Dead Run gage (USGS station ID 01589312) for the 17 sampling periods in this study in (A) Summer 2017, (B) Fall 2017, and (C) Winter/Spring 2018 (data available at U. S. Geological Survey, 2019). Fig. 3. Summary of flow events at Dead Run gage (USGS station ID 01589312) for the 17 sampling periods in this study in Winter/Spring 2018 (data available at U. S. Geological Survey, 2019). ad Run gage (USGS station ID 01589312) for the 17 sampling periods in this study in (A) Summer 2017, (B) Fall 2017, and (C) t U. S. Geological Survey, 2019). rainfall, runoff, and suspended and bed sediment from one or more rainfall events. A sampling period had as many as one to five peak flows, depending on how closely spaced the rainfall events were (Table 2). The decision to collect samples was based on rainfall and discharge. The lower sediment tube at Dead Run gage filled at a dis- charge of about 0.19 m3/s. When it rained, we examined the on-line real time discharge at Dead Run; if it exceeded 0.19 m3/s the decision was made to retrieve SS samples, if conditions permitted (low flow). 3.1. Rainfall and flow during the study Street sweeps were collected only when conditions were dry. Additional rainfall event(s) did occur before it was possible to get street sweeps during some sampling periods, and thus a sampling period may have had multiple rainfall and SS samples but only one set of sweep samples. Table 2 Seventeen runoff samples (9 stormwater samples at Alexander Culvert and 8 stormwater samples from roads) were collected during 4 of the 17 sampling periods (Table 2). Of the 9 runoff samples collected at Alexander Culvert, 7 were used for whole-water analysis (Bq/L), and 6 were used for dry sediment analysis (Bq/g). Of the 8 runoff samples collected on roads, 7 were used for whole water analysis (Bq/L) and 5 were used for dry sediment analysis (Bq/g) (Clifton et al., 2019). Often because of high-flow conditions it was not possible to access the suspended sampler immediately at the end of the event, and in some of these cases a second flow event occurred (Table 2) during the same sampling period and the sampler composited suspended sediment over more than one closely spaced rainfall event. Rain samples were col- lected during 16 of the 17 sampling periods and analyzed for radio- nuclides. Seventeen runoff samples (9 stormwater samples at Alexander Culvert and 8 stormwater samples from roads) were collected during 4 of the 17 sampling periods (Table 2). Of the 9 runoff samples collected at Alexander Culvert, 7 were used for whole-water analysis (Bq/L), and 6 were used for dry sediment analysis (Bq/g). Of the 8 runoff samples collected on roads, 7 were used for whole water analysis (Bq/L) and 5 were used for dry sediment analysis (Bq/g) (Clifton et al., 2019). To determine if any of the seasons sampled were drier or wetter than average, rainfall for each sampling season was compared to the historic rainfall for the same season measured at the National Weather Service gage at the Baltimore/Washington International Airport (USW00093721), 16.2 km from the Dead Run gage (Table S3). The three seasons had rainfall that was 179% (summer), 137% (fall), and 70% (winter/spring) of normal rainfall for that season (Table S3). The wetter summer and fall seasons could affect the results by supplying more sediment than might occur during average rainfall conditions or could cause higher flows that exhaust sediment in channel storage. Conversely, samples collected during the drier winter/spring season might reflect lower sediment flux than might occur during average rainfall conditions. Fig. 5. Comparison of 210Pbex in Midwest topsoil, Dead Run soil, and street sweeps. parts of the Dead Run watershed were developed between the 1950s and 1970s. Table 2 The median year for homes built in the Dead Run watershed was 1962 (Barnes et al., 2018), which is one year earlier than the peak fallout of 137Cs. Thus topsoil mobilized during the build out of the area would have contained relatively high 137Cs, which could have been deposited on the floodplain. l In contrast to 137Cs, 210Pbex and 7Be are continually deposited on the land surface by atmospheric fallout, resulting in distinct signatures of these two radionuclides in the three sediment sources. Activities of 210Pbex and 7Be were substantially higher in samples of pavement se- diment than in samples of upland soil and streambank material (Fig. 4A,B). The difference between activities in pavement sediment and streambank material is consistent with the limited exposure of streambanks to modern atmospheric fallout (Gellis et al., 2017b; Hancock et al., 2014; Matisoff et al., 2005). The soil activities of 210Pbex in Dead Run watershed are similar to those reported for agricultural topsoil in the Midwest (Gellis et al., 2017b) (Fig. 5). The difference between activities in pavement sediment and upland soil (Fig. 5) re- flects the difference in the mass of sediment exposed to rain. On pa- vement, a thin veneer of sediment is exposed to a relatively large vo- lume of rain whereas for soil, the top several centimeters are exposed. As a result, on a per gram basis, pavement sediment receives greater amounts of radionuclides (resulting in higher activities) during a rain event than does soil. Table 2 Table 2 Hydrologic summary of sampled events at Alexander Culvert and Dead Run Gage. * Indicates a stormwater runoff sample(s) was taken at Alexander Culvert and ǂ indicates a road runoff sample(s) was taken during a given sampling period. Sampling period start date Sampling period start time Sampling period end date Sampling period end time Total Rain (mm) Number of flow events in this period at Dead Run gage Highest peak flow during sample period, (m3/s) Total Suspended Sediment Load, (Mg) Summer 2017 7/14/2017 13:40 7/27/2017 14:35 21.3 4 0.96 1.3 7/27/2017 14:35 8/1/2017 13:30 96.2 4 2.81 3.4 8/1/2017 13:30 8/3/2017 12:30 5.4 1 1.75 0.59 8/3/2017 12:30 8/4/2017 10:30 7.5 1 2.86 2.1 8/4/2017* 10:30 8/10/2017 10:20 25.3 3 1.16 1.1 8/10/2017 10:20 8/17/2017 09:30 51.8 3 2.54 7.0 Fall 2017 10/20/2017 13:00 10/24/2017 13:45 6.3 2 0.46 1.0 10/24/2017ǂ 13:45 10/31/2017 11:30 37.3 4 1.84 5.1 10/31/2017ǂ 11:30 11/10/2017 11:46 39.4 2 0.61 4.2 Spring 2018 2/27/2018 12:00 3/6/2018 12:50 16.6 2 0.90 1.2 3/6/2018 12:50 3/19/2018 12:00 13.1 1 0.21 0.19 3/19/2018 12:00 3/26/2018 11:40 snow 5 0.62 0.79 3/26/2018* 11:40 4/4/2018 13:05 12.7 3 1.32 0.52 4/4/2018 13:05 4/18/2018 13:55 53.5 1 14.0 19.9 4/18/2018 13:55 4/26/2018 13:45 32.1 3 1.31 2.7 4/26/2018 13:45 5/2/2018 15:40 8.41 1 0.44 0.22 5/2/2018 15:40 5/14/2018 15:50 28.8 3 3.20 2.9 Alexander Culvert and Dead Run Gage. * Indicates a stormwater runoff sample(s) was taken at Alexander Culvert and ǂ n during a given sampling period. Table 2 Hydrologic summary of sampled events at Alexander Culvert and Dead Run Gage. * Indicates a stormwater runoff sampl indicates a road runoff sample(s) was taken during a given sampling period. 6 A.C. Gellis, et al. Journal of Hydrology 588 (2020) 124855 Fig. 5. Comparison of 210Pbex in Midwest topsoil, Dead Run soil, and street sweeps. Often because of high-flow conditions it was not possible to access the suspended sampler immediately at the end of the event, and in some of these cases a second flow event occurred (Table 2) during the same sampling period and the sampler composited suspended sediment over more than one closely spaced rainfall event. Rain samples were col- lected during 16 of the 17 sampling periods and analyzed for radio- nuclides. ry of radionuclide activity in sediment (Bq/g) for all samples (A) 7Be, (B)210Pbex, and (C) 137Cs Table S2; Clifton et al., 2019 3.2. Radionuclide activities in source and collected sediment samples We interpret the lack of correlation between 210Pbex and 137Cs activities in pavement sediment as resulting from tagging of pavement sediment by recent fallout of 210Pbex (and 7Be) but not of 137Cs.f sediment samples can be quite large, as indicated by the ratios of these activities for the four runoff periods sampled to pavement sediment- sample activities (Fig. 6). We hypothesize that the difference in activ- ities provides critical insight into the dynamics of 210Pbex and 7Be particle tagging at the scale of an urban watershed and explains why the fluvial sample 210Pbex and 7Be activities are not bracketed by the source activities.f Rainfall-runoff entrains a relatively small mass of particles in sus- pension, which comes in contact with a relatively large volume of precipitation, leading to enrichment in particle-associated radio- nuclides carried by the rainfall/runoff. The mass of particles that re- mains on the pavement also is exposed to rainfall, but the ratio of rainfall volume to pavement sediment mass is smaller, resulting in less enrichment on a per particle-mass basis. By extension, soil, with its much greater mass of particles and downward diffusion of rainwater through infiltration, is even less enriched by a given rainfall event than are pavement particles (Fig. 5). One of the four rainfall events sampled (2 August, 2017) included runoff samples collected in Alexander Cul- vert shortly after the onset of rainfall (Figs. S3Aix-xi; S4); for that event, 7Be activity of sediment in runoff samples decreased through the event (12.6, 10.4, 8.81, 3.24 Bq/g) (Fig. S3-Ax). A similar pattern was ob- served for 210Pbex (2.65, 2.41, 1.66, 0.65 Bq/g) (Fig. S3Axi). Delivery of 7Be has been observed to decrease rapidly during a rain event, a process attributed to scavenging and washout of radionuclides from the atmo- sphere (Caillet et al., 2001; Ioannidou and Papastefanou, 2006; Gourdin et al., 2014). Thus, the earlier part of the runoff period would have higher radionuclide tagging of sediment than later in the event. De- creasing activities of 7Be and 210Pbex relative to the onset of rainfall could be related to the washout of those radionuclides during an event (Fig. S5). However, the composite rainfall samples collected by this study do not allow for an evaluation of rainfall activities over the course of the event. 3.2. Radionuclide activities in source and collected sediment samples The three types of source material—pavement sediment, upland soil, and streambanks—have characteristic activities of 7Be, 210Pbex, and 137Cs that reflect differences in initial (pre-rain) activities and the timing and intensity of radionuclide tagging and dilution (Clifton et al., 2019). Activities of 137Cs were higher in soils than in streambank ma- terial, and similar to, or higher than, those in pavement sediment (Fig. 4). Fallout of 137Cs for North America largely ceased by the late 1960s (Ritchie and McHenry, 1990), and soil now is a reservoir of le- gacy 137Cs from past fallout. Variable 137Cs activities in pavement se- diment likely reflect varying contributions of upland soil to the sedi- ment on the impervious surface. Significant differences in activities of 137Cs (as well as 210Pbex and 7Be) among some of the pavement sedi- ment sites (Mann-Whitney Rank Sum test (p < 0.05)) (Table S4) likely reflect different source contributions to sediment at a site. The detection of 137Cs activity in some streambanks indicates that these samples do not represent older alluvial deposits, which should be devoid of 137Cs, but may indicate deposition from eroded topsoil that occurred when Activities of 210Pbex and 7Be in pavement sediment are correlated (r2 = 0.51; Fig. S2A) because they both are delivered to the Earth’s Fig. 4. Summary of radionuclide activity in sediment (Bq/g) for all samples (A) 7Be, (B)210Pbex, and (C) 137Cs Table S2; Clifton et al., 2019). f radionuclide activity in sediment (Bq/g) for all samples (A) 7Be, (B)210Pbex, and (C) 137Cs Table S2; Clifton et al., 2019). 7 7 A.C. Gellis, et al. Journal of Hydrology 588 (2020) 124855 surface by rainfall and both associate with particles (McNeary and Baskaran, 2003; Matisoff et al., 2005). In contrast, 210Pbex in pavement sediment is not correlated with activities of 137Cs (Fig. S2B (r2 = 0.12)) and suggests a fundamental difference between pavement sediment and soils (Fig. 5). However, 210Pbex and 137Cs are strongly correlated in soils (Wallbrink and Murray, 1996; Huh and Su, 2004; Porto et al., 2010; Teramage et al., 2013). Porto et al. (2010) suggested that the close relation of 210Pbex and 137Cs activities in soils reflects a similar and consistent response to erosion and sediment redistribution processes. Pavement sediment, which is transported away and replenished over short time scales, does not undergo such processes. 3.3. Radionuclide activity by event Activities of 210Pbex and 7Be are highest in particles in runoff (Alexander and road runoff), followed by SS at Alexander Culvert and pavement sediment (Figs. S3A–C). The amount of enrichment of 7Be and 210Pbex in runoff and Alexander Culvert SS relative to the pavement Fig. 6. Activity ratios of (A) 7Be (B) and 210Pbex in sediment from runof samples (Alexander Avenue Culvert runoff and road runoff) to Alexande Culvert suspended sediment and pavement sediment. Solid gray bars represen rainfall activity. Another potential explanation for the higher activities in the Alexander Culvert runoff and SS samples versus other sources is grain size and organic content differences among the sample types. The ac- tivity of radionuclides has been shown to increase with decreasing se- diment size (Taylor et al., 2014; He and Walling, 1996) and increasing organic content (Navas et al., 2011; Motha et al., 2002). Examination of grain size (D50) and organic content (total organic carbon %; TOC) among sample types does not indicate that the Alexander Culvert SS has finer grain sizes or higher TOC content (Fig. S6), and thus neither grain size nor organic content explain the higher activities in the Alexander Culvert runoff and SS samples relative to sediment sources. Froger et al. (2018) also did not observe any relation between sediment grain size and 7Be and 210Pbex activities for an urban area in France. 3.2. Radionuclide activities in source and collected sediment samples Furthermore, fallout radionuclide activity during a storm can be influenced by other factors, such as cloud height, storm types and duration, and seasonality (Todd et al. 1989; Kaste et al., 2002; Karwan et al., 2016). Activities of 7Be, 210Pbex, and 137Cs in runoff, SS, and bed samples are expected to be bracketed by those in the sediment sources (pave- ment sediment, soils, and, for Dead Run, streambanks). Activities of 137Cs in fluvial sediments collected at Alexander Culvert and Dead Run are within the range of source sediment (pavement sediment, upland soil, and streambank material) (Fig. 4C). Activities of 7Be and 210Pbex in SS at Alexander Culvert, however, are similar to or greater than those in the assumed source materials (Fig. 4A,B). The explanation for this is presented in section 3.3. Table 3 Table 3 Summary of source contributions to fluvial sediment samples using an unmixing model (Eqs. (3) and (4)) (SI Table 5). Values are mean percentage and standard deviation in parenthesis. * The fallout radionuclide (FRN) activity of fluvial sediment draining to the Alexander Culvert is similar to FRN activity at Dead Run. Fluvial sediment type Pavement Soil Banks and other FRN free material Alexander suspended sediment 45(25) 22(19) 32(35)* Dead Run suspended sediment 15(9) 28(7) 57(15) Dead Run bed sediment 4(2) 39(11) 57(12) the median activities from runoff for 210Pbex (1.638 Bq/g) and 7Be (4.121 Bq/g), assuming that these runoff particles more reasonably represent activities of pavement sediment that is transported to the stream. Because pavement sediment is not currently subject to fallout of 137Cs, we believed it adequately reflected 137Cs activities in runoff. Median radionuclide activities were used for the soil endmembers (137Cs = 0.0071 Bq/g; 210Pbex = 0.1070 Bq/g; 7Be = 0.0611 Bq/g) and values were set to one-half the detection levels for the streambank (and other fallout-radionuclide-free material) endmember (210Pbex = 0.0033 Bq/g; 7Be = 0.0050 Bq/g; 137Cs = 0.0002 Bq/g). Low fallout radionuclide activities in some target samples indicate that a low-activity, or fallout-radionuclide-free, endmember is supplying sediment in spite of the detection of 137Cs, and to lesser extent 210Pbex, in bank samples collected for this study. results in a mean pavement contribution at Alexander Culvert of 45 ± 25% in SS samples, with a contribution from older channel ma- terial of 32 ± 35%, and the remainder coming from soils (22 ± 19%) (Table 3). These relatively large contributions of pavement sediment are consistent with the ranges of 210Pbex and 7Be in pavement sediment, runoff, and Alexander Culvert samples (Fig. 7) and with the nature of the contributing watershed. The Dead Run gage site sampled the open channel portion, thus all three potential sources are expected. Based on the unmixing model, the mean fraction of pavement sediment is 15 ± 9% in Dead Run SS and 4 ± 2% in bed sediment. The decrease in the pavement-sediment contribution from Alexander Culvert to Dead Run indicates relatively rapid dilution of the pavement-sediment fraction downstream from where buried channels emerge into open channels. The dilution is from streambanks, which contributed 57 ± 15% of the Dead Run SS and 57 ± 12% of the Dead Run bed sediment. Table 3 Large variation in activities of the endmember and target samples resulted in large variability (>20%) in source proportions for the Alexander SS samples (Table 3). Target samples should be bracketed by the source sample activities (within 10% of the minimum and > 10% of maximum activity) (Gellis and Gorman Sanisaca, 2018). In 4 of 57 target samples, the sample activities of 210Pbex (n=1) and 7Be (n = 4) were outside the range of endmember activities in a direction that implies a mix of soil and pavement sediment, the two higher activity sources (Fig. 7). All four target samples were Alexander Culvert SS, which had relatively high activities of 7Be and 210Pbex. One possible explanation for the activities in these samples is that the soil in sus- pension in runoff is being tagged by rainfall radionuclides in the runoff, similar to the tagging of the pavement sediment. Counterintuitively, the model results indicate an average of 32 ± 35% streambank contribution to Alexander Culvert SS, with very high variability between sampled events. Because the culvert emerges from buried channels with no exposed streambanks, there should be only two major sources possible: soil and pavement sediment. We in- terpret the modeled component of streambank material at the culvert as older channel material and subsoil eroded from within the culvert system. In many urban areas, stream channels have been buried in pipes, culverts, and combined sewer systems (Broadhead and Lerner, 2013; Broadhead et al., 2015; Elmore and Kaushal, 2008). The breakup, cracking, and wear of these underground pipes and culverts is common and leads to openings that allow the surrounding soil to enter the pipe (infiltration) (Tang et al., 2018; Sato and Kuwano, 2015; Ellis, 2001). The continued erosion of the soil forms a cavity and in some cases leads to sinkholes (Ali and Choi, 2019; Sato and Kuwano, 2015; Tang et al., 2018). We hypothesize that the bank source contributions observed in our model output at Alexander Culvert reflect buried channel sediment entering the culvert through openings. Further studies that examine sediment transport and the condition of underground pipes in areas of buried channels may help to resolve this question, and radionuclide analyses, such as done here, could indicate culvert systems subject to underground failures. 3.4. Sediment sources A two-endmember model using 137Cs (Eq. (2)) determined the sources of pavement sediment and indicates a soil contribution to the pavement sediment samples (n = 85) of 26 ± 23%. The high varia- bility within the source contributions reflect the heterogeneity of pa- vement sediment and is consistent with other studies that have reported a wide range of soil and/or inorganic sediment on impervious surfaces (Loganathan et al., 2013; Sartor et al., 1974; Fergusson and Ryan, 1984). Sediment sources to suspended sediment collected at Alexander Culvert and suspended and bed sediment collected at Dead Run were apportioned using an unmixing model (Eqs. (3) and (4)) with three tracers (7Be, 210Pbex, 137Cs) and the results averaged (Table 3; Table S5). Several metrics (mean, median, 90th percentile) were tested for each endmember. The pavement sediment endmember was assigned the median pavement sediment activities for 137Cs (0.0015 Bq/g), andf Fig. 6. Activity ratios of (A) 7Be (B) and 210Pbex in sediment from runoff samples (Alexander Avenue Culvert runoff and road runoff) to Alexander Culvert suspended sediment and pavement sediment. Solid gray bars represent rainfall activity. 8 Journal of Hydrology 588 (2020) 124855 A.C. Gellis, et al. 3.5. Potential radionuclide tagging of pavement sediment Based on Eq. (1), the potential activity of 7Be and 210Pbex added to pavement sediment by tagging from rain events is more than sufficient to account for the sediment activity measured in the runoff and the Alexander Culvert SS samples (Fig. 8). Activities in runoff samples and rain samples are also compared using whole-water units of measure- ment (Bq/L; activity/volume) (Table S7), which avoids the large un- certainty in the pavement-sediment yields. Activities of 210Pbex and 7Be in rain are substantially greater than those in whole-water samples of runoff for the same event (Table S7). Although the rainfall samples reflect rainfall collected over a longer time than represented by the runoff and the runoff samples represent a single point in time, the data are consistent with the hypothesis that a substantial part of the rainfall load of 7Be and 210Pbex does not exit the system but is retained within watershed soils, pavement surface, or other solid surfaces. To determine the potential sediment load and yield available on impervious services, the median total and fine yields from the vacuum samples were extrapolated to the total impervious area of roads and parking lots in the Dead Run gage (0.42 km2) (Table 1). The results indicate that about 6.4 and 0.65 Mg of total and fine sediment, re- spectively, are present on impervious surfaces in the Dead Run wa- tershed at any given time. The average annual SS load at the Dead Run gage (2013–2016) is 127 Mg (Kemper et al., 2019). Three events sampled for percent fines in Dead Run at Catonsville and surrounding gages (Table S8) indicated that on average 91% of the suspended se- diment is < 63 µm.i If the same ratio of fines and sand in suspended sediment is applied to the impervious surface sediment, then 0.71 Mg of sediment is available for transport, which is a very small percentage (0.6%) of the total annual suspended-sediment load measured at the Dead Run gage. However, this relatively small mass represents the potential sediment available at any one time on roads and parking lots and it is likely that only a part of this impervious sediment is mobilized in a given flow event. suspended sediment in some samples at the Dead Run gage. suspended sediment in some samples at the Dead Run gage. suspended sediment in some samples at the Dead Run gage. sediment source areas. Contrary to this expected decline in sediment yield as urban areas expand, Gellis et al. (2017a), Cashman et al. (2018) found that sediment yields for Difficult Run, an urban/suburban wa- tershed in Virginia outside of Washington, D.C., remained high even after 30 or more years had passed since peak rates of urbanization. Both studies attributed the high sediment yields to streambank erosion. 3.5. Potential radionuclide tagging of pavement sediment To estimate the total mass of impervious sediment mobilized in a given year from all events, we first defined an event as when peak flow exceeds the 90th percentile of all 5-minute discharges for the 2017 and 2018 study period (0.032 m3/s). Using this definition, 68 flow events occurred. Sediment delivery in urban watersheds can be high (Russell et al., 2019), and in watersheds with impervious areas similar to Dead Run (50%) the sediment delivery ratio is ~50% (Heathcoate, 2009). If we assume that 50% of the impervious sediment is mobilized and de- livered to the stream, the estimated total contribution of the 68 events is 24 Mg, or 19% of the annual sediment load. Our results suggest that the sediment available for transport from roads and parking lots has the potential to be an important contributor to the total sediment load in an urban stream as well as an important contributor of contaminants. We recognize that values assigned to define an event and to estimate the percentage of sediment mobilized on impervious surfaces were arbi- trary. The actual mass available for transport, mobilized from im- pervious areas, and delivered to the stream would require systematic Table 3 The model results indicate that pavement sediment is the dominant sediment source in the buried channel part of the watershed at Alexander Culvert, and that downstream at Dead Run, where the channel is open, streambank sediment dominates. The unmixing model Fig. 7. Mean 137Cs, 210Pbex, and 7Be source endmembers (banks, soil, pave- ment) plotted with fluvial (target) samples at Alexander Avenue Culvert and Dead Run gage. [SS = suspended sediment] Sediment budgets from the literature that quantify sediment sources in urban watersheds indicate that the contribution from impervious areas ranges from 4 to 46% (Table S6), covering the range in pavement sediment contributions to fluvial sediments from the Alexander Culvert (45%) and Dead Run gage (4 to 15%) (Table 3). Contributions from soil (22 to 39%) and streambank (and other fallout-radionuclide-free ma- terial, i.e., older channel deposits) (32 to 57%) sources at Dead Run are also within the range reported for other urban areas (Table S6). Because pavement sediment is a vector for metals, PAHs, and other urban contaminants (Van Metre and Mahler, 2003; Mahler et al., 2005; Callender and Rice, 2000), its proportion in fluvial sediment is an in- dicator of the contribution of pavement sediment to sediment con- tamination. The high percentage of pavement sediment at Alexander Culvert indicates the potential for contaminant “hot spots” where cul- verts and buried channels discharge to urban streams. Although the fraction of pavement sediment decreases rapidly with distance down- stream, it nonetheless accounts for about one-third or more of Fig. 7. Mean 137Cs, 210Pbex, and 7Be source endmembers (banks, soil, pave- ment) plotted with fluvial (target) samples at Alexander Avenue Culvert and Dead Run gage. [SS = suspended sediment] 9 Fig. 8. Potential tagging of pavement sediment for (A) 7Be and (B) 210Pbex compared for Alexander Avenue Culvert SS samples and runoff samples. A.C. Gellis, et al. Journal of Hydrology 588 (2020) 124855 A.C. Gellis, et al. Journal of Hydrology 588 (2020) 124855 3.7. Conceptual model The results of this study indicate that the proportion of pavement sediment in suspended sediment in the buried-channel part of the urban watershed is substantially higher than in the open-channel reaches. As a result, concentrations of urban pavement-related contaminants asso- ciated with suspended sediment also are likely to be higher in buried channels. The part of an urban watershed that is directly connected to the stream by stormwater drains and buried channels has been termed “effective imperviousness” (Booth and Jackson, 1997), and has been reported to be a better predictor of ecologically relevant geomorphic indicators (Vietz et al., 2014) and bedload yield (Russell et al., 2018) than the percentage of total impervious cover over the watershed. Our results indicate that the better prediction capability may be because “effective imperviousness” better represents areas of potential con- taminant transport to the stream than does total impervious area. De- lineating the part of an urban watershed that contains buried channels may provide insight into how sediment and contaminants are trans- ported in urban environments. We propose a conceptual model based on the activities of 7Be and 210Pbex in rain, runoff samples, pavement sediment, topsoil, and fluvial samples, that describes how activities of 7Be, 210Pbex, 137Cs in sediment change as sediment is transported in an urban setting (Fig. 9). Ante- cedent conditions control the initial 7Be and 210Pbex activity of pave- ment sediment before a rainfall-runoff event occurs, as reflected by the activities of the pavement-sediment samples collected prior to rain events. This initial activity is a function of 7Be and 210Pbex activity tagging from previous events, dilution from deposition of other material with lower activities of those radionuclides during and between events, and decay of 7Be since the last event (decay is negligible for 210Pbex over the short period between rainfall events). We assume that the highest activities of 7Be and 210Pbex in rainfall occur early in the rain event (initial runoff) and decrease exponentially (Ioannidou and Papastefanou, 2006) (Fig. S5). Pavement-sediment particles transported by initial rainfall-runoff receive the highest amounts of 7Be and 210Pbex because sediment mass is small relative to rainwater volume. These particles are transported to the stream where they contribute to the radionuclide activities of SS and, to a lesser de- gree, bed sediment (Fig. 9). This initial runoff of tagged sediment is reflected in elevated 7Be and 210Pbex activities (Figs. 3.7. Conceptual model S3A–C) and SSC in the runoff samples (Table S7).f Fig. 9. Conceptual diagram illustrating how fallout radionuclide activities change along the transport cycle of sediment through the urban environment. The different colors of the sediment indicate changes in radionuclide labelling over time. g how fallout radionuclide activities change along the transport cycle of sediment through the urban environment. The different es in radionuclide labelling over time. in runoff to rainfall volume (i.e., runoff water), and (3) dilution of the remaining pavement sediment with soil during the event as soil is eroded and transported to adjacent impervious surfaces. Fallout radionuclide analysis confirms that pavement sediment contains a component of soil. The suspended sediment collected at the Alexander Culvert integrates activities over the storm hydrograph, which includes the higher activities of 7Be and 210Pbex in the initial runoff and lower activities of 7Be and 210Pbex from later runoff and from topsoil and bank material (Fig. 9). event-based monitoring over time, which was beyond the scope of this study. However, this estimate of pavement sediment determined using the sediment yield provided by the vacuum cleaner results (19%) is similar to the pavement sediment contribution results from the un- mixing model (15%) for Dead Run SS (Table 3), indicating our as- sumption of 50% mobility in a given event is reasonable. 3.6. Urban pavement sediment yields The total potential sediment yield determined from vacuuming (sand and fines) averaged 55.6 ± 97.2 g/m2 and the fine-sediment potential yield averaged 5.9 ± 11.7 g/m2 (n = 39). The high varia- bility and skewness (figs. S7A,B) are consistent with those reported for other studies (Sartor et al., 1974). The median total sediment yield 15.4 g/m2 and fine sediment yield of 1.57 g/m2 were used in further analysis. Parking lot samples had significantly lower total and fine se- diment yields than road samples (Mann-Whitney Rank Sum test, p = 0.003) (Figs. S7A, B). Pavement sediment yields determined for this study are comparable to those reported by Waschbush (2003) and by Selbig and Bannerman (2007) but are greater than those reported by Mahler et al. (2005) (Figs. S7-C). Most of the pavement sediment mass is sand sized (mean percentage of material < 63 μm of 16.1 ± 16.9%), a grain size commonly found in urban environments (Sartor et al., 1974; Waschbush, 2003; Selbig and Bannerman, 2007; Taylor and Owens, 2009). Could pavement sediment constitute a substantial part of stream sediment in an urban environment? Wolman (1967) presented a con- ceptual model where sediment yields in an urban environment were projected to decrease after development, related to the loss of upland 10 A.C. Gellis, et al. Journal of Hydrology 588 (2020) 124855 Fig. 9. Conceptual diagram illustrating how fallout radionuclide activities change along the transport cycle of sediment through the urban environment. The d colors of the sediment indicate changes in radionuclide labelling over time. Acknowledgements Devereux, O.H., Prestegaard, K.L., Needelman, B.A., Gellis, A.C., 2010. Suspended-sedi- ment sources in an urban watershed, Northeast Branch Anacostia River, Maryland. Hydrol. Process. 24, 1391–1403. https://doi.org/10.1002/hyp.7604. The authors acknowledge assistance in this study from USGS per- sonnel Zachary Grzywacz for field and laboratory assistance, Shannon Jackson for GIS support, and Jeff Klein for construction of the passive samplers. We would like thank Dr. Joel Moore, Towson University, for suggestions and review, and three anonymous reviewers for helpful comments, all which greatly improved this paper. Data generated during this study are available as a USGS data release (Clifton et al., 2019). Dominik, J., Burrus, D., Vernet, J.P., 1987. Transport of the environmental radionuclides in an alpine watershed. Earth Planetary Sci. Lett. 84, 165–180. https://doi.org/10. 1016/0012-821X(87)90083-5.f Duncan, J.M., Welty, C., Kemper, J.T., Groffman, P.M., Band, L.E., 2017. Dynamics of nitrate concentration-discharge patterns in an urban watershed. Water Resour. Res. 53, 7349–7365. https://doi.org/10.1002/2017WR020500.i 53, 7349–7365. https://doi.org/10.1002/2017WR020500. Eckley, C.S., Branfireun, B., 2008. Gaseous mercury emissions from urban surfaces: controls and spatiotemporal trends. Appl. Geochem. 23, 369–383.ii Ellis, J.B., 2001. Sewer Infiltration/Exfiltration and Interactions with Sewer Flows and Groundwater Quality,” Interurba II, Lisbon, 19-22 February 2001, p. 311-319. This research was funded by the U.S. Geological Survey National Water Quality Program. Funding for operation and maintenance of USGS stream gage 01589312 and the rain gage located at that site, as well as time of C. Welty, A.J. Miller, and J.T. Kemper was provided in part by NSFGrants # EAR-1427150 and DEB-1637661; NSF cooperative agreement #CBET-1444758; and Chesapeake Bay Trust Grant #12507.i Elmore, A.J., Kaushal, S.S., 2008. Disappearing headwaters: patterns of stream burial due to urbanization. Front. Ecol. Environ. 6, 308–312. Evrard, O., Némery, J., Gratiot, N., Duvert, C., Ayrault, S., Lefèvre, I., Poulenard, J., Prat, C., Bonté, P., Esteves, M., 2010. Sediment dynamics during the rainy season in tro- pical highland catchments of central Mexico using fallout radionuclides. Geomorphology 124, 42–54. Fergusson, J.E., Ryan, D.E., 1984. The elemental composition of road dust from large and small urban areas related to city type, source and particle size. Sci. Total Environ. 34, 101–116. Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government. 4. Summary and conclusion Our results indicate that material washed from impervious surfaces (pavement sediment) is an important contributor to urban stream se- diment, especially in areas with large amounts of impervious surface and buried channels. Pavement sediment can be a vector for many common urban contaminants and can be an especially important con- tributor to receiving streams where networks of buried channels exit into open channels. Activities of 7Be and 210Pbex in fluvial sediment differ between buried and open channels. Fallout of 7Be and 210Pbex in f Throughout the runoff period (Fig. 9), pavement sediment continues to be tagged by rain, a process that is cumulative, but activities in pa- vement sediment are lower than those in runoff sediment for three possible reasons: (1) lower rainfall activities of 7Be and 210Pbex later in the event, (2) the relatively greater ratio of residual pavement sediment mass to rainfall volume compared to that of suspended sediment mass 11 Journal of Hydrology 588 (2020) 124855 A.C. Gellis, et al. rainfall and radionuclide tagging of pavement sediment increases the activities on the sediment, which also contains relic activities from previous events. We hypothesize that the low ratio of sediment mass to rainfall volume in entrained sediment results in higher activities in the runoff sediment than in the pavement sediment, which in turn is—for the same reason—more highly tagged than soils. As runoff particles move through buried channels, activities of 7Be and 210Pbex remain high. With the exception of material entering buried channels through cracks and breaks, there is limited opportunity for dilution by soils and streambank erosion. Once the suspended sediment moves into the open- channel parts of the watershed, 7Be and 210Pbex activities are diluted by contributions of lower-activity sediment from stream beds and streambanks. These findings support the hypothesis that sediment from urban impervious surfaces is substantially enriched in 210Pbex and 7Be from rainfall but not in 137Cs, resulting in a unique radionuclide sig- nature compared to other sediment-source settings. This difference in source activity provides a tool for identifying the sediment contribution to streams from heavily developed urban areas and suggests the need to define source terms by land use in regional stream sediment source and age studies using fallout radionuclide tracers. prognoses. Northwest Environ. J. 7, 93–118. Booth, D.B., Jackson, C.R., 1997. Urbanization of aquatic systems: degradation thresh- olds, stormwater detection, and the limits of mitigation. J. Am. Water Resour. Assoc. 33, 1077–1090. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influ- ence the work reported in this paper. Conaway, C.H., Storlazzi, C.D., Draut, A.E., Swarzenski, P.W., 2013. Short-term varia- bility of 7Be atmospheric deposition and watershed response in a Pacific coastal Conaway, C.H., Storlazzi, C.D., Draut, A.E., Swarzenski, P.W., 2013. Short-term varia- bility of 7Be atmospheric deposition and watershed response in a Pacific coastal stream, Monterey Bay, California, USA. J. Environ. Radioact. 120, 94e103. C l W P 1976 Th l f h lli k B l i d i b i Crowley, W.P., 1976. The geology of the crystalline rocks near Baltimore and its bearing on the evolution of the eastern Maryland Piedmont: Maryland Geological Survey. Report of Investigations 27, 40 p. Acknowledgements Froger, C., Ayrault, S., Evrard, O., Monvoisin, G., Bordier, L., Lefèvre, I., Quantin, C., 2018.Tracing the sources of suspended sediment and particle-bound trace metal elements in an urban catchment coupling elemental and isotopic geochemistry, and FRNs: Environmental Science and Pollution Research, published online, https://doi. org/10.1007/s11356-018-2892-3. 4. Summary and conclusion Broadhead, A.T., Lerner, D.N., 2013. www.daylighting.org.uk: case study website sup- porting research into daylighting urban rivers. Hydrol. Processes 27 (12), 1840–1842. Broadhead, A.T., Horn, R., Lerner, D., 2015. Finding lost streams and springs captured in combined sewers: a multiple lines of evidence approach. Water Environ. J. 29, 288–297. Caillet, S., Arparagaus, P., Monna, F., Dominik, J., 2001. Factors controlling 7Be and 210Pb atmospheric deposition as revealed by sampling individual rain events in the region of Geneva. J. Environ. Radioact. 53, 241–256.l g , Callender, E., Rice, K.C., 2000. The urban environmental gradient: anthropogenic influ- ences on the spatial and temporal distributions of lead and zinc in sediments. Environ. Sci. Technol. 34 (2), 232–238. Cannon, W.F., Horton, J.D., 2009. Soil geochemical signature of urbanization and in- dustrialization – Chicago, Illinois, USA. Appl. Geochem. 24, 1590–1601. Cashman, M.J., Gellis, A.C.. Gorman Sanisaca, L., Noe, G., Cogliandro, V., Baker., A., 2018. Bank-derived sediment dominates in a suburban Chesapeake Bay watershed, Upper Difficult Run, Virginia, USA. River Research and Applications, published on line, DOI: 10.1002/rra.3325. https://onlinelibrary.wiley.com/doi/epdf/10.1002/ rra.3325=. Clifton, Z.J., Gellis, A.C., Malen, J.M., Grzywacz, Z.T., Fuller C.C., and Van Metre P.C., 2019. Urban sediment and fallout radionuclide input characteristics of Dead Run watershed in Catonsville, Maryland for 2017-2018 (Version 1.1, January, 2020) : U.S. Geological Survey data release, https://doi.org/10.5066/P9K86P4Q. Collins, A.L., Zhang, Y., McChesney, D., Walling, D.E., Haley, S.M., Smith, P., 2011. Sediment source tracing in a lowland agricultural catchment in southern England using a modified procedure combining statistical analysis and numerical modelling. Sci. Tot. Environ. 414, 301–317. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jhydrol.2020.124855. Fuller, C.C., van Green, A., Baskaran, M., Anima, R., 1999. Sediment chronology in San Francisco Bay, California defined by 210Pb, 234Th, 137Cs, and 239,240Pu. Mar. Chem 64, 7–27.i l Benmansour, M., Mabit, L., Owens, P.N., Tarjan, S., Walling, D.E., 2014. The use of excess 210Pb (210Pbex) as a soil and sediment tracer measurements. In: Guidelines for Using Fallout Radionuclides to Assess Erosion and Effectiveness of Soil Conservation Strategies, IAEA-TECDOC-1741. IAEA, Vienna p, pp. 3–26. Boonyatumanond, R., Murakami, M., Wattayakorn, G., Togo, A., Takada, H., 2007. Sources of polycyclic aromatic hydrocarbons (PAHs) in street dust in a tropical Asian mega-city, Bangkok, Thailand. Sci. Total Environ. 384, 420–432. g y g Booth, D.B., 1990. Stream-channel incision following drainage-basin urbanization. Water Resour. Res. 26, 407–417. https://doi.org/10.1111/j.1752-1688.1990.tb01380.x. Booth D B 1991 Urbanization and the natural drainage system impacts solutions and Ali, H., Choi, J.-H., 2019. A review of underground pipeline leakage and sinkhole mon- itoring methods based on wireless sensor networking. Sustainability 11 (15), 4007. Bain, D.J., Yesilonis, I.D., Pouyat, R.V., 2012. Metal concentrations in urban riparian sediments along an urbanization gradient. Biogeochemistry 107, 67–79. Barnes, M.L., Welty, C., Miller, A.J., 2018. Impacts of development pattern on urban groundwater flow regime. Water Resour. Res. 54 (8), 5198–5212. Benmansour, M., Mabit, L., Owens, P.N., Tarjan, S., Walling, D.E., 2014. The use of excess 210Pb (210Pbex) as a soil and sediment tracer measurements. In: Guidelines for Using Fallout Radionuclides to Assess Erosion and Effectiveness of Soil Conservation Strategies, IAEA-TECDOC-1741. IAEA, Vienna p, pp. 3–26. Boonyatumanond, R., Murakami, M., Wattayakorn, G., Togo, A., Takada, H., 2007. Sources of polycyclic aromatic hydrocarbons (PAHs) in street dust in a tropical Asian mega-city, Bangkok, Thailand. Sci. Total Environ. 384, 420–432. Booth, D.B., 1990. Stream-channel incision following drainage-basin urbanization. Water Resour. Res. 26, 407–417. https://doi.org/10.1111/j.1752-1688.1990.tb01380.x. Booth, D.B., 1991. Urbanization and the natural drainage system-impacts, solutions, and Ali, H., Choi, J.-H., 2019. A review of underground pipeline leakage and sinkhole mon- itoring methods based on wireless sensor networking. Sustainability 11 (15), 4007. Bain, D.J., Yesilonis, I.D., Pouyat, R.V., 2012. Metal concentrations in urban riparian sediments along an urbanization gradient. Biogeochemistry 107, 67–79. Barnes, M.L., Welty, C., Miller, A.J., 2018. Impacts of development pattern on urban groundwater flow regime. Water Resour. Res. 54 (8), 5198–5212. Benmansour M Mabit L Owens P N Tarjan S Walling D E 2014 The use of excess Resour. Res. 26, 407–417. https://doi.org/10.1111/j.1752-1688.1990.tb01380.x. Booth, D.B., 1991. Urbanization and the natural drainage system-impacts, solutions, and References Suspended sediment source and propagation during monsoon events across nested sub-catchments with con- trasted land uses in Laos. J. Hydrol. Reg. Stud. 9, 69–84. 137Cs and 210Pbex and other sediment source fingerprints to document suspended sediment sources in small forested catchments in south-central Chile. J Environ Rad 124, 147–159. i sediment sources in small forested catchments in south-central Chile. J Environ Rad 124, 147–159. Selbig, W.R., Bannerman, R.T., 2007. Evaluation of street sweeping as a stormwater- quality-management tool in three residential basins in Madison, Wisconsin: U.S. Geological Survey Scientific Investigations Report 2007–5156, 103 p. Ioannidou, A., Papastefanou, C., 2006. Precipitation scavenging of Be-7 and (CS)-C-137 radionuclides in air. J. Environ. Radioact. 85 (1), 121–136.ff Jorgenson, B.C., Young, T.M., 2010. Formulation effects and the off-target transport of pyrethroid insecticides from urban hard surfaces. Environ. Sci. Tech. 44, 4951–4957. i Slimane, A.B., Raclot, D., Evrard, O., Sanaa, M., Lefevre, I., Bissonnais, Y., 2016. Relative contribution of rill/interrill and gully/channel erosion to small reservoir siltation in Mediterranean environments. Land Degrad. Dev. 27 (3), 785–797. py Kaste, J.M., Norton, S.A., Hess, C.T., 2002. Environmental Chemistry of Beryllium-7. Mineralogical Society of America, Special Volume 50, 271-289. Southworth, G., Mathews, T., Greeley, M., Peterson, M., Brooks, S., Ketelle, D., 2013. Sources of mercury in a contaminated stream—implications for the timescale of re- covery. Environ. Toxicol. Chem. 32, 764–772.i Karwan, D.L., Siegert, C.M., Levia, D.F., Pizzuto, J., Marquard, J., Aalto, R., Aufdenkampe, A.K., 2016. Beryllium-7 wet deposition variation with storm height, synoptic classification, and tree canopy state in the mid-Atlantic USA. Hydrol. Process. 30, 75–89. Tang, Y., Zhu, D.Z., and Chan, D.H., 2018, Modeling soil loss by water infiltration through sewer pipe defects. Kamojjala, S., ed., Hydraulics and Waterways, Water Distribution Systems Analysis, and Smart Water, World Environmental and Water Resources Congress 2018, 254-262. Kemper, J.T., Miller, A.J., Welty, C.W., 2019. Spatial and temporal patterns of suspended sediment transport in nested urban watersheds. Geomorphology 336, 95–106. gy Leopold, L.B., 1968. Hydrology for Urban Land Planning - A Guidebook on the Hydrologic Effects of Urban Land Use. U.S Geol. Survey Circ. 55, 18. Taylor, K.G., Owens, P.N., 2009. Sediments in urban river basins: a review of sediment- contaminant dynamics in an environmental system conditioned by human activities. J Soils Sediments 9, 281–303. f Loganathan, P., Vigneswaran, S., Kandasamy, J., 2013. Road-deposited sediment pollu- tants: a critical review of their characteristics, source apportionment, and manage- ment Crit. Rev. Environ. References Urban stream deserts: mapping a legacy of ur- banization in the United States. Appl. Geogr. 67, 128–129. Van Metre, P.C., Wilson, J.T., Fuller, C.C., Callender, E., Mahler, B.J., 2004. Collection, analysis, and age-dating of sediment cores from 56 U.S. lakes and reservoirs sampled by the U.S. Geological Survey, 1992–2001: U.S. Geological Survey Scientific Investigations Report 2004–5184, 180 p. Navas, A., Gaspar, L., López-Vicente, M., Machín, J., 2011. Spatial distribution of natural and artificial radionuclides at the catchment scale (South Central Pyrenees). Radiat. Meas. 46 (2), 261–269. Vietz, G.J., Sammonds, M.J., Walsh, C.J., Fletcher, T.D., Rutherfurd, I.D., Stewardson, M.J., 2014. Ecologically relevant geomorphic attributes of streams are impaired by even low levels of watershed effective imperviousness. Geomorphology 206, 67–78. Olley, J., Brooks, A., Spencer, J., Pietsch, T., Borombovits, D., 2013a. Subsoil erosion dominates the supply of fine sediment to rivers draining into Princess Charlotte Bay, Australia. J. Environ. Radioact. 124, 121–129. f Wallbrink, P.J., Murray, A.S., 1996. Determining soil loss using the inventory ratio of excess lead-210 to cesium-137. Soil Sci. Soc. Am. J. 60, 1201–1208.i Olley, J., Burton, J., Smolders, K., Pantus, F., Pietsch, T., 2013b. The application of fallout radionuclides to determine the dominant erosion process in water supply catchments of subtropical south-east Queensland, Australia. Hydrol. Process. 27, 885–895. Wallbrink, P., Olley, J.M., Hancock, G., 2002. Estimating residence times of fine sediment in river channels using fallout 210Pb. In: The Structure, Function and Management Implications of Fluvial Sedimentary Systems, Fiona J. Dyer, Martin C. Thoms, Jon M. Olley, eds., Proceedings of an international symposium held at Alice Springs, Australia. September 2002, IAHS Publ. no. 276, p. 425-432. p y Paul, M.J., Meyer, J.L., 2001. Streams in the Urban Landscape. Annu. Rev. Ecol. Syst. 32, 333–365.l Phillips, J.M., Russell, M.A., Walling, D.E., 2000. Time-integrated sampling of fluvial suspended sediment: a simple methodology for small catchments. Hydrol. Process. 14, 2589–2602. Walling, D.E., 2005. Tracing suspended sediment sources in catchments and river sys- tems. Sci. Total Environ. 344 (1–3), 159–184.f Porto, P., Walling, D.E., Callegari., G., La Spada, C., 2010. Exploring the relationship between sediment and FRN output for two small Calabrian catchments. In: Banasik (Ed.), Sediment dynamics for a changing future, vol. 273. IAHS PRESS, Wallingford (GBR), pp. 163–171. Walsh, C.J., Roy, A.H., Feminella, J.W., Cottingham, P.D., Groffman, P.M., Morgan, R.P., 2005. The urban stream syndrome: current knowledge and the search for a cure. J. North Am. Benthol. Soc. References Gellis, A.C., Fuller, C.C., Van Metre, P.C., 2017b. Sources and ages of fine-grained sedi- ment to streams using FRNs in the Midwestern United States. J. Environ. Manage. 194 (1), 73–85.i Gellis, A.C., Gorman Sanisaca, L., 2018. Sediment fingerprinting to delineate sources of sediment in the agricultural and forested Smith Creek watershed, Virginia, USA. J. Am. Water Resour. Assoc. 54 (6), 1197–1221. Gellis, A.C., Myers, M.K., Noe, G.B., Hupp, C.R., Schenk, E.R., Myers, L., 2017a. Storms, channel changes, and a sediment budget for an urban-suburban stream, Difficult Run, Virginia, USA. Geomorphology 278, 128–148. Gourdin, E., Evrard, O., Huon, S., Reyss, J.L., Ribolzi, O., Bariac, T., Sengtaheuanghoung, O., Ayrault, S., 2014. Spatial and temporal variability of 7Be and 210Pb wet deposition during four successive monsoon storms in a catchment of northern Laos. J. Environ. Radioact. 136, 195–205. Hancock, G.J., Wilkinson, S.N., Hawdon, A.A., Keen, R.J., 2014. Use of fallout tracers 7Be, 210Pb and 137Cs to distinguish the form of sub-surface soil erosion delivering sediment to rivers in large catchments. Hydrol. Process. 28, 3855–3874. Hatcher, C.L., Filippelli, G.M., 2010. Mercury cycling in an urbanized watershed: the influence of wind distribution and regional subwatershed geometry in Central Indiana, USA. Water Air Soil Pollut 219, 251–261. 12 A.C. Gellis, et al. Journal of Hydrology 588 (2020) 124855 He, Q., Walling, D.E., 1996. Interpreting particle size effects in the adsorption of 137Cs and unsupported 210Pb by mineral soils and sediments. J. Environ. Radioact. 30, 117–137. Sansalone, J.J., Cristina, C.M., 2004. First flush concepts for suspended and dissolved solids in small impervious watersheds. J. Environ. Eng. 130 (11), 1301–1314. p g Sartor, J., Boyd, G.B., Agardy, F.J., 1974. Water pollution aspects of street surface con- taminants. J. Water Pollut. Contr. Federation 46 (3), 458–465.l Heathcoate, I.W., 2009. Integrated Watershed Management: Principles and Practice. 2009. 2nd Edition. John Wiley & Sons, Inc., New Jersey. Sato, M., Kuwano, R., 2015. Influence of location of subsurface structures on development of underground cavities induced by internal erosion. Soils Found. 55, 829–840. Huh, C.A., Su, C.C., 2004. Distribution of FRNs (7Be, 137Cs, 210Pb and 239,240Pu) in soils of Taiwan. J. Environ. Radioact. 77, 87–100. Schuller, P., Walling, D.E., Iroumé, A., Quilodrán, C., Castillo, A., Navas, A., 2013. Using 137Cs and 210Pbex and other sediment source fingerprints to document suspended Huon, S., Evrard, O., Gourdin, E., Lefévre, I., Bariac, T., Reyss, J.L., des Tureaux, T.H., Sengtaheuanghoung, O., Ayrault, S., Ribolzi, O., 2017. References Sci. Technol. 43 (13), 1315–1348. Taylor, A., Blake, W.H., Keith-Roach, M.J., 2014. Estimating Be-7 association with soil particle size fractions for erosion and deposition modelling. J. Soils Sediments 14, 1886–1893. Mabit, L., Benmansour, M., Abril, J.M., Walling, D.E., Meusburger, K., Iurian, A.R., Bernard, C., Tarján, S., Owens, P.N., Blake, W.H., Alewell, C., 2014. Fallout 210Pb as a soil and sediment tracer in catchment sediment budget investigations - A review. Earth Sci Rev 138, 335–351. Taylor, A., Blake, W.H., Couldrick, L., Keith-Roach, M.J., 2012. Sorption behaviour of beryllium-7 and implications for its use as a sediment tracer. Geoderma 187–188, 16–23. Mahler, B.J., Van Metre, P.C., Bashara, T.J., Wilson, J.T., Johns, D.A., 2005. Parking lot sealcoat: An unrecognized source of urban PAHs. Environ. Sci. Technol. 39 (15), 5560–5566.f Teramage, M.T., Onda, Y., Kato, H., Wakiyama, Y., Mizugaki, S., Hiramatsu, S., 2013. The relationship of soil organic carbon to 210Pbex and 137Cs during surface soil erosion in a hillslope forested environment. Geoderma 192, 59–67. Matisoff, G., Wilson, C.G., Whiting, P.J., 2005. The 7Be/210Pbxs ratio as an indicator of suspended sediment age or fraction new sediment in suspension. Earth Surf. Process. Landforms 30, 1191–1201. Thapalia, A., Borrok, D.M., Van Metre, P.C., Musgrove, M., Landa, E.R., 2010. Zn and Cu isotopes as tracers of anthropogenic contamination in a sediment core from an urban lake. Environ. Sci. Technol. 44, 1544–1550. McNeary, D., Baskaran, M., 2003. Depositional characteristics of 7Be and 210Pb in southeastern Michigan. J. Geophys. Res. 108 (D7), 4210. https://doi.org/10.1029/ 2002JD003021. Todd, J.F., Wong, G.T.F., Olsen, C.R., Larsen, I.L., 1989. Atmospheric depositional characteristics of beryllium 7 and lead 210 along the southeastern Virginia Coast. J. Geophys. Res. 94, 11106–11116. Motha, J.A., Walbrink, P.J., Hairsine, P.B., Grayson, R.B., 2002. Tracer properties of eroded sediment and source material. Hydrol Process 16, 1983–2000. U.S. Geological Survey, 2019, USGS 01589312 Dead Run near Catonsville, MD in USGS water data for the Nation: U.S. Geological Survey National Water Information System database, accessed December 21, 2018, at https://doi.org/10.5066/F7P55KJN. [Site information directly accessible at https://waterdata.usgs.gov/nwis/dv?referred_ module=sw&site_no=01589312.]. Nakano, Y., Inoue, M., Komura, K., 2008. Simple coprecipitation method combined with low background γ-spectrometry: determination of 7Be, 137Cs, 210Pb, and radium and thorium isotopes in small volume coastal water samples. J. Oceanogr. 64 (5), 713–717. Van Metre, P.C., Mahler, B.J., 2003. The contribution of particles washed from rooftops to contaminant loading to urban streams. Chemosphere 52, 1727–1741. Napieralski, J.A., Carvalhaes, T., 2016. References 24 (3), 706–723. Ritchie, J.C., McHenry, J.R., 1990. Application of radioactive fallout cesium-137 for measuring soil erosion and sediment accumulation rates and patterns: a review. J. Environ. Quality 19, 215–233. Waschbush, R.J., 2003. Data and methods of a 1999-2000 road sweeping study on an urban freeway in Milwaukee County, Wisconsin. U.S. Geological Survey Open-File Report 03-93, 41p. Russell, K.L., Vietz, G.F., Fletcher, T.D., 2019. A suburban sediment budget: Coarse- grained sediment flux through hillslopes, stormwater systems and streams. Earth Surf. Proc. Land. 44, 2600–2614.f Watts, A.W., Ballestero, T.P., Roseen, R.M., Houle, J.P., 2010. Polycyclic aromatic hy- drocarbons in stormwater runoff from sealcoated pavements. Environ. Sci. Technol 44 (23), 8849–8854. Russell, K.L., Vietz, G.F., Fletcher, T.D., 2018. Urban catchment runoff increases bedload sediment yield and particle size in stream channels. Anthropocene 23, 53–66. Wolman, M.G., 1967. A cycle of sedimentation and erosion in urban river channels. Geogr. Ann. Ser. A Phys. Geogr. 49, 385–395. 13 13
https://openalex.org/W4292707429	https://www.frontiersin.org/articles/10.3389/fbioe.2022.954707/pdf	English	null	The context matrix: Navigating biological complexity for advanced biodesign	Frontiers in bioengineering and biotechnology	2,022	cc-by	7,336	OPEN ACCESS EDITED BY Mattheos Koffas, Rensselaer Polytechnic Institute, United States Camillo Moschner, Charlie Wedd and Somenath Bakshi Control Group, Department of Engineering, University of Cambridge, Cambridge, United Kingdom Synthetic biology offers many solutions in healthcare, production, sensing and agriculture. However, the ability to rationally engineer synthetic biosystems with predictable and robust functionality remains a challenge. A major reason is the complex interplay between the synthetic genetic construct, its host, and the environment. Each of these contexts contains a number of input factors which together can create unpredictable behaviours in the engineered biosystem. It has become apparent that for the accurate assessment of these contextual effects a more holistic approach to design and characterisation is required. In this perspective article, we present the context matrix, a conceptual framework to categorise and explore these contexts and their net effect on the designed synthetic biosystem. We propose the use and community-development of the context matrix as an aid for experimental design that simpliﬁes navigation through the complex design space in synthetic biology. synthetic biology, context, biodesign, function-centric, design of experiments, standardisation synthetic biology, context, biodesign, function-centric, design of experiments, standardisation COPYRIGHT © 2022 Moschner, Wedd and Bakshi . 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. The context matrix: Navigating biological complexity for advanced biodesign OPEN ACCESS EDITED BY Mattheos Koffas, Rensselaer Polytechnic Institute, United States REVIEWED BY Chris John Myers, University of Colorado Boulder, United States *CORRESPONDENCE Camillo Moschner , camillo.moschner@gmail.com Somenath Bakshi , sb2330@cam.ac.uk SPECIALTY SECTION This article was submitted to Synthetic Biology, a section of the journal Frontiers in Bioengineering and Biotechnology RECEIVED 07 June 2022 ACCEPTED 29 June 2022 PUBLISHED 23 August 2022 CITATION Moschner C, Wedd C and Bakshi S (2022), The context matrix: Navigating biological complexity for advanced biodesign. Front. Bioeng. Biotechnol. 10:954707. doi: 10.3389/fbioe.2022.954707 TYPE Perspective PUBLISHED 23 August 2022 DOI 10.3389/fbioe.2022.954707 TYPE Perspective PUBLISHED 23 August 2022 DOI 10.3389/fbioe.2022.954707 FIGURE 1 The three contexts of an engineered biosystem’s function (A) Construct context includes factors intrinsic to the design of the synthetic genetic circuit which can affect performance, and can be broadly divided into intra and inter-transcription unit construct contexts. An example of an intra-TU context is the composition of the construct itself, which can be tuned with different parts to achieve different outputs. Relative gene orientation is an example of an inter-TU context, which can signiﬁcantly affect expression (Yeung et al., 2017). (B) The host context concerns all factors where the host organism affects the performance of the biosystem, and can be divided into the contexts of genetic factors, resource competition and the state in which the cell is growing. Resource competition (top, middle panel), is the phenomenon of a synthetic circuit competing with the host genome for shared resources. For genome-integrated circuits differing transcriptional propensities (indicated by the height of the green region) specify how the location of the circuit in the chromosome will affect performance (Scholz et al., 2019). (C) The environmental context in which the biosystem operates is deﬁned by physical, chemical and ecological factors (reducing to just physical and chemical factors for cell-free systems). A selection of cultivation processes are illustrated, the differences between which are likely to have signiﬁcant effects on gene expression in individual cells and shape the growth of the population as a whole. Acroynms: TU = transcription unit. RNAP = RNA polymerase. RBS = Ribosomal binding site. 1 Introduction Synthetic biology is deﬁned by the design and construction of biological systems for useful purposes. This approach typically follows a Design-Build-Test-Learn cycle. Various tools and pipelines have been created to standardise, enhance, or automate this construction cycle or speciﬁc segments of it (Carbonell et al., 2018; Jessop-Fabre and Sonnenschein, 2019). However, the design process in particular remains elusive to standardisation approaches. This is attributed to various factors, in particular the difﬁculty of choosing between vast numbers of design principles that could yield the desired function. Therefore, design typically relies on the experimenter’s knowledge of the biosystem they are working with. Acquisition of this knowledge typically relies on mining the literature. However, this is a time-consuming, labour-intensive and inefﬁcient process. From a materials perspective, this bottleneck led to a focus on the creation and characterisation of parts and repositories like the iGEM Registry of Standardised Biological Parts or the SynBioHub (Canton et al., 2008; Kwok, 2010; McLaughlin et al., 2018). However, it has become clear that the performance of these parts is highly context-dependent. For example, the same construct- host combination will likely exhibit different behaviours in different growth media, pH or 01 Frontiers in Bioengineering and Biotechnology frontiersin.org Moschner et al. 10.3389/fbioe.2022.954707 FIGURE 1 The three contexts of an engineered biosystem’s function (A) Construct context includes factors intrinsic to the design of the synthetic genetic circuit which can affect performance, and can be broadly divided into intra and inter-transcription unit construct contexts. An example of an intra-TU context is the composition of the construct itself, which can be tuned with different parts to achieve different outputs. Relative gene orientation is an example of an inter-TU context, which can signiﬁcantly affect expression (Yeung et al., 2017). (B) The host context concerns all factors where the host organism affects the performance of the biosystem, and can be divided into the contexts of genetic factors, resource competition and the state in which the cell is growing. Resource competition (top, middle panel), is the phenomenon of a synthetic circuit competing with the host genome for shared resources. For genome-integrated circuits differing transcriptional propensities (indicated by the height of the green region) specify how the location of the circuit in the chromosome will affect performance (Scholz et al., 2019). Frontiers in Bioengineering and Biotechnology 1 Introduction (C) The environmental context in which the biosystem operates is deﬁned by physical, chemical and ecological factors (reducing to just physical and chemical factors for cell-free systems). A selection of cultivation processes are illustrated, the differences between which are likely to have signiﬁcant effects on gene expression in individual cells and shape the growth of the population as a whole. Acroynms: TU = transcription unit. RNAP = RNA polymerase. RBS = Ribosomal binding site. FIGURE 1 2 The context matrix The chosen combination of all input factors (orange or pink outlines) completely deﬁnes the context of an engineered biosystem, and can be thought of as an input landscape. Each input landscape will produce an input-output mapping to outputs such as performance and ﬁtness. (B) Emergent properties arising from overlapping contexts. (C) Integrating the context matrix into the Design-Build-Test-Learn cycle for context-aware synthetic biology. DoE = Design of Experiments. FIGURE 2 The context matrix and its applications. (A) A representation of the context matrix. The three primary contexts (construct = C, host = H, en ironment E) are each composed of a n mber of inp t factors (C F E F ) hich are described in Fig re 1 The inp t factors are f rther FIGURE 2 The context matrix and its applications. (A) A representation of the context matrix. The three primary contexts (construct = C, host = H, environment = E), are each composed of a number of input factors (C.F1, E.F2, . . . ), which are described in Figure 1. The input factors are further FIGURE 2 The context matrix and its applications. (A) A representation of the context matrix. The three primary contexts (construct = C, host = H, environment = E), are each composed of a number of input factors (C.F1, E.F2, . . . ), which are described in Figure 1. The input factors are further subdivided into levels (C.F1. L1, C.F1. L2, . . . ). Continuous input factors (such as temperature or glucose concentration) can take any feasible ﬁnite level, whereas categorical input factors (such as species or gene orientation) are restricted to discrete values. The chosen combination of all input factors (orange or pink outlines) completely deﬁnes the context of an engineered biosystem, and can be thought of as an input landscape. Each input landscape will produce an input-output mapping to outputs such as performance and ﬁtness. (B) Emergent properties arising from overlapping contexts. (C) Integrating the context matrix into the Design-Build-Test-Learn cycle for context-aware synthetic biology. DoE = Design of Experiments. choice is an intergenic design factor that can be utilised to create “transcriptional valves” (Tarnowski and Gorochowski, 2022). While this strategy purposefully utilises intergenic interactions, a lot of attention has focused on the creation and characterisation of orthogonal parts to avoid intergenic cross-talk. For example, Meyer et al. 2 The context matrix temperatures (Boo et al., 2019; Chory et al., 2021). Similarly, in a ﬁxed environment, the same construct can display dramatically different performance in different hosts. Many context-deﬁning factors have been identiﬁed and characterised (Cardinale and Arkin, 2012) but have so far lacked a connection into one user- friendly framework for design and troubleshooting. The context matrix is a multi-dimensional list of input factors that enables decision making about what factors are important for the function of the engineered biosystem because of their combined effect on the output performance (Figure 2A), and helps to quickly identify factors previously unknown to the experimenter. Our approach considers a function-centric rather than the more traditional construct- centric view of synthetic biology. The biosystem function is seen in the contexts of synthetic construct, host, and environment composition. These contexts have been chosen based on their relatively independent preparation in the design process. For example, a genetic construct can be created outside of a host, and a host can be grown in various different environmental conditions. Each context is further divided into unique factors which can be quantitative or qualitative in nature. Understanding the current position of Here we present the “context matrix,” a database of previously encountered input factors categorised based on the contexts of either synthetic genetic construct, host or environment (Figure 1) which is intended to help experimenters identify their key input factors and enable the full utilisation of biological complexity. We ﬁrst explain the structure of the matrix, then use speciﬁc examples to highlight each context’s potential implication in system performance, how contexts overlapping with each other create emergent behaviours, and ﬁnally discuss how this information could be utilised to inform advanced design strategies. Frontiers in Bioengineering and Biotechnology 02 frontiersin.org Moschner et al. 10.3389/fbioe.2022.954707 FIGURE 2 The context matrix and its applications. (A) A representation of the context matrix. The three primary contexts (construct = C, host = H, environment = E), are each composed of a number of input factors (C.F1, E.F2, . . . ), which are described in Figure 1. The input factors are further subdivided into levels (C.F1. L1, C.F1. L2, . . . ). Continuous input factors (such as temperature or glucose concentration) can take any feasible ﬁnite level, whereas categorical input factors (such as species or gene orientation) are restricted to discrete values. 2 The context matrix (2019) showed the extensive part engineering required to create 12 transcription factors that can be used in the same cell without interfering with one another. Brophy and Voigt (2014) provides a holistic review of principles of genetic circuit design. Recently, various other potential construct design input factors have been identiﬁed. For example, the relative orientation of two mutually-repressive TUs has been shown to yield up to a 400% difference in maximal expression between convergent and divergent TU arrangements (Figure 1A) and has been associated with DNA supercoiling events inside the construct itself (Yeung et al., 2017). Furthermore, advances in sequencing technology have enabled the identiﬁcation of cryptic, anti-sense promoters inside coding sequences of a multi-TU logic gate that interfere with the desired biosystem function (Gorochowski et al., 2017). While this exempliﬁes anti-sense transcription interference as a failure mode, for a different engineered biosystem it can be harnessed as a construct input the engineered biosystem within this context space is essential for the design of novel biosystems, and for understanding and troubleshooting their failure modes. The input space from the context matrix maps to a speciﬁc, emergent phenotypic output state, such as performance of the engineered biosystem, or the ﬁtness of the host (Figure 2A). Frontiers in Bioengineering and Biotechnology 2.3 Environmental context Complex media such as lysogeny broth (LB) (Bertani, 1951, 2004) and trypticase soy broth (TSB) (FDA, 2001) are frequently used in bacterial studies. However, these complex nutrient broths include poorly deﬁned components (for example, LB contains yeast extract and protein digests), and therefore the use of complex media makes it impossible for researchers to know the precise chemical composition of their biosystem environment. This calls into question the reproducibility of physiological studies utilising LB or other complex media, and signiﬁcant differences in gene expression between different brands of LB have been observed (Sridhar and Steele-Mortimer, 2016). Host input factors are closely associated with the notion of “host-aware synthetic biology” (Boo et al., 2019). For example, synthetic biologists have increasingly become aware of the effects of plasmid-based versus genome-integrated constructs on output performance and have extensively investigated genome location effects (Block et al., 2012; Bryant et al., 2014; Sauer et al., 2016; Englaender et al., 2017; Goormans et al., 2020). Analysis of genome wide RNA polymerase activity lead to the mapping of “transcriptional propensities”, a measure of a genomic region’s likelihood to be transcribed (Figure 1B), across the entire 4.6 Mb E. coli genome (Scholz et al., 2019). These transcriptional propensities are the product of a complex interplay between nucleoid-associated proteins and the chromosome leading to actively and passively silenced regions, similar to the well-known mechanisms of chromosome compaction in eukaryotic heterochromatin (Wang et al., 2011). Knowledge of transcriptional propensities have been utilised to identify genome locations well-suited to accommodate heterologous gene circuits (Goormans et al., 2020; Park et al., 2020). This problem has long been recognised in microbiology. Neidhardt et al. (1974) published a fully deﬁned media formulation optimised for the growth of enterobacteria, noting that the ill-deﬁned components and autoclave sterilisation (as opposed to ﬁlter sterilisation) used in complex media preparation can introduce variability and uncertainty in results. Wherever possible, fully deﬁned media should be used in characterisation studies, such that the chemical context of an engineered biosystem can be controlled and optimised. A recent systematic mapping of the concentration of phosphorus, carbon and nitrogen sources in a deﬁned media to the growth rate and protein expression of E. coli cultures revealed media formulations which give high protein expression without sacriﬁcing growth (Chory et al., 2021). This exempliﬁes the importance of the environmental context in achieving the desired biosystem function. 2.1 Synthetic construct context The ﬁrst component of the context matrix, the synthetic genetic construct, captures contextual effects and failure modes intrinsic to the design of the synthetic system. This is closely associated with the term “genetic circuit design” in synthetic biology (Brophy and Voigt, 2014). The factors of the construct context can conceptually be divided into intra- and intergenic categories. In the ﬁrst instance, part tuning of a transcription unit (TU) might be considered which often includes the testing of various promoter and ribosome binding site strength levels (Figure 1A). This represents an example of intragenic design. Transcription read-through from one TU into another based on terminator 03 frontiersin.org 10.3389/fbioe.2022.954707 Moschner et al. Srivastava, 2020), stressed (Rodrigues and Rodrigues, 2018) and even spore states (Mohsin et al., 2021; Quijano and Sahin, 2021). Consideration of the cell state will be vital in the ongoing transition towards real-world applications of engineered biosystems. factor to tune gene expression (Brophy and Voigt, 2016). If such tuning is not desired, codon optimisation strategies or double terminators between TUs can be used as input factors to mitigate anti-sense transcription interference. 2.3 Environmental context The second component of the context matrix concerns the host, and captures advantages and disadvantages behind different host choices and their implications for the performance of an engineered biosystem. Input factors in this context include host-speciﬁc structural, metabolic or pre-existing genetic characteristics and includes knowledge of global gene regulatory mechanisms (Figure 1B). One of the ﬁrst host input factors to consider is what domain of life and speciﬁc species is most suitable for a given application. For example, eukaryotic cell engineering is often preferred for the production of humanized therapeutic proteins due to their already existing post- translational modiﬁcation machinery (Dumont et al., 2016). More recently, bioengineers have started to harness subcellular compartmentalisation as a means to eliminate metabolic pathway cross-talk and utilise organelle-speciﬁc micro- environments for particular biochemical reactions (Hammer and Avalos, 2017; Gassler et al., 2020; Grewal et al., 2021). The third component of the context matrix concerns the environment, capturing physical and chemical variables, and ecological interactions (Figure 1C). To create a particular functional output from an engineered biosystem, the environmental composition must either be carefully selected and maintained, or the biosystem should be designed such that its function is robust to changes in environmental context. As designers, we have the opportunity to precisely control the initial chemical composition of a biosystem. Complex media such as lysogeny broth (LB) (Bertani, 1951, 2004) and trypticase soy broth (TSB) (FDA, 2001) are frequently used in bacterial studies. However, these complex nutrient broths include poorly deﬁned components (for example, LB contains yeast extract and protein digests), and therefore the use of complex media makes it impossible for researchers to know the precise chemical composition of their biosystem environment. This calls into question the reproducibility of physiological studies utilising LB or other complex media, and signiﬁcant differences in gene expression between different brands of LB have been observed (Sridhar and Steele-Mortimer, 2016). The third component of the context matrix concerns the environment, capturing physical and chemical variables, and ecological interactions (Figure 1C). To create a particular functional output from an engineered biosystem, the environmental composition must either be carefully selected and maintained, or the biosystem should be designed such that its function is robust to changes in environmental context. that its function is robust to changes in environmental context. As designers, we have the opportunity to precisely control the initial chemical composition of a biosystem. Frontiers in Bioengineering and Biotechnology 2.3 Environmental context This reasoning extends beyond bacteria, and media optimisation through measurements of the changing quantities of metabolites present in the culture has been used to double antibody titres from mammalian cells (Sellick et al., 2011). Another crucial host input factor is the cell state in which it is supposed to be used. Most synthetic biology studies using E. coli, for example, test synthetic constructs in nutrient-rich, exponential growth phase. The resulting cell state is known to heavily utilise the growth-related σ70 global transcriptional regulator and hence the majority of characterised promoters are based on this sigma factor. More recently other cell states have been utilised in the design of engineered biosystems that operate in stationary (Gefen et al., 2014; Jaishankar and In many applications precise control of the environmental composition may not be attainable, and a biosystem function which is robust to environmental perturbations is desirable. For example, various applications do not allow control of 04 frontiersin.org Moschner et al. 10.3389/fbioe.2022.954707 10.3389/fbioe.2022.954707 temperature, a key physical input factor which can affect biosystem function due to its effect on the rates of biochemical reactions (Peterson et al., 2007). A synthetic gene circuit known as the dual-feedback oscillator exhibits a period which decreases with increasing temperatures (Stricker et al., 2008). For this system, a temperature-stable period was achieved by using a temperature-sensitive lac repressor protein (Hussain et al., 2014), demonstrating how a simple change in the construct context can give a robust functional performance in different environmental contexts. Another example of a synthetic construct yielding environmentally robust performance is the repressilator, a different genetic oscillator (Elowitz and Leibier, 2000). Potvin-Trottier et al. (2016) found that removing degradation tags and using protein titration in an improved design created an oscillator with a stable period of 14 cell generations across a range of temperatures. This design was later veriﬁed to be robust to changes in growth-phases using a microﬂuidic setup for complex growth conditions (Bakshi et al., 2021) and was also applied to quantify bacterial growth dynamics in the mouse gut, demonstrating the robustness of the desired function in an unknown and changing environmental context (Riglar et al., 2019). Interference between construct and host factors has also been recognised as an important emergent property of engineered biosystems (Del Vecchio et al., 2018). For example, Cookson et al. (2011) showed that two synthetic proteins which are both targeted for degradation by the E. 2.3 Environmental context coli ClpXP machinery can saturate the degradation system. This results in protein queuing of the synthetic proteins and stochastic accumulation of one of the proteins, resulting in failure of the designed function of the biosystem. Potvin-Trottier et al. (2016) identiﬁed a similar speciﬁc interference effect in the repressilator. The reporter protein in this oscillator construct contained an endogenous ClpXP degradation tag that caused ﬂuctuations in the degradation of the circuit’s repressor proteins and had to be removed to create regular oscillations. One potential mitigation strategy to avoid this speciﬁc interference is to engineer the construct to be as orthogonal as possible to the host. Various tools for orthogonal protein degradation in bacteria (Cameron and Collins, 2014) and eukaryotes (Pedone et al., 2019) have been created for this purpose. A further important consideration for successful design is evolution. Largely due to the effects of burden, the function performed by a synthetic construct is rarely stable in a population, and will be lost over time as ﬁtter, non-functional mutants arise (Sleight et al., 2010). Castle et al. (2021) developed a powerful conceptual framework for integrating evolutionary considerations into the design process. They deﬁne the “design type” as the engineered biosystem at the point of design. They then describe in detail the “evotype”, the set of evolutionary dispositions of the design type, and different ways in which the evotype can be shaped to change the probabilities of certain evolutionary outcomes. Consideration of the evotype is essential where prolonged or changing function is required. Efforts to properly characterise and understand contextual effects in synthetic biology will help to more completely deﬁne the design type, and should aid efforts to characterise and understand the long-term evolution of a biosystem. 3 Emergent properties of context effects While the context matrix is generally divided into the three contexts of synthetic construct, host and environment, some properties of an engineered biosystem cannot be usefully ﬁt into just one of these contexts but rather emerge from the interplay between two or even all three contexts simultaneously (Figure 2B). One well-studied example is the emergence of genetic burden. In synthetic biology, burden is generally deﬁned as an unnatural load on the host by the synthetic construct (Ceroni et al., 2015; Borkowski et al., 2016; Lee et al., 2016; Gorochowski et al., 2019). Many studies have identiﬁed resource competition between construct and host as the cause of burden (Kim et al., 2020). Qian et al. (2017) showed that a simple two node repression cascade can be rescued through a decrease in plasmid-copy number and RBS strengths of the circuit, indicating burden effects due to ribosome limitation (Figure 1B). Furthermore, the effects of the system’s environment have been shown to be equally important: By modifying rather than deleting endogenous genes Keren et al. (2016) showed that burden effects for the same expression level of a gene signiﬁcantly varied in different media. While genetic burden is not a design input factor itself but an emergent property of the engineered biosystem, awareness of the phenomenon has been cleverly utilised to create burden-driven feedback loops limiting the burden effects themselves (Ceroni et al., 2018). Frontiers in Bioengineering and Biotechnology 4 Context-aware biodesign Even in cases where some of this information seems irrelevant, it is still worth reporting, as it may be relevant to researchers in allied ﬁelds. As the ﬁeld of synthetic biology grows, complete and rigorous data reporting will help ensure results can be reproduced and understood, and will aid in standardisation efforts (Endy, 2005; Arkin, 2008, 2013). The value of standardisation in synthetic biology is demonstrated by the success of SBOL as a tool to aid the visualisation and design of synthetic biological parts (Galdzicki et al., 2014). Other standards, such as for data acquisition (Sainz De Murieta et al., 2016), have also been developed. However, there is currently no standardised way to report or characterise contextual effects in synthetic biology, and the context matrix is a step towards this goal. parts. This idea ﬁts into the statistical ﬁeld of Design of Experiments (DoE), in which multiple input factors are designed and tested simultaneously to identify the ones signiﬁcant to the desired application (Gilman et al., 2021). A common desired biosystem function is a simple induction system in which a gene of interest (GOI) is expressed after the cell receives a chemical or optical signal (Ong and Tabor, 2018; Groseclose et al., 2020). Traditionally this is implemented through a constitutively expressed, protein-based repressor inhibiting the GOI until the signal leads to a conformational change in the repressor, relieving its inhibition. However, when designing such a simple system the ﬁrst question is which repressors to choose from. An array of part repositories and characterisation experiments allows informed decision making about what repressor characteristics are available (Meyer et al., 2019). However, thus far no database of further design strategies exists. Once a repressor has been chosen, the question emerges about how to arrange the two transcription units of GOI and repressor with respect to each other. Most induction systems desire a low leakiness in the inhibited state. Looking at the context matrix (see GitHub repository) we learn that a convergent orientation is likely to increase inhibitory effects on the GOI through supercoiling of its DNA, and therefore decreases leakiness (Yeung et al., 2017). However, if this is still not sufﬁcient one might consider changing the construct to an RNA-based riboregulator or even a mixed RNA-plus protein- based multi-level controller which has shown reduced leakiness and improved dynamic range (Greco et al., 2021). 6 Conclusion Since the inception of synthetic biology, a myriad of different design strategies have been developed, utilising all biomolecules, various model and non-model organisms, and complex environmental and growth conditions. Here we present a holistic design framework for the categorisation of the learned design principles called the context matrix. As the ﬁeld continues to advance, we envision the context matrix to evolve from a list of design strategies to a complete database which maps any given combination of input factors to experimental outputs. Such a database could then be queried with a design brief and return candidate designs for testing, and explanations of the mechanisms behind these recommendations. Ultimately, the context matrix aims to help facilitate synthetic biology’s transition to a more context-aware future. The limited examples presented here exemplify how even a simple induction system can beneﬁt from various advanced design strategies and how the context matrix can help the experimenter identify which ones are suitable for their application. 5 Community development We envisage the context matrix as a community-built resource to aid in the design, characterisation, understanding and standardisation of engineered biosystems and their applications. 4 Context-aware biodesign If the desired function can be hosted in a eukaryotic organism additional design strategies for induction system improvements can include physical separation of the activating element from the GOI through protein localisation out of the nucleus (Di Ventura and Kuhlman, 2016) or designed, epigenetic-based silencing of the GOI (Sgro and Blancafort, 2020). The context matrix is hosted on GitHub (https://github.com/ camos95/context_matrix). We welcome content suggestions (adding new input factors or expanding and clarifying existing ones) and feedback or code submissions to improve the user experience via GitHub or email. Frontiers in Bioengineering and Biotechnology 4 Context-aware biodesign The context matrix represents an easily navigable database of knowledge of design strategies available to an experimenter, and lists strategies that might not have been known or considered before. Its emphasis on a function-centric view means it aims to achieve the desired biosystem function while being agnostic towards any particular construct, host or environment context. A consequence from this is that multiple engineered biosystems with the same function but very different contexts can be created. We call such systems analogous engineered biosystems. It is our goal to allow for simpliﬁed comparison between analogous systems to quickly identify the most appropriate design strategy based, for example, on available material, time constraints or even patent-protected genetic Frontiers in Bioengineering and Biotechnology Frontiers in Bioengineering and Biotechnology 05 frontiersin.org Moschner et al. 10.3389/fbioe.2022.954707 10.3389/fbioe.2022.954707 system (for example, the conditions under which cells were grown overnight prior to the experiment), and the DNA sequence data of all synthetic constructs and host genomes within the biosystem studied, except in cases where publishing such data could pose a biosecurity risk (Smith and Sandbrink, 2022). The reporting of all this data should be sufﬁcient to allow the biosystem to be fully and accurately recreated. Even in cases where some of this information seems irrelevant, it is still worth reporting, as it may be relevant to researchers in allied ﬁelds. As the ﬁeld of synthetic biology grows, complete and rigorous data reporting will help ensure results can be reproduced and understood, and will aid in standardisation efforts (Endy, 2005; Arkin, 2008, 2013). The value of standardisation in synthetic biology is demonstrated by the success of SBOL as a tool to aid the visualisation and design of synthetic biological parts (Galdzicki et al., 2014). Other standards, such as for data acquisition (Sainz De Murieta et al., 2016), have also been developed. However, there is currently no standardised way to report or characterise contextual effects in synthetic biology, and the context matrix is a step towards this goal. system (for example, the conditions under which cells were grown overnight prior to the experiment), and the DNA sequence data of all synthetic constructs and host genomes within the biosystem studied, except in cases where publishing such data could pose a biosecurity risk (Smith and Sandbrink, 2022). The reporting of all this data should be sufﬁcient to allow the biosystem to be fully and accurately recreated. Author contributions Doctoral Training in Sensor Technologies for a Healthy and Sustainable Future. CM and CW designed the database and prepared the manuscript. SB supervised the manuscript preparation and revisions. References Arkin, A. P. (2013). A wise consistency: Engineering biology for conformity, reliability, predictability. Curr. Opin. Chem. Biol. 17, 893–901. doi:10.1016/j.cbpa. 2013.09.012 Cardinale, S., and Arkin, A. P. (2012). Contextualizing context for synthetic biology - Identifying causes of failure of synthetic biological systems. Biotechnol. J. 7, 856–866. doi:10.1002/biot.201200085 Castle, S. D., Grierson, C. S., and Gorochowski, T. E. (2021). Towards an engineering theory of evolution. Nat. Commun. 12, 3326. doi:10.1038/s41467- 021-23573-3 Arkin, A. (2008). Setting the standard in synthetic biology. Nat. Biotechnol. 26, 771–774. doi:10.1038/nbt0708-771 Arkin, A. (2008). Setting the standard in synthetic biology. Nat. Biotechnol. 26, 771–774. doi:10.1038/nbt0708-771 Bakshi, S., Leoncini, E., Baker, C., Cañas-Duarte, S. J., Okumus, B., Paulsson, J., et al. (2021). Tracking bacterial lineages in complex and dynamic environments with applications for growth control and persistence. Nat. Microbiol. 6, 783–791. doi:10.1038/s41564-021-00900-4 Ceroni, F., Algar, R., Stan, G. B., and Ellis, T. (2015). Quantifying cellular capacity identiﬁes gene expression designs with reduced burden. Nat. Methods 12, 415–418. doi:10.1038/NMETH.3339 Bertani, G. (2004). Lysogeny at mid-twentieth century: P1, P2, and other experimental systems. J. Bacteriol. 186, 595–600. doi:10.1128/JB.186.3.595-600. 2004 Ceroni, F., Boo, A., Furini, S., Gorochowski, T. E., Borkowski, O., Ladak, Y. N., et al. (2018). Burden-driven feedback control of gene expression. Nat. Methods 15, 387–393. doi:10.1038/NMETH.4635 Bertani, G. (1951). Studies on lysogenesis: I. The mode of phage liberation by lysogenic Escherichia coli. J. Bacteriol. 62, 293–300. doi:10.1128/JB.62.3.293-300. 1951 Chory, E. J., Gretton, D. W., DeBenedictis, E. A., and Esvelt, K. M. (2021). Enabling high-throughput biology with ﬂexible open-source automation. Mol. Syst. Biol. 17, e9942. doi:10.15252/msb.20209942 Block, D. H., Hussein, R., Liang, L. W., and Lim, H. N. (2012). Regulatory consequences of gene translocation in bacteria. Nucleic Acids Res. 40, 8979–8992. doi:10.1093/NAR/GKS694 Cookson, N. A., Mather, W. H., Danino, T., Mondragõn-Palomino, O., Williams, R. J., Tsimring, L. S., et al. (2011). Queueing up for enzymatic processing: Correlated signaling through coupled degradation. Mol. Syst. Biol. 7, 561. doi:10.1038/MSB. 2011.94 Boo, A., Ellis, T., and Stan, G. B. (2019). Host-aware synthetic biology. Curr. Opin. Syst. Biol. 14, 66–72. doi:10.1016/J.COISB.2019.03.001 Del Vecchio, D., Qian, Y., Murray, R. M., and Sontag, E. D. (2018). Future systems and control research in synthetic biology. Annu. Rev. Control 45, 5–17. doi:10.1016/ j.arcontrol.2018.04.007 Borkowski, O., Ceroni, F., Stan, G. B., and Ellis, T. (2016). Overloaded and stressed: Whole-cell considerations for bacterial synthetic biology. Curr. Opin. Microbiol. 33, 123–130. doi:10.1016/J.MIB.2016.07.009 Di Ventura, B., and Kuhlman, B. (2016). Go in! 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. Funding This research in the SB laboratory was supported by the Wellcome Trust Award (grant number RG89305), a University Startup Award for Lectureship in Synthetic Biology (grant number NKXY ISSF3/46) and a Royal Society Research Grant Award (Award number G109931). CM was supported by the United Kingdom Biotechnology and Biological Sciences (BBSRC) University of Cambridge Doctoral Training Partnership 2 (BB/M011194/1). CW was supported by the United Kingdom Engineering and Physical Sciences Research Council (EPSRC) grant EP/S023046/1 for the EPSRC Centre for 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. Data availability statement To make continuous progress in populating the context matrix and for the ﬁeld to develop a broader and deeper understanding of the role of contextual effects in synthetic biology, it is important that all relevant contextual factors are reported in studies. This should include all physical and chemical factors, some accounting of the history of the A continuously updated, community-driven context matrix and additional information about how to contribute to the development can be found on our GitHub page: https:// github.com/camos95/context_matrix. 06 frontiersin.org frontiersin.org Moschner et al. 10.3389/fbioe.2022.954707 References Go out! Inducible control of nuclear localization. Curr. Opin. Chem. Biol. 34, 62–71. doi:10.1016/j.cbpa.2016.06.009 Brophy, J. A., and Voigt, C. A. (2016). Antisense transcription as a tool to tune gene expression. Mol. Syst. Biol. 12, 854. doi:10.15252/MSB.20156540 Dumont, J., Euwart, D., Mei, B., Estes, S., and Kshirsagar, R. (2016). Human cell lines for biopharmaceutical manufacturing: History, status, and future perspectives. Crit. Rev. Biotechnol. 36, 1110–1122. doi:10.3109/07388551.2015.1084266 Brophy, J. A., and Voigt, C. A. (2014). Principles of genetic circuit design. Nat. Methods 11, 508–520. doi:10.1038/nmeth.2926 Elowitz, M. B., and Leibier, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature 403, 335–338. doi:10.1038/35002125 Bryant, J. A., Sellars, L. E., Busby, S. J., and Lee, D. J. (2014). Chromosome position effects on gene expression in Escherichia coli K-12. Nucleic Acids Res. 42, 11383–11392. doi:10.1093/NAR/GKU828 Endy, D. (2005). Foundations for engineering biology. Nature 438, 449–453. doi:10.1038/nature04342 Cameron, D. E., and Collins, J. J. (2014). Tunable protein degradation in bacteria. Nat. Biotechnol. 32, 1276–1281. doi:10.1038/nbt.3053 Englaender, J. A., Jones, J. A., Cress, B. F., Kuhlman, T. E., Linhardt, R. J., Koffas, M. A. G., et al. (2017). Effect of genomic integration location on heterologous protein expression and metabolic engineering in E. coli. ACS Synth. Biol. 6, 710–720. doi:10.1021/acssynbio.6b00350 Canton, B., Labno, A., and Endy, D. (2008). Reﬁnement and standardization of synthetic biological parts and devices. Nat. Biotechnol. 26, 787–793. doi:10.1038/ nbt1413 Canton, B., Labno, A., and Endy, D. (2008). Reﬁnement and standardization of synthetic biological parts and devices. Nat. Biotechnol. 26, 787–793. doi:10.1038/ nbt1413 [Dataset] FDA (2001). Laboratory methods - BAM media M154: Trypticase (tryptic) soy broth. Washington, DC: FDA. Carbonell, P., Jervis, A. J., Robinson, C. J., Yan, C., Dunstan, M., Swainston, N., et al. (2018). An automated Design-Build-Test-Learn pipeline for enhanced microbial production of ﬁne chemicals. Commun. Biol. 1, 66. doi:10.1038/ s42003-018-0076-9 Galdzicki, M., Clancy, K. P., Oberortner, E., Pocock, M., Quinn, J. Y., Rodriguez, C. A., et al. (2014). The Synthetic Biology Open Language (SBOL) provides a 07 Frontiers in Bioengineering and Biotechnology frontiersin.org Moschner et al. 10.3389/fbioe.2022.954707 Neidhardt, F. C., Bloch, P. L., and Smith, D. F. (1974). Culture medium for enterobacteria. J. Bacteriol. 119, 736–747. doi:10.1128/jb.119.3.736-747.1974 community standard for communicating designs in synthetic biology. Nat. Biotechnol. 32, 545–550. doi:10.1038/nbt.2891 Ong,N.T., and Tabor,J.J.(2018).Aminiaturized Escherichia coligreen light sensorwith high dynamic range. ChemBioChem 19, 1255–1258. doi:10.1002/cbic.201800007 Gassler, T., Sauer, M., Gasser, B., Egermeier, M., Troyer, C., Causon, T., et al. (2020). References The industrial yeast Pichia pastoris is converted from a heterotroph into an autotroph capable of growth on CO2. Nat. Biotechnol. 38, 210–216. doi:10.1038/ s41587-019-0363-0 Park, Y., Espah Borujeni, A., Gorochowski, T. E., Shin, J., and Voigt, C. A. (2020). Precision design of stable genetic circuits carried in highly-insulated E. coli genomic landing pads. Mol. Syst. Biol. 16, e9584. doi:10.15252/msb.20209584 Gefen, O., Fridman, O., Ronin, I., and Balaban, N. Q. (2014). Direct observation of single stationary-phase bacteria reveals a surprisingly long period of constant protein production activity. Proc. Natl. Acad. Sci. U. S. A. 111, 556–561. doi:10.1073/ pnas.1314114111 Pedone, E., Postiglione, L., Aulicino, F., Rocca, D. L., Montes-Olivas, S., Khazim, M., et al. (2019). A tunable dual-input system for on-demand dynamic gene expression regulation. Nat. Commun. 10, 4481. doi:10.1038/s41467-019-12329-9 Gilman, J., Walls, L., Bandiera, L., and Menolascina, F. (2021). Statistical design of experiments for synthetic biology. ACS Synth. Biol. 10, 1–18. doi:10.1021/acssynbio. 0c00385 Peterson, M. E., Daniel, R. M., Danson, M. J., and Eisenthal, R. (2007). The dependence of enzyme activity on temperature: Determination and validation of parameters. Biochem. J. 402, 331–337. doi:10.1042/BJ20061143 Goormans, A. R., Snoeck, N., Decadt, H., Vermeulen, K., Peters, G., Coussement, P., et al. (2020). Comprehensive study on Escherichia coli genomic expression: Does position really matter? Metab. Eng. 62, 10–19. doi:10.1016/j.ymben.2020.07.007 Potvin-Trottier, L., Lord, N. D., Vinnicombe, G., and Paulsson, J. (2016). Synchronous long-term oscillations in a synthetic gene circuit. Nature 538, 514–517. doi:10.1038/nature19841 Gorochowski, T. E., Chelysheva, I., Eriksen, M., Nair, P., Pedersen, S., Ignatova, Z., et al. (2019). Absolute quantiﬁcation of translational regulation and burden using combined sequencing approaches. Mol. Syst. Biol. 15, e8719. doi:10.15252/ msb.20188719 Qian, Y., Huang, H. H., Jiménez, J. I., and Del Vecchio, D. (2017). Resource competition shapes the response of genetic circuits. ACS Synth. Biol. 6, 1263–1272. doi:10.1021/acssynbio.6b00361 Quijano, J. F., and Sahin, O. (2021). Genetically intact bioengineered spores of Bacillus subtilis. ACS Synth. Biol. 10, 778–785. doi:10.1021/acssynbio.0c00578 Gorochowski, T. E., Espah Borujeni, A., Park, Y., Nielsen, A. A., Zhang, J., Der, B. S., et al. (2017). Genetic circuit characterization and debugging using RNA -seq. Mol. Syst. Biol. 13, 952. doi:10.15252/msb.20167461 Riglar, D. T., Richmond, D. L., Potvin-Trottier, L., Verdegaal, A. A., Naydich, A. D., Bakshi, S., et al. (2019). Bacterial variability in the mammalian gut captured by a single-cell synthetic oscillator. Nat. Commun. 10, 4665. doi:10.1038/s41467-019-12638-z Greco, F. V., Pandi, A., Erb, T. J., Grierson, C. References S., and Gorochowski, T. E. (2021). Harnessing the central dogma for stringent multi-level control of gene expression. Nat. Commun. 12, 1738. doi:10.1038/s41467-021-21995-7 Rodrigues, J. L., and Rodrigues, L. R. (2018). Potential applications of the Escherichia coli heat shock response in synthetic biology. Trends Biotechnol. 36, 186–198. doi:10.1016/j.tibtech.2017.10.014 Grewal, P. S., Samson, J. A., Baker, J. J., Choi, B., and Dueber, J. E. (2021). Peroxisome compartmentalization of a toxic enzyme improves alkaloid production. Nat. Chem. Biol. 17, 96–103. doi:10.1038/s41589-020-00668-4 Sainz De Murieta, I., Bultelle, M., and Kitney, R. I. (2016). Toward the ﬁrst data acquisition standard in synthetic biology. ACS Synth. Biol. 5, 817–826. doi:10.1021/ acssynbio.5b00222 Groseclose, T. M., Rondon, R. E., Herde, Z. D., Aldrete, C. A., and Wilson, C. J. (2020). Engineered systems of inducible anti-repressors for the next generation of biological programming. Nat. Commun. 11, 4440. doi:10.1038/s41467-020-18302-1 Sauer, C., Syvertsson, S., Bohorquez, L. C., Cruz, R., Harwood, C. R., Van Rij, T., et al. (2016). Effect of genome position on heterologous gene expression in Bacillus subtilis: An unbiased analysis. ACS Synth. Biol. 5, 942–947. doi:10.1021/acssynbio.6b00065 Hammer, S. K., and Avalos, J. L. (2017). Harnessing yeast organelles for metabolic engineering. Nat. Chem. Biol. 13, 823–832. doi:10.1038/nchembio.2429 Scholz, S. A., Diao, R., Wolfe, M. B., Fivenson, E. M., Lin, X. N., Freddolino, P. L., et al. (2019). High-resolution mapping of the Escherichia coli chromosome reveals positions of high and low transcription. Cell. Syst. 8, 212–225. e9. doi:10.1016/j.cels. 2019.02.004 Hussain, F., Gupta, C., Hirning, A. J., Ott, W., Matthews, K. S., Josic, K., et al. (2014). Engineered temperature compensation in a synthetic genetic clock. Proc. Natl. Acad. Sci. U. S. A. 111, 972–977. doi:10.1073/pnas.1316298111 Jaishankar, J., and Srivastava, P. (2020). Strong synthetic stationary phase promoter-based gene expression system for Escherichia coli. Plasmid 109, 102491. doi:10.1016/j.plasmid.2020.102491 Sellick, C. A., Croxford, A. S., Maqsood, A. R., Stephens, G., Westerhoff, H. V., Goodacre, R., et al. (2011). Metabolite proﬁling of recombinant CHO cells: Designing tailored feeding regimes that enhance recombinant antibody production. Biotechnol. Bioeng. 108, 3025–3031. doi:10.1002/bit.23269 Jessop-Fabre, M. M., and Sonnenschein, N. (2019). Improving reproducibility in synthetic biology. Front. Bioeng. Biotechnol. 7, 18. doi:10.3389/fbioe.2019.00018 Sgro, A., and Blancafort, P. (2020). Epigenome engineering: New technologies for precision medicine. Nucleic Acids Res. 48, 12453–12482. doi:10.1093/nar/gkaa1000 Keren, L., Hausser, J., Lotan-Pompan, M., Vainberg Slutskin, I., Alisar, H., Kaminski, S., et al. (2016). Massively parallel interrogation of the effects of gene expression levels on ﬁtness. Cell. References 166, 1282–1294. e18. doi:10.1016/j.cell.2016.07.024 Sleight, S. C., Bartley, B. A., Lieviant, J. A., and Sauro, H. M. (2010). Designing and engineering evolutionary robust genetic circuits. J. Biol. Eng. 4, 12. doi:10.1186/ 1754-1611-4-12 Kim, J., Darlington, A., Salvador, M., Utrilla, J., and Jiménez, J. I. (2020). Trade- offs between gene expression, growth and phenotypic diversity in microbial populations. Curr. Opin. Biotechnol. 62, 29–37. doi:10.1016/j.copbio.2019.08.004 Smith, J. A., and Sandbrink, J. B. (2022). Biosecurity in an age of open science. PLoS Biol. 20, e3001600. doi:10.1371/journal.pbio.3001600 Kwok, R. (2010). Five hard truths for synthetic biology. Nature 463, 288–290. doi:10.1038/463288a Sridhar, S., and Steele-Mortimer, O. (2016). Inherent variability of growth media impacts the ability of Salmonella Typhimurium to interact with host cells. PLoS ONE 11, e0157043. doi:10.1371/journal.pone.0157043 Lee, J. W., Gyorgy, A., Cameron, D. E., Pyenson, N., Choi, K. R., Way, J. C., et al. (2016). Creating single-copy genetic circuits. Mol. Cell. 63, 329–336. doi:10.1016/j. molcel.2016.06.006 Stricker, J., Cookson, S., Bennett, M. R., Mather, W. H., Tsimring, L. S., Hasty, J., et al. (2008). A fast, robust and tunable synthetic gene oscillator. Nature 456, 516–519. doi:10.1038/nature07389 McLaughlin, J. A., Myers, C. J., Zundel, Z., Mısırlı, K., Zhang, M., Oﬁteru, I. D., et al. (2018). SynBioHub: A standards-enabled design repository for synthetic biology. ACS Synth. Biol. 7, 682–688. doi:10.1021/acssynbio.7b00403 Tarnowski, M. J., and Gorochowski, T. E. (2022). Massively parallel characterization of engineered transcript isoforms using direct RNA sequencing. Nat. Commun. 13, 434. doi:10.1038/s41467-022-28074-5 Meyer, A. J., Segall-Shapiro, T. H., Glassey, E., Zhang, J., and Voigt, C. A. (2019). Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors. Nat. Chem. Biol. 15, 196–204. doi:10.1038/s41589-018-0168-3 Wang, W., Li, G.-W., Chen, C., Xie, X. S., and Zhuang, X. (2011). Chromosome organization by a nucleoid-associated protein in live bacteria. Science 333, 1445–1449. doi:10.1126/science.1204697 Mohsin, M. Z., Omer, R., Huang, J., Mohsin, A., Guo, M., Qian, J., et al. (2021). Advances in engineered Bacillus subtilis bioﬁlms and spores, and their applications in bioremediation, biocatalysis, and biomaterials. Synthetic Syst. Biotechnol. 6, 180–191. doi:10.1016/j.synbio.2021.07.002 Yeung, E., Dy, A. J., Martin, K. B., Ng, A. H., Del Vecchio, D., Beck, J. L., et al. (2017). Biophysical constraints arising from compositional context in synthetic gene networks. Cell. Syst. 5, 11–24. e12. doi:10.1016/j.cels.2017.06.001 08 frontiersin.org Frontiers in Bioengineering and Biotechnology Frontiers in Bioengineering and Biotechnology
https://openalex.org/W3083237116	https://e-journal.upr.ac.id/index.php/eej/article/download/1553/1402	Indonesian	null	IMPLEMENTASI GERAKAN LITERASI SEKOLAH	Equity in Education Journal	2,019	cc-by	3,636	Eric Santosa, Piter Joko Nugroho, Reddy Siram FKIP, Universitas Palangka Raya E-mail: esantosa203@gmail.com Abstrak: Penelitian kualitatif dengan rancangan studi kasus ini bertujuan untuk mendeskripsikan tentang Implementasi GLS di SDN 5 Menteng Palangka Raya, dilihat dari aspek: (1) tahapan implementasi GLS, (2) Mekanisme implementasi GLS, dan (3) Faktor pendukung dan kendala dalam implementasi GLS. Sumber data dalam penelitian ini adalah kepala sekolah, tenaga pustakawan, dan 2 orang guru kelas. Teknik pengumpulan data dilakukan dengan cara observasi, wawancara, dan dokumentasi. Analisis data menggunakan pola interaktif data dari Miles dan Huberman (1994). Hasil penelitian menunjukan bahwa: (1) Tahapan GLS yang dilaksanakan baru pada tahap awal (pembiasaan berliterasi) melalui aktivitas membaca dan menulis, (2) Mekanisme implementasi GLS dilaksanakan dengan mendasarkan pada prinsip dasar manajemen yaitu: perencanaan GLS, pengorganisasian GLS, penggerakan GLS, serta pengawasan dan evaluasi GLS, (3) Faktor pendukung implementasi GLS meliputi minat yang tinggi dari siswa untuk berliterasi, komitmen dan semangat guru mengajar, program literasi dilaksanakan secara terjadwal, tersedianya sarana prasarana sekolah yang mendukung pelaksanaan literasi; Sedangkan faktor penghambatnya adalah guru masih belum menerima pembinaan dalam bentuk pelatihan untuk melaksanakan GLS, kondisi koleksi buku bacaan yang rusak, serta fungsi evaluasi GLS yang belum dilaksanakan secara menyeluruh. Kata Kunci: Implementasi, Gerakan Literasi Sekolah, SDN 5 Menteng. Kata Kunci: Implementasi, Gerakan Literasi Sekolah, SDN 5 Menteng. Abstract: Qualitative research with a case study design aims to describe the implementation of GLS in SDN 5 Menteng Palangka Raya, viewed from aspects: (1) stages of GLS implementation, (2) mechanism of GLS implementation, and (3) supporting factors and constraints in GLS implementation. The data sources in this study are the principal, librarian, and 2 class teachers. Data collection techniques carried out by observation, interviews, and documentation. Data analysis uses interactive data patterns from Miles and Huberman (1994). Eric Santosa, Piter Joko Nugroho, Reddy Siram FKIP, Universitas Palangka Raya E-mail: esantosa203@gmail.com The results showed that: (1) The stages of GLS were carried out only in the initial stages (habituation of iteration) through reading and writing activities, (2) The mechanism of GLS implementation was carried out based on basic management principles, namely: GLS planning, GLS organizing, GLS mobilization, and GLS monitoring and evaluation, (3) Supporting factors for the implementation of GLS include the high interest of students to titrate, commitment and enthusiasm of teaching teachers, literacy programs carried out on a scheduled basis, the availability of school infrastructure that supports literacy; While the inhibiting factor is the teacher still has not received coaching in the form of training to implement GLS, the condition of the collection of damaged reading books, as well as the evaluation function of GLS that has not been implemented thoroughly. Keywords: Implementation, School Literacy Movements, SDN 5 Menteng. Keywords: Implementation, School Literacy Movements, SDN 5 Menteng. IMPLEMENTASI GERAKAN LITERASI SEKOLAH Eric Santosa, Piter Joko Nugroho, Reddy Siram FKIP, Universitas Palangka Raya E-mail: esantosa203@gmail.com Equity in Education Journal (EEJ), Vol. 1, No. 1, Oktober 2019 Equity in Education Journal (EEJ), Vol. 1, No. 1, Oktober 2019 PENDAHULUAN dalam pembangunan bangsa Indonesia yang terencana untuk mengembangkan kemampuan dalam membentuk watak pribadi seseorang dan segenap potensi yang ada pada diri manusia, agar menjadi PENDAHULUAN Pendidikan merupakan aspek mendasar yang menjadi tolak ukur kemajuan suatu bangsa. Pendidikan memiliki peranan yang sangat penting 56 Equity in Education Journal (EEJ), Vol. 1, No. 1, Oktober 2019 manusia yang utuh secara jasmani dan rohani, serta mampu mengembangkan kemampuan yang ada dalam diri seorang manusia. Indonesia sendiri juga sangat memperhatikan tentang pendidikan. Cita- cita pendidikan bangsa Indonesia tersebut tertuang dalam Undang-Undang Dasar 1945, yaitu “untuk mencerdaskan kehidupan bangsa”. Berdasarkan hasil survey yang dilakukan oleh PISA (Programme for International Student Assessment) 2009 menunjukkan peserta didik Indonesia berada pada peringkat ke- 57 dengan skor 396 (skor rata-rata OECD 493), sedangkan PISA 2012 menunjukkan peserta didik Indonesia berada pada peringkat ke-64 dengan skor 396 (skor rata-rata OECD 496). Sebanyak 65 negara berpartisipasi dalam PISA 2009 dan 2012. Peserta didik kesulitan untuk menguasai materi bacaan dan sulit untuk menjawab soal uraian yang memerlukan analisis logika, hal ini merupakan gambaran tentang bagaimana rendahnya kualitas literasi peserta didik Indonesia pada jenjang pendidikan dasar. Kurangnya minat membaca siswa merupakan penyebab utama rendahnya kualitas literasi peserta didik di Indonesia, siswa masih sangat bergantung pada guru sebagai sumber ilmu pengetahuan, sehingga minat untuk melakukan kegiatan membaca sangat rendah. Dari kedua hasil survey PISA tersebut, dapat dikatakan bahwa praktik pendidikan yang dilaksanakan di sekolah belum memperlihatkan fungsi sekolah sebagai organisasi pembelajaran yang berupaya menjadikan peserta didik terampil membaca untuk mendukung mereka sebagai pembelajar sepanjang hayat (Kemendikbud, 2016:1). Berdasarkan fakta tersebut, maka pemerintah melalui Permendikbud Nomor 23 tahun 2015 tentang budi pekerti menggalakkan program literasi yang dinamakan Gerakan Literasi Sekolah (GLS). Pengertian literasi dalam konteks GLS adalah kemampuan mengakses memahami dan melalui berbagai aktivitas, antara lain membaca, melihat, menyimak, menulis dan/berbicara. Melalui adanya program GLS ini pemerintah berusaha meningkatkan minat baca peserta didik yang menjurus pada pengembangan budi pekerti dan kemampuan peserta didik untuk dapat memahami materi bacaan secara utuh dalam rangka mempersiapkan diri mereka sebagai pembelajar sepanjang hayat. manusia yang utuh secara jasmani dan rohani, serta mampu mengembangkan kemampuan yang ada dalam diri seorang manusia. Indonesia sendiri juga sangat memperhatikan tentang pendidikan. Cita- cita pendidikan bangsa Indonesia tersebut tertuang dalam Undang-Undang Dasar 1945, yaitu “untuk mencerdaskan kehidupan bangsa”. PENDAHULUAN 1, Oktober 2019 yaitu melalui kegiatan 15 menit membaca bagi semua peserta didik. Pentingnya kegiatan membaca dalam GLS tentu memberi hal positif bagi peserta didik, beberapa manfaat dari kegiatan membaca, yaitu: (1) meningkat kadar intelektul; (2) memperoleh berbagai pengetahuan hidup; (3) memiliki cara pandang dan pola pikir yang luas; (4) memperkaya perbendaharaan kata; (5) mengetahui berbagai peristiwa yang terjadi di berbagai belahan dunia; (6) meningkatkan keimanan; (7) mendapatkan hiburan (Rachmawati, 2008:4). Pada SDN 5 Menteng Palangka Raya, kepala sekolah bertanggung jawab langsung dalam proses berjalannya GLS secara menyeluruh di lingkungan sekolah dengan memberikan arahan, saran, dan bimbingan kepada guru kelas maupun tenaga pustakawan dalam melaksanakan kegiatan literasi untuk mengarahkan, membimbing, dan memotivasi siswa agar menjadi tertarik dalam kegiatan berliterasi, khususnya membaca dan menulis. Kegiatan GLS, 15 menit membaca, di SDN 5 Menteng disebut dengan kegiatan literasi kelas yang juga selain itu didukung dengan program kegiatan kunjung perpustakaan. Sehingga, berjalannya GLS di SDN 5 Menteng dilakukan melalui dua macam kegiatan, yaitu kegiatan literasi kelas dan kegiatan kunjung perpustakaan. pelajaran yang tersedia diperpustakaan. Pada kegiatan ini peran guru dibantu oleh tenaga pustakawan dalam memberikan arahan dan bimbingan kegiatan membaca. Seperti kegiatan literasi kelas, selesai kegiatan kunjung perpustakaan siswa diarahkan untuk mengekspresikan pendapatnya terhadap buku bacaan yang telah disimaknya baik secara lisan maupun tertulis. Kedua kegiatan tersebut telah terlaksana dalam menunjang program GLS untuk menumbuhkan minat baca siswa di SDN 5 Menteng Palangka Raya. Selain itu, lingkungan sekolah yang kaya literasi juga telah tercipta di SDN 5 Menteng, yang nampak dari banyaknya media baca seperti mading, poster, atau kliping, atau gambar kartun yang merupakan hasil karya siswa terpampang di berbagai sudut dinding ruang kelas maupun luar kelas, karya literasi yang penuh warna dan menarik sehingga memotivasi siswa yang lain untuk membaca/melihat isi bacaan yang disampaikan. yaitu melalui kegiatan 15 menit membaca bagi semua peserta didik. Pentingnya kegiatan membaca dalam GLS tentu memberi hal positif bagi peserta didik, beberapa manfaat dari kegiatan membaca, yaitu: (1) meningkat kadar intelektul; (2) memperoleh berbagai pengetahuan hidup; (3) memiliki cara pandang dan pola pikir yang luas; (4) memperkaya perbendaharaan kata; (5) mengetahui berbagai peristiwa yang terjadi di berbagai belahan dunia; (6) meningkatkan keimanan; (7) mendapatkan hiburan (Rachmawati, 2008:4). PENDAHULUAN Pada SDN 5 Menteng Palangka Raya, kepala sekolah bertanggung jawab langsung dalam proses berjalannya GLS secara menyeluruh di lingkungan sekolah dengan memberikan arahan, saran, dan bimbingan kepada guru kelas maupun tenaga pustakawan dalam melaksanakan kegiatan literasi untuk mengarahkan, membimbing, dan memotivasi siswa agar menjadi tertarik dalam kegiatan berliterasi, khususnya membaca dan menulis. Kegiatan GLS, 15 menit membaca, di SDN 5 Menteng disebut dengan kegiatan literasi kelas yang juga selain itu didukung dengan program kegiatan kunjung perpustakaan. Sehingga, berjalannya GLS di SDN 5 Menteng dilakukan melalui dua macam kegiatan, yaitu kegiatan literasi kelas dan kegiatan kunjung perpustakaan. PENDAHULUAN Berdasarkan hasil survey yang dilakukan oleh PISA (Programme for International Student Assessment) 2009 menunjukkan peserta didik Indonesia berada pada peringkat ke- 57 dengan skor 396 (skor rata-rata OECD 493), sedangkan PISA 2012 menunjukkan peserta didik Indonesia berada pada peringkat ke-64 dengan skor 396 (skor rata-rata OECD 496). Sebanyak 65 negara berpartisipasi dalam PISA 2009 dan 2012. Peserta didik kesulitan untuk menguasai materi bacaan dan sulit untuk menjawab soal uraian yang memerlukan analisis logika, hal ini merupakan gambaran tentang bagaimana rendahnya kualitas literasi peserta didik Indonesia pada jenjang pendidikan dasar. Kurangnya minat membaca siswa merupakan penyebab utama rendahnya kualitas literasi peserta didik di Indonesia, siswa masih sangat bergantung pada guru sebagai sumber ilmu pengetahuan, sehingga minat untuk melakukan kegiatan membaca sangat rendah. Dari kedua hasil survey PISA tersebut, dapat dikatakan bahwa praktik pendidikan yang dilaksanakan di sekolah belum memperlihatkan fungsi sekolah sebagai organisasi pembelajaran yang berupaya menjadikan peserta didik terampil membaca untuk mendukung mereka sebagai pembelajar sepanjang hayat (Kemendikbud, 2016:1). Berdasarkan fakta tersebut, maka pemerintah melalui Permendikbud Nomor 23 tahun 2015 tentang budi pekerti menggalakkan program literasi yang dinamakan Gerakan Literasi Sekolah (GLS). Pengertian literasi dalam konteks GLS adalah kemampuan mengakses, memahami, dan menggunakan sesuatu secara cerdas Program GLS sejatinya memiliki tiga tahapan yang disesuaikan dengan kesiapan sekolah, yaitu tahap pembiasaan, tahap pengembangan, dan tahap pembelajaran (Kemendikbud, 2016:5). Surabaya menjadi contoh keberhasilan dalam melaksanakan program literasi. Adanya dukungan kebijakan dari pemerintah kota Surabaya dengan membuat program tantangan membaca yang mewajibkan kegiatan membaca bagi seluruh siswa setiap hari, fenomena yang nampak pada setiap sekolah di Surabaya dimana siswa diwajibkan membaca 2 buku dalam satu bulan dengan target capaian sekolah membaca 3000 buku setiap tahunnya. Dengan diwajibkannya kegiatan membaca ini memacu para siswa di kota Surabaya untuk lebih mengasah kemampuan dalam berliterasi, yang mana siswanya tidak hanya terampil membaca namun telah bisa membuat karya tulisan sendiri. Melalui program literasi para siswa di kota Surabaya telah mampu mengakses dan memahami informasi melalui membaca serta menyampaikan kembali informasi tersebut melalui karya tulisan mereka sendiri. Kota Palangka Raya juga telah melaksanakan program GLS, salah satu sekolah yang telah melaksanakan program GLS yang telah berjalan dengan baik yaitu pada jenjang pendidikan dasar di SDN 5 Menteng. Kegiatan literasi di SDN 5 Menteng telah diprogramkan dalam salah satu misi sekolah yang menunjukan keseriusan SDN 5 Menteng dalam mewujudkan tujuan GLS di sekolah untuk menumbuhkembangkan budaya membaca 57 Equity in Education Journal (EEJ), Vol. 1, No. METODE Penelitian ini menggunakan pendekatan kualitatif dengan rancangan studi kasus. Studi kasus yang pada hakekatnya meneliti kasus, dimana kasus tersebut diperlukan dan penting untuk menguji suatu teori yang telah tersusun dengan baik. Studi kasus juga dimaknai kasus organisasi, yaitu studi kasus untuk mendapatkan informasi tentang keterangan-keterangan organisasi dimana peneliti ingin mengetahui bagaimana kehidupan orang-orang dalam organisasi tersebut. Penelitian dilakukan di SDN 5 Menteng Kota Palangka Raya. Penentuan informan dilakukan dengan teknik pengambilan sampel secara purposive sampling dan snowball sampling dengan maksud agar diperoleh data dan informasi dari orang-orang yang benar-benar mengetahui secara mendalam terkait fokus penelitian sehingga data yang diterima dapat lebih akurat. Dari informan pertama, maka akan berkembang ke Kegiatan literasi kelas dilakukan selama 15 menit setiap hari, dimana siswa diarahkan oleh guru kelas untuk melakukan aktivitas baik di dalam maupun di luar kelas seperti membaca, mengamati, atau menulis. Pada akhir kegiatan siswa diarahkan kembali oleh guru kelas untuk menyampaikan tanggapannya terhadap aktivitas yang dilakukannya dapat secara langsung maupun tertulis dalam bentuk rangkuman. Sedangkan kegiatan kunjung perpustakaan dilakukan seminggu sekali secara terjadwal untuk satu kelas per minggunya dengan durasi 30 menit untuk membaca maupun menyimak buku bergambar, baik buku cerita maupun buku 58 Equity in Education Journal (EEJ), Vol. 1, No. 1, Oktober 2019 hal ini mengingat bahwa program GLS di SDN 5 Menteng Palangka Raya belum lama berjalan, juga karena ditunjang oleh kemampuan sekolah dalam menyediakan sarana dan prasarana literasi yang hanya mencukupi untuk melakukan literasi pada tahapan pembiasaan. Tujuan GLS di SDN 5 Menteng Palanga Raya pada tahapan pembiasaan adalah untuk menumbuhkembangkan budaya berliterasi melalui berbagai aktivitas seperti membaca, menulis, bernyanyi, bercerita, menyimak, dan melihat, yang dilakukan selama 15 menit sebelum belajar setiap hari sekolah. Pada tahapan pembiasaan ini tidak ada tagihan wajib yang berpengaruh pada nilai akademik oleh guru kepada siswa yang telah melakukan kegiatan literasi. informan kedua dan seterusnya sehingga diibaratkan seperti bola salju. Hal itu sejalan dengan yang dikatakan oleh Ulfatin (2014:181), begitu informan kunci pertama diwawancarai secukupnya, ia diminta untuk menunjukkan satu atau lebih sumber lain yang dianggapnya memiliki informasi yang dianggap relevan dan memadai, sehingga dapat dijadikan sebagai informan berikutnya. Dari informan kedua yang ditunjuk oleh informan pertama, kemudian ia diminta untuk menyebutkan sumber lain yang dapat dijadikan informan berikutnya lagi. Dengan cara inilah, informasi yang diperoleh peneliti menjadi semakin besar dengan melibatkan beberapa orang yang menurut Bogdan dan Biklen (1992) diibaratkan seperti bola salju (snowball sampling). METODE Teknik pengumpulan data yang digunakan adalah (a) observasi, (b) wawancara mendalam, dan (c) studi dokumentasi. Informasi yang terkumpul dari ketiga teknik tersebut dianalisis secara berulang dengan menggunakan alur pola interaktif. Analisis data penelitian Miles dan Huberman yang meliputi reduksi data, penyajian data, dan penarikan kesimpulan. Pengecekan kredibilitas data dilakukan dengan teknik triangulasi (sumber dan teknik/metode), member checks, dan kecukupan bahan referensi; pengecekan dependabilitas data penelitian dilakukan oleh peneliti mulai dari melakukan penelitian, penyusunan transkrip wawancara sampai dengan penulisan laporan hasil penelitian dan pelaksanaan penelitian sesuai dengan jadwal yang telah disepakati bersama; dan pengecekan konfirmabilitas digunakan untuk melihat bahwa hasil penelitian yang dilakukan menunjukkan adanya proses penelitian di lapangan. Hasil penelitian tentang implementasi GLS di SDN 5 Menteng Palangka Raya yang baru dilaksanakan pada tahap awal yaitu tahap pembiasaan melalui berbagai aktivitas seperti membaca, menulis, bernyanyi, bercerita, menyimak, dan melihat, yang dilakukan selama 15 menit sebelum belajar setiap hari sekolah di atas sejalan dengan Kemendikbud (2016) bahwa program literasi dapat diawali dengan melalukan kegiatan pembiasaan yang antara lain bertujuan untuk menumbuhkan minat peserta didik terhadap bacaan dan terhadap kegiatan membaca. 2. Mekanisme Implementasi GLS Berdasarkan hasil penelitian diketahui bahwa implementasi GLS yang dilaksanakan di SDN 5 Menteng Palangka Raya dilaksanakan dengan mendasarkan pada prinsip dasar manajemen, yaitu perencanaan, pengorganisasi, penggerakan dan motoring dan evaluasi. Perencanaan GLS dilaksanakan dengan menetapkan tujuan GLS untuk menumbuhkembangkan budaya literasi yang dilakukan melalui rapat kerja yang diikuti oleh seluruh elemen sekolah baik guru, kepala sekolah dan seluruh karyawan. Rapat kerja ini dilakukan Berdasarkan hasil penelitian diketahui bahwa implementasi GLS yang dilaksanakan di SDN 5 Menteng Palangka Raya dilaksanakan dengan mendasarkan pada prinsip dasar manajemen, yaitu perencanaan, pengorganisasi, penggerakan dan motoring dan evaluasi. Perencanaan GLS dilaksanakan dengan menetapkan tujuan GLS untuk menumbuhkembangkan budaya literasi yang dilakukan melalui rapat kerja yang diikuti oleh seluruh elemen sekolah baik guru, kepala sekolah dan seluruh karyawan. Rapat kerja ini dilakukan penggerakan dan motoring dan evaluasi. Perencanaan GLS dilaksanakan dengan menetapkan tujuan GLS untuk menumbuhkembangkan budaya literasi yang dilakukan melalui rapat kerja yang diikuti oleh seluruh elemen sekolah baik guru, kepala sekolah dan seluruh karyawan. Rapat kerja ini dilakukan 3. Faktor Pendukung dan Kendala Faktor pendukung dan kendala dalam implementasi GLS di SDN 5 Menteng Palangka Raya adalah: (1) Faktor pendukung: (a) adanya upaya untuk mensosialisasikan dan mengembangkan program GLS dari sekolah. Upaya tersebut diantaranya melalui rapat kerja guru maupun pertemuan dengan orang tua siswa, (b) adanya alokasi waktu dari sekolah untuk melakukan literasi secara terjadwal, (c) semangat dan komitmen mengajar yang baik dari guru-guru dalam mengarahkan siswa untuk berliterasi, (d) tingginya minat siswa untuk berliterasi, (e) tersedianya koleksi buku bacaan, dan (f) lingkungan sekolah yang kaya akan literasi, seperti banyaknya poster bergambar, tulisan-tulisan indah, serta suasana yang rindang. Sedangkan kendala yang dihadapi dalam implementasi GLS di sekolah, meliputi: (a) guru masih belum memperoleh pendidikan dan pelatihan secara komprehensif tentang Gerakan Literasi Sekolah, sehingga masih bergantung pada arahan dari kepala sekolah, (b) buku bacaan bergambar yang kaya akan nilai-nilai masih sedikit jumlah koleksinya, sehingga siswa menjadi mudah bosan terhadap sumber bacaan, (c) kondisi buku bacaan yang kurang baik seperti robek dan dicoret siswa, (d) teknis pelaksanaan GLS masih monoton, dan (e) belum adanya evaluasi secara menyeluruh. HASIL DAN PEMBAHASAN 1. Tahapan Implementasi GLS Sedangkan pengawasan dilakukan dengan menentukan standar ketercapaian program GLS untuk menegukur sejauhmana keterlaksanaan dari program GLS dan bilamana perlu dilakukan upaya perbaikan agar implementasi GLS sesuai dengan yang direncanakan. Pengawasan terhadap implementasi GLS dilakukan oleh guru kelas melalui pengamatan langsung terhadap perubahan sikap dan perkembangan kemampuan literasi siswa, termasuk pula oleh kepala sekolah guna melihat kesesuaiaan hasil yang sudah dicapai dalam kegiatan literasi. HASIL DAN PEMBAHASAN 1. Tahapan Implementasi GLS 1. Tahapan Implementasi GLS Berdasarkan hasil penelitian diketahui bahwa implementasi GLS di SDN 5 Palangka Raya baru dilaksanakan pada tahap awal yaitu tahap pembiasaan, 59 Equity in Education Journal (EEJ), Vol. 1, No. 1, Oktober 2019 diawal semester. Rapat kerja berfungsi untuk menentukan program-program yang akan dilakukan dan mensosialisasikan apa saja peran dari masing-masing warga sekolah. Pengorganisasian GLS dilaksanakan dengan mengacu pada struktur organisasi sekolah yang sudah ada dalam menjalankan GLS. Pengorganasiaan dalam program GLS terstruktur dalam lingkup sekolah dari tingkat yang tertinggi yaitu kepala sekolah sebagai pengarah dan penanggungjawab program GLS secara menyeluruh, guru- guru kelas yang telah menerima arahan dari kepala sekolah untuk melakukan kegiatan literasi di ruang kelas, tenaga pustakawan, serta warga sekolah lainnya yang mendukung berjalannya literasi. Penggerakan GLS dilaksanakan dengan menciptakan hubungan individual yang baik yang melibatkan unsur pimpinan dan bawahan yaitu arahan kerja dari kepala sekolah dengan gaya kepemimpinan terbuka atas segala masukan dan saran yang disambut positif sehingga memotivasi guru dalam melakukan literasi agar tujuan GLS dapat tercapai. Sedangkan pengawasan dilakukan dengan menentukan standar ketercapaian program GLS untuk menegukur sejauhmana keterlaksanaan dari program GLS dan bilamana perlu dilakukan upaya perbaikan agar implementasi GLS sesuai dengan yang direncanakan. Pengawasan terhadap implementasi GLS dilakukan oleh guru kelas melalui pengamatan langsung terhadap perubahan sikap dan perkembangan kemampuan literasi siswa, termasuk pula oleh kepala sekolah guna melihat kesesuaiaan hasil yang sudah dicapai dalam kegiatan literasi. Hasil penelitian tentang mekanisme implementasi GLS di atas pengawasan sebagai upaya untuk mencapai tujuan sekolah. diawal semester. Rapat kerja berfungsi untuk menentukan program-program yang akan dilakukan dan mensosialisasikan apa saja peran dari masing-masing warga sekolah. Pengorganisasian GLS dilaksanakan dengan mengacu pada struktur organisasi sekolah yang sudah ada dalam menjalankan GLS. Pengorganasiaan dalam program GLS terstruktur dalam lingkup sekolah dari tingkat yang tertinggi yaitu kepala sekolah sebagai pengarah dan penanggungjawab program GLS secara menyeluruh, guru- guru kelas yang telah menerima arahan dari kepala sekolah untuk melakukan kegiatan literasi di ruang kelas, tenaga pustakawan, serta warga sekolah lainnya yang mendukung berjalannya literasi. Penggerakan GLS dilaksanakan dengan menciptakan hubungan individual yang baik yang melibatkan unsur pimpinan dan bawahan yaitu arahan kerja dari kepala sekolah dengan gaya kepemimpinan terbuka atas segala masukan dan saran yang disambut positif sehingga memotivasi guru dalam melakukan literasi agar tujuan GLS dapat tercapai. SIMPULAN Berdasarkan hasil penelitian dan pembahasan yang telah diuraikan sebelumnya, dapat dirumuskan kesimpulan sebagai berikut: Berdasarkan hasil penelitian dan pembahasan yang telah diuraikan sebelumnya, dapat dirumuskan kesimpulan sebagai berikut: Hasil penelitian tentang mekanisme implementasi GLS di atas sejalan dengan pendapat Hendrawati (2018) bahwa dalam melaksanakan program sekolah hendaknya dilaksanakan dengan menerapkan fungsi-fungsi prinsip dasar manajamen, yaitu perencanaan, pengorganisasian, penggerakan dan p g 1. Implementasi GLS di SDN 5 Menteng Palangka Raya baru dilaksanakan pada tahap awal yaitu tahap pembiasaan, hal ini mengingat bahwa program GLS di SDN 5 Menteng Palangka Raya belum 60 Equity in Education Journal (EEJ), Vol. 1, No. 1, Oktober 2019 lama berjalan, juga karena ditunjang oleh kemampuan sekolah dalam menyediakan sarana dan prasarana literasi yang hanya mencukupi untuk melakukan literasi pada tahapan pembiasaan. Tujuan GLS di SDN 5 Menteng Palanga Raya pada tahapan pembiasaan adalah untuk menumbuhkembangkan budaya berliterasi melalui berbagai aktivitas seperti membaca, menulis, bernyanyi, bercerita, menyimak, dan melihat, yang dilakukan selama 15 menit sebelum belajar setiap hari sekolah. Pada tahapan pembiasaan ini tidak ada tagihan wajib yang berpengaruh pada nilai akademik oleh guru kepada siswa yang telah melakukan kegiatan literasi. Literasi Sekolah, sehingga masih bergantung pada arahan dari kepala sekolah, (2) buku bacaan bergambar yang kaya akan nilai-nilai masih sedikit jumlah koleksinya, sehingga siswa menjadi mudah bosan terhadap sumber bacaan, (3) kondisi buku bacaan yang kurang baik seperti robek dan dicoret siswa, (4) teknis pelaksanaan GLS masih monoton, dan (5) belum adanya evaluasi secara menyeluruh. UCAPAN TERIMA KASIH Terima kasih yang sebesar- besarnya diucapkan kepada pihak SDN 5 Menteng yang telah memberikan ijin serta berbagai fasilitas sehingga penulis dapat menyelesaikan penelitian ini, serta pihak Redaksi Equity in Education Journal (EEJ) yang telah memberikan kesempatan artikel saya dapat dimuat dalam jurnal ini. y g g 2. Mekanisme Implementasi GLS di SDN 2. Mekanisme Implementasi GLS di SDN 5 Menteng Palangka Raya, dilaksanakan dengan mendasarkan pada prinsip dasar manajemen, yaitu perencanaan, pengroganisasian, penggerakan dan pengawasan. DAFTAR PUSTAKA Bogdan, R. C., & Biklen, S. K. (1992). Qualitative Research for Education, second edition. USA: Allyn and Bacon. p gg p g 3. Faktor pendukung dan kendala dalam implementasi GLS di SDN 5 Menteng Palangka Raya adalah: (a) Faktor pendukung: (1) adanya upaya untuk mensosialisasikan dan mengembangkan program GLS dari sekolah. Upaya tersebut diantaranya melalui rapat kerja guru maupun pertemuan dengan orang tua siswa, (2) adanya alokasi waktu dari sekolah untuk melakukan literasi secara terjadwal, (3) semangat dan komitmen mengajar yang baik dari guru-guru dalam mengarahkan siswa untuk berliterasi, (4) tingginya minat siswa untuk berliterasi, (5) tersedianya koleksi buku bacaan, dan (6) lingkungan sekolah yang kaya akan literasi, seperti banyaknya poster bergambar, tulisan-tulisan indah, serta suasana yang rindang. Sedangkan kendala yang dihadapi dalam implementasi GLS di sekolah, meliputi: (1) guru masih belum memperoleh pendidikan dan pelatihan secara komprehensif tentang Gerakan y Hendrawati, S. (2018). Fungsi-Fungsi Manaemen Sekolah. Diakses 19 Maret 2018, dari: https://www.academia.edu/11289 318/Fungsi_Manajemen_Sekolah. g _ j _ Kemendikbud. (2016). Desain Induk Gerakan Literasi Sekolah. Jakarta: Direktorat Jenderal Pendidikan Dasar dan Menengah Kementerian Pendidikan dan Kebudayaan Republik Indonesia. Peraturan Menteri Pendidikan dan Kebudayaan Nomor 23 Tahun 2015 tentang Penumbuhan Budi Pekerti. Rachmawati, F. (2008). Dunia di Balik Kata (Pintar Membaca). Yogyakarta: Citra Aji Parama. Ulfatin, N. (2014). Metode Penelitian Kualitatif di Bidang Pendidikan: Teori dan Aplikasinya. Malang: Bayumedia Publishing. y g Undang-Undang Dasar Tahun 1945. 61
https://openalex.org/W4386501899	https://ejtas.com/index.php/journal/article/download/264/222	English	null	Possibilistic Aggregate Production Planning Considering Dynamic Workforce with Fuzzy Demand	European journal of theoretical and applied sciences	2,023	cc-by	10,227	Abstract: Suggested Citation Chineh, U.O. (2023). Possibilistic Aggregate Production Planning Considering Dynamic Workforce with Fuzzy Demand. European Journal of Theoretical and Applied Sciences, 1(5), 129-147. DOI: 10.59324/ejtas.2023.1(5).10 This Aggregate Production Planning (APP) is a combination of Possibilistic Linear Programming (PLP), Fuzzy Goal Programming (FGP), and the Perfume Accounting System (PAS) to maximize profit. APP involves strategic decisions on Production levels, Inventory management, and Resource allocation to meet client demand while maximizing profit. Traditional planning models face significant challenges due to the uncertainties and complexities inherent in real world production environments. In this paper, there is an integration of PLP, Fuzzy Goal Programing, and throughput accounting system to overcome these challenges. At the very end of the paper, based on the data received from the company, the derived findings were by using Lingo Version 18 software. The model includes possibility distributions of the input parameters. Decision-makers can take into account the uncertainty and imprecision in demand forecasts and dynamic workforce in maximizing profit while taking into account risk tolerance. gg Chineh, U.O. (2023). Possibilistic Aggregate Production Planning Considering Dynamic Workforce with Fuzzy Demand. European Journal of Theoretical and Applied Sciences, 1(5), 129-147. DOI: 10.59324/ejtas.2023.1(5).10 Keywords: Aggregate production planning, fuzzy demands, capacity utilization, Decision maker, throughput accounting dynamics, businesses may effectively match labor resources with changing production demands. This work is licensed under a Creative Commons Attribution 4.0 International License. The license permits unrestricted use, distribution, and reproduction in any medium, on the condition that users give exact credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if they made any changes. Possibilistic Aggregate Production Planning Considering Dynamic Workforce with Fuzzy Demand Possibilistic Aggregate Production Planning Considering Dynamic Workforce with Fuzzy Demand UgoChukwu O. Chineh  Department of Mathematics/Statistics, Federal Polytechnic Oko, Anambra State, Nigeria UgoChukwu O. Chineh  Department of Mathematics/Statistics, Federal Polytechnic Oko, Anambra State, Nigeria Introduction Aggregate Production Planning (APP) is a crucial component of manufacturing and production management that entails choosing the best production levels, labor distribution, and inventory management to satisfy the uncertain demand while minimizing costs and maximizing efficiency. Fuzzy logic must be incorporated into the APP process when dealing with concerns about a dynamic workforce and unpredictable, imprecise demand. Adjusting the number of employees, their abilities, and shifts in response to shifting demand levels is known as dynamic workforce management. Traditional methods frequently take into account a static workforce, which results in ineffective resource use and potential labor shortages during peak demand. By taking into account workforce Review of Related Works Aggregate production planning (APP) is a medium-term production decision in a manufacturing organization that establishes the production rate, inventory level, amount of subcontractors, and workforce level in a particular time according to a number of constraints. "Aggregate" refers to the preparation done for two or more manufacturing categories. Determining output levels across all categories to meet current, specific demands is the goal of aggregate production planning. APP governs the best way to meet forecast demand in the intermediate future, often from 6 to 24 months ahead, by uncertain data and imprecise information. This enhances the reliability and effectiveness of decisions. For the planning process to fully address the dynamic nature of both labor and demand issues, further research is required. This work investigates the optimal profit level, using proper account system in the APP system under an imprecise demand and a dynamic workforce scenario using Possibilistic Programming model. adjusting regular and overtime production rates, inventory levels, labor levels, subcontracting and backordering rates, and other controllable variables (Wang et al., 2005). The primary inputs of APP are market demands and the manufacturing plan to meet those expectations. (Leung et al., 2003). The traditional models for APP often assume deterministic demand and a fixed workforce, which might not be realistic in today's volatile market conditions. For the purpose of production-distribution planning, Du, M., and Leung, S. Y. S. (2007) provide a fuzzy multi-objective model that takes into account workforce dynamics, unpredictable demand, and production capacity. The goal of the concept is to balance consumer happiness and production costs as efficiently as possible. In their study on the make-to-order sector, Yen, B. P. C., and Liu (2009) provide a fuzzy APP model that takes into account both imprecise demand and dynamic workforce modifications. The study shows how well fuzzy logic captures demand uncertainty and its influence on production choices. Fuzzy multi-objective linear programming approaches are introduced for issues involving aggregate production planning by Taleizadeh, A. A., and Pentico, D. W. (2012). In order to help decision-makers make sound and reliable planning decisions, the research tackles labor dynamics, production capacity, and ambiguous demand. A fuzzy multi-objective model that incorporates workforce allocation choices into the process of aggregate production planning is presented in a study by Guneri, A. F., and Olgun, H. (2018). Review of Related Works The research optimizes for both profit and customer pleasure while taking a variety of elements, such as labor prices, production rates, and ambiguous demand, into consideration. Method and Procedure The Financial Aspects of the Theory of Constraints The Financial Aspects of the Theory of Constraints The amount of money spent on a system to increase its capacity is known as investment, and throughput accounting lays a lot of attention on it. The following formulae are used by throughput accounting in conjunction with throughput, entirely variable costs, and operational expenditures for a variety of accounting decisions: 𝑇𝑇𝑅 • Throughput (𝑇𝑇𝑇𝑇) = Revenue (𝑅𝑅) − Totally Variable Expenses(TVE) 𝑇𝑇 • Net Profit(NP) = Throughput(𝑇𝑇𝑇𝑇) − Operating Expenses(OE) • Net Profit(NP) = Throughput(𝑇𝑇𝑇𝑇) − Operating Expenses(OE) • Return on Investment (RoI) = Net Profit / Investment • Return on Investment (RoI) = Net Profit / Investment • Productivity(Pr) = Throughput / Operational Expense • Inventory Turns(IT) = Throughput / Inventory Value = Throughput / Inventory Value Goldratt's strategy demanded a shift in emphasis in order to redefine accounting principles. The goal for managers should be to increase throughput while lowering operating costs and inventories. The latter two, however, must be maintained at a certain minimum level to prevent a decrease in throughput, thus there is very little room for reduction in those two areas. These might be viewed as restrictions on the suggested model. The weakest link in the system chain is a constraint (Dettemer, 1997). There are three different kinds of constraints: material, resource, and policy (paradigm) (Woeppel, 2001). Considering dynamic labor management with fuzzy demand modeling makes the already difficult process of aggregate production planning considerably more difficult. The literature study emphasizes the significance of including these components in planning models to improve alignment between production capabilities and changing consumer needs. In the context of planning collective production, fuzzy logic offers a useful tool for dealing with 130 2023 \| Volume 1 \| Number 5 2023 \| Volume 1 \| Number 5 www.ejtas.com www.ejtas.com EJTAS Moreover, financial limitations are crucial for real-world issues (Fung, et al., 2003). Moreover, financial limitations are crucial for real-world issues (Fung, et al., 2003). Moreover, financial limitations are crucial for real-world issues (Fung, et al., 2003). • The impreciseness and uncertainty of real-world problem and confliction of different objectives are modeled using fuzzy goals. Assumptions and Problem Definition Assumptions and Problem Definition • Linear membership functions are defined for fuzzy goals. Following the findings of a real-world case study, the following presumptions are made for the mathematical model of the suggested APP problem. 𝑡𝑇 Parameters, Indices, Decision Variables and Notations • Production planning is done in a time horizon of T time periods (∀ 𝑡𝑡= 1,2, …, 𝑇𝑇). 𝑁 Table 1. Set of indices 𝑇 Table 1. Set of indices 𝑡𝑡 Number of periods in the planning horizon; 𝑡𝑡= 1,2, … , 𝑇𝑇 𝑖𝑖 Number of product types; 𝑖𝑖= 1,2, … , 𝐼𝐼 𝑚𝑚 Raw material type; 𝑚𝑚= 1,2, … , 𝑀𝑀 𝑞𝑞 Types of shifts; 𝑞𝑞∈1,2 𝑤𝑤 Types of warehouse; 𝑤𝑤= 1,2, … , 𝑊𝑊 𝑘𝑘 Skill levels of workers; 𝑘𝑘= 1,2, … , 𝐾𝐾 𝑗𝑗 Number of objective Functions; 𝑗𝑗= 1,2,3 • There is a Batch production system capable of producing all kinds of 𝑁𝑁 types of products. 𝑡 • Market demand can be fulfilled or backordered, however no backorder in the last 𝑡𝑡 is allowed. • There are two working shifts; Regular time production and Over time production • There are two working shifts; Regular time production and Over time production Table 2. Notation for parameters Parameter Definition 𝑃𝑃𝑃𝑃𝑖𝑖 Fraction of the Product 𝑖𝑖 wasted during production in period 𝑡𝑡 𝐼𝐼𝐼𝐼𝑖𝑖 Fraction of the Product 𝑖𝑖 wasting in inventory in period 𝑡𝑡 𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖 Cost of Overhead(fixed) Production; for product 𝑖𝑖 in shift 𝑞𝑞 𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 Demand of product 𝑖𝑖 in period 𝑡𝑡 𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖 Cost of Backordering; for product 𝑖𝑖 in period 𝑡𝑡 𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖 Sales Revenue for product 𝑖𝑖 (₦/unit) 𝐸𝐸𝑡𝑡 cumulative investment in tools and equipment in period t (currency unit) 𝑃𝑃𝑃𝑃𝑃𝑃𝑡𝑡 Process time of product 𝑖𝑖 in period 𝑡𝑡 𝐵𝐵𝐵𝐵𝐵𝐵𝑡𝑡 The Budget upper limit in period 𝑡𝑡 AsP𝑖𝑖𝑖𝑖 Allowable shortage of product 𝑖𝑖 in period 𝑡𝑡 𝐴𝐴𝐴𝐴𝐴𝐴𝑡𝑡 Available Maximum workforce in period 𝑡𝑡 𝐴𝐴𝐴𝐴𝐴𝐴𝑡𝑡 Available Minimum workforce in period 𝑡𝑡 𝑊𝑊𝑊𝑊𝑊𝑊 workforce that are available for overtime (in percentage) 𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 Cost of workforce of level k in period 𝑡𝑡 𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 Cost of Hiring workforce of level k in period 𝑡𝑡 𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 Cost of firing workforce of level k in period 𝑡𝑡 𝐶𝐶𝐶𝐶𝐶𝐶𝑚𝑚𝑚𝑚𝑚𝑚 Cost for raw material type 𝑚𝑚 in period 𝑡𝑡 in warehouse 𝑤𝑤 Table 2. Notation for parameters 𝑃𝑃𝑖 • A warehouse is allowed for holding final products. • A warehouse is allowed for holding final products. Assumptions and Problem Definition • In advance, the holding cost of inventories are determined and well known. 𝑘𝑙𝑙𝑙𝑙𝑙𝑙 • The workforce accommodates various skill levels (𝑘𝑘−𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙). • Workers salary is independent of unit production cost. • At each period T, Production quantity is considered more of the safety stock for finished products. • Hiring and firing of Manpower based on product demand is eligible and there is an allowable limit. • In each period T, the shortage of production is recovered by overtime production in each shift. • In each period T, the nominal and actual capacity of production machines is not the same due to unforeseen failures. • If an unforeseen failure occurs during a shift the repair process is completed in the next. This may stop, reduce, or decrease the production rate during maintenance actions • If an unforeseen failure occurs during a shift the repair process is completed in the next. This may stop, reduce, or decrease the production rate during maintenance actions 131 EJTAS 2023 \| Volume 1 \| Number 5 www.ejtas.com www.ejtas.com EJTAS 𝐶𝐶𝐶𝐶𝐶𝐶𝑚𝑚𝑚𝑚𝑚𝑚 Holding cost for raw material type 𝑚𝑚 in period 𝑡𝑡 in warehouse 𝑤𝑤 𝐶𝐶𝐶𝐶ℎ𝑃𝑃𝑖𝑖𝑖𝑖𝑖𝑖 Holding cost of unit of product 𝑖𝑖 in period 𝑡𝑡 𝐹𝐹𝐹𝐹𝐹𝐹𝑡𝑡 fraction of the workforce variation in period 𝑡𝑡 𝑀𝑀𝑀𝑀𝑖𝑖𝑖𝑖 Machine hours needed to produce unit of product 𝑖𝑖 in period 𝑡𝑡 𝑀𝑀𝑀𝑀𝑀𝑀𝑡𝑡 Machine capacity that is lost due to interruption in period 𝑡𝑡 (in percentage) 𝑀𝑀𝑀𝑀𝑀𝑀𝑡𝑡 Machine capacity that is lost due to repairs in period 𝑡𝑡 (in percentage) 𝑀𝑀𝑀𝑀𝑀𝑀𝑞𝑞𝑞𝑞 The maximum of machine capacity that is available in shift 𝑞𝑞 in period 𝑡𝑡 𝑀𝑀𝑀𝑀𝑀𝑀 The machine capacity that is available for overtime (in percentage) 𝐴𝐴𝐴𝐴𝐴𝐴𝑖𝑖𝑖𝑖 Available Regular time in both shifts in period 𝑡𝑡 𝑢𝑢𝑢𝑢𝑢𝑢𝑖𝑖𝑖𝑖 The units of type 𝑚𝑚 raw material required to produce unit of product 𝑖𝑖 𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖 product 𝑖𝑖 safety stock 𝑆𝑆𝑆𝑆𝑆𝑆𝑚𝑚 Raw material type 𝑚𝑚 safety stock 𝑀𝑀𝑆𝑆𝑆𝑆𝑚𝑚 The maximum available space of warehouse w 𝑊𝑊ℎ𝐶𝐶𝐶𝐶𝑤𝑤𝑤𝑤𝑤𝑤 The capacity of warehouse 𝑤𝑤 for storage of raw-material type 𝑚𝑚 in period 𝑡𝑡 𝑊𝑊ℎ𝐶𝐶𝐶𝐶𝑤𝑤𝑤𝑤𝑤𝑤 The capacity of warehouse 𝑤𝑤 for storage of finished-product 𝑖𝑖 in period 𝑡𝑡 𝒟𝒟𝒟𝒟𝑖𝑖 The Due date of product 𝑖𝑖 ℬ𝑖𝑖 Batch size of product 𝑖𝑖 𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖 Finished product 𝑖𝑖 Defect rate 𝑃𝑃𝑃𝑃 Production Capacity Table 3. Decision variable Notation Decision variable Definition 𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 Number of product i produced in shift q of period t 𝑋𝑋𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 Number batches of product i produced in shift q of period t 𝐵𝐵𝑖𝑖𝑖𝑖 Backorder level of product i in period t 𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 Number of available workers of level k in period t 𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 Number of hired workers of level k in period t 𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 Number of fired workers of level k in period t 𝑋𝑋𝑋𝑋𝑚𝑚𝑚𝑚𝑚𝑚 Inventory level of raw material type m at the end of period t in warehouse w 𝑋𝑋𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 Inventory level of finished-product i in period t in warehouse w Table 3. Decision variable Notation Model Formulation Total income less the cost of the materials you purchased is your throughput (TP). Equation (1) can be used to numerically express the throughput (TP). 𝑇𝑇𝑇𝑇= ෍෍𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 −෍෍𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖𝐵𝐵𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 −෍෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑚𝑚𝑚𝑚𝑚𝑚𝑋𝑋𝑖𝑖𝑖𝑖𝑡𝑡 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝑀𝑀 𝑚𝑚=1 (1) 𝑇𝑇𝑇𝑇= ෍෍𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 −෍෍𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖𝐵𝐵𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 −෍෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑚𝑚𝑚𝑚𝑚𝑚𝑋𝑋𝑖𝑖𝑖𝑖𝑡𝑡 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝑀𝑀 𝑚𝑚=1 (1) (1) The definition of inventory is "the entire money the system invests," which includes the money spent on all assets (such as structures, machinery, and fixtures) as well as on raw materials and parts (Woeppel, 2001). TOC inventory differs from traditional inventory in that it encompasses all assets in addition to raw materials, work-in- progress, and finished goods. The first two terms reflect total sales revenue based on total demands and lost sales at the conclusion of the planned horizon. The last term denotes the cost of materials, which includes the cost of materials needed for both regular and overtime production. 𝐼𝐼𝐼𝐼= 1 𝑇𝑇൥෍෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑚𝑚𝑚𝑚𝑚𝑚𝑋𝑋𝑋𝑋𝑚𝑚𝑚𝑚𝑚𝑚 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝑀𝑀 𝑚𝑚=1 + ෍𝐸𝐸𝑡𝑡 𝑇𝑇 𝑡𝑡=1 ൩ (2) 132 www.ejtas.com EJTAS 2023 \| Volume 1 \| Number 5 132 2023 \| Volume 1 \| Number 5 The APP can take assets like small machines, material handling equipment and other instruments into account. Strategic or long-range planning takes into account structures and huge machineries. The capacity of a machine or system can be increased and a bottleneck reduced by investing in tools and equipment. Equation (2) represents the typical inventory investment (IN) in terms of TOC. Whereas the second term indicates the investment in tools and equipment, the first term represents the investment in raw materials. OE = ෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍𝐶𝐶𝑜𝑜𝑜𝑜𝑘𝑘𝑘𝑘𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍෍𝐶𝐶𝐶𝐶ℎ𝑃𝑃𝑖𝑖𝑖𝑖𝑖𝑖𝑋𝑋𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝐼𝐼 𝑖𝑖=1 + ෍෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑚𝑚𝑚𝑚𝑚𝑚𝑋𝑋𝑋𝑋𝑚𝑚𝑚𝑚𝑚𝑚 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝑀𝑀 𝑚𝑚=1 + ෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖𝐵𝐵𝑙𝑙𝑙𝑙 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 + ෍෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑞𝑞∈{1,2} 𝐼𝐼 𝑖𝑖=1 (𝟑𝟑) (𝟑𝟑) function. The profit function is the most desirable of these objective functions (Phruksaphanrat et al., 2006). Thus, the objective function of the suggested APP model is the Net Profit (NP). Throughput (TP) minus Operational expenses(OE) equals Net Profit (NP). The profit function includes two TOC metrics. Inventory is the final factor, which should also be taken into account. Model Formulation According to TOC, inventory refers to all financial investments made by the system, including those made in tools and equipment. It is incorporated into the model as constraints. The entire amount of money needed to convert inventory into throughput is referred to as operating expenses. All direct and indirect payroll expenses, purchases, overhead and time related expenses are involved. Equation (3) represents operating expense (OE). It covers all labour, overtime, holding expenses for inventory, backordering and fixed overhead costs. Aggregate Production Planning Considering Throughput Accounting Aggregate Often, the objective function of APP problems is chosen to be the revenue, cost, or profit Maximize Net Profit (NP) 𝑍𝑍= ෍෍𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 −෍෍𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖𝐵𝐵𝑖𝑖𝑡𝑡 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 −෍෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑚𝑚𝑚𝑚𝑚𝑚𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝑀𝑀 𝑚𝑚=1 −൥෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍෍𝐶𝐶𝐶𝐶ℎ𝑃𝑃𝑖𝑖𝑖𝑖𝑖𝑖𝑋𝑋𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝐼𝐼 𝑖𝑖=1 + ෍෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑚𝑚𝑚𝑚𝑚𝑚𝑋𝑋𝑋𝑋𝑚𝑚𝑚𝑚𝑚𝑚 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝑀𝑀 𝑚𝑚=1 + ෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖𝐵𝐵𝑙𝑙𝑙𝑙 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 + ෍෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑞𝑞∈{1,2} 𝐼𝐼 𝑖𝑖=1 ቏ (4) Maximize Net Profit (NP) 𝑍𝑍= ෍෍𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 −෍෍𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖𝐵𝐵𝑖𝑖𝑡𝑡 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 −෍෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑚𝑚𝑚𝑚𝑚𝑚𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝑀𝑀 𝑚𝑚=1 −൥෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍෍𝐶𝐶𝐶𝐶ℎ𝑃𝑃𝑖𝑖𝑖𝑖𝑖𝑖𝑋𝑋𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝐼𝐼 𝑖𝑖=1 + ෍෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑚𝑚𝑚𝑚𝑚𝑚𝑋𝑋𝑋𝑋𝑚𝑚𝑚𝑚𝑚𝑚 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝑀𝑀 𝑚𝑚=1 + ෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖𝐵𝐵𝑙𝑙𝑙𝑙 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 + ෍෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑞𝑞∈{1,2} 𝐼𝐼 𝑖𝑖=1 ቏ (4) Maximize Net Profit (NP) (4) Constraints Constraints Constraints The Labor-force Constraints are considered as follows: 𝑋𝑋𝐾𝐴𝐴𝐴𝑡 The Labor-force Constraints are considered as follows: 𝑋𝑋𝐾𝐴𝐴𝐴𝑡 ෍𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝐾𝐾 𝑘𝑘=1 ≤𝐴𝐴𝐴𝐴𝐴𝐴𝑡𝑡 , ∀𝑡𝑡 (5) ෍𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝐾𝐾 𝑘𝑘=1 ≤𝐴𝐴𝐴𝐴𝐴𝐴𝑡𝑡 , ∀𝑡𝑡 (5) (5) 133 2023 \| Volume 1 \| Number 5 2023 \| Volume 1 \| Number 5 www.ejtas.com EJTAS ෍𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝐾𝐾 𝑘𝑘=1 ≥𝐴𝐴𝐴𝐴𝐴𝐴𝑡𝑡 , ∀𝑡𝑡 (6) 𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘= 𝑋𝑋𝑋𝑋𝑘𝑘(𝑡𝑡−1) + 𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘−𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 , ∀𝑘𝑘, ∀𝑡𝑡, 𝑡𝑡> 1 (7) 𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘−𝑋𝑋𝑋𝑋𝑘𝑘(𝑡𝑡−1) ≤𝐹𝐹𝐹𝐹𝐹𝐹𝑡𝑡∗𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 , ∀𝑘𝑘, ∀𝑡𝑡, 𝑡𝑡> 1 (8) ෍𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝐾𝐾 𝑘𝑘=1 ≥𝐴𝐴𝐴𝐴𝐴𝐴𝑡𝑡 , ∀𝑡𝑡 (6) 𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘= 𝑋𝑋𝑋𝑋𝑘𝑘(𝑡𝑡−1) + 𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘−𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 , ∀𝑘𝑘, ∀𝑡𝑡, 𝑡𝑡> 1 (7) 𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘−𝑋𝑋𝑋𝑋𝑘𝑘(𝑡𝑡−1) ≤𝐹𝐹𝐹𝐹𝐹𝐹𝑡𝑡∗𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 , ∀𝑘𝑘, ∀𝑡𝑡, 𝑡𝑡> 1 (8) (6) (8) given period is equal to the workforce with the same skill level k during the previous period plus the change in workforce level during the current period. Model Formulation The change in workforce level in each planning period cannot be greater than a benchmark number of workers in the present period, according to constraint number seven. Constraints (5) attests that the total labor utilized during period t does not exceed the total workforce that is available. In a similar vein, (6) guarantees that in period t, the employed workforce exceeds the available minimum workforce. Set of Constraints (7) is a workforce level balance equation that assures that the workforce with skill level k available during a Time Constraints𝐼𝐾 ෍𝑃𝑃𝑃𝑃𝑃𝑃𝑖𝑖𝑖𝑖∗ 𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝐼𝐼 𝑖𝑖=1 ≤෍𝐴𝐴𝐴𝐴𝐴𝐴𝑞𝑞𝑞𝑞∗ 𝑋𝑋𝑋𝑋𝐾𝐾𝐾𝐾 𝐾𝐾 𝑘𝑘=1 , ∀𝑡𝑡, 𝑞𝑞= 1 (9) ෍𝑃𝑃𝑃𝑃𝑃𝑃𝑖𝑖𝑖𝑖∗ 𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝐼𝐼 𝑖𝑖=1 ≤෍𝐴𝐴𝐴𝐴𝐴𝐴𝑞𝑞𝑞𝑞∗𝑊𝑊𝑊𝑊𝑊𝑊∗ 𝑋𝑋𝑋𝑋𝐾𝐾𝐾𝐾 𝐾𝐾 𝑘𝑘=1 , ∀𝑡𝑡, 𝑞𝑞= 2 (10) ෍𝑃𝑃𝑃𝑃𝑃𝑃𝑖𝑖𝑖𝑖∗ 𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝐼𝐼 𝑖𝑖=1 ≤෍𝐴𝐴𝐴𝐴𝐴𝐴𝑞𝑞𝑞𝑞∗ 𝑋𝑋𝑋𝑋𝐾𝐾𝐾𝐾 𝐾𝐾 𝑘𝑘=1 , ∀𝑡𝑡, 𝑞𝑞= 1 (9) ෍𝑃𝑃𝑃𝑃𝑃𝑃𝑖𝑖𝑖𝑖∗ 𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝐼𝐼 𝑖𝑖=1 ≤෍𝐴𝐴𝐴𝐴𝐴𝐴𝑞𝑞𝑞𝑞∗𝑊𝑊𝑊𝑊𝑊𝑊∗ 𝑋𝑋𝑋𝑋𝐾𝐾𝐾𝐾 𝐾𝐾 𝑘𝑘=1 , ∀𝑡𝑡, 𝑞𝑞= 2 (10) ෍𝑃𝑃𝑃𝑃𝑃𝑃𝑖𝑖𝑖𝑖∗ 𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝐼𝐼 𝑖𝑖=1 ≤෍𝐴𝐴𝐴𝐴𝐴𝐴𝑞𝑞𝑞𝑞∗ 𝑋𝑋𝑋𝑋𝐾𝐾𝐾𝐾 𝐾𝐾 𝑘𝑘=1 , ෍𝑃𝑃𝑃𝑃𝑃𝑃𝑖𝑖𝑖𝑖∗ 𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝐼𝐼 𝑖𝑖=1 ≤෍𝐴𝐴𝐴𝐴𝐴𝐴𝑞𝑞𝑞𝑞∗𝑊𝑊𝑊𝑊𝑊𝑊∗ 𝑋𝑋𝑋𝑋𝐾𝐾𝐾𝐾 𝐾𝐾 𝑘𝑘=1 , (9) ∀𝑡𝑡, 𝑞𝑞= 2 (10) (10) The relationships mentioned above make sure hat each working shift's necessary production time is less than or equal to the available regular production time and overtime. time is less than or equal to the available regular production time and overtime. time is less than or equal to the available regular production time and overtime. The relationships mentioned above make sure that each working shift's necessary production time is less than or equal to the available regular production time and overtime. The relationships mentioned above make sure that each working shift's necessary production Inventory Constraints 𝑋𝑋𝑖𝑖𝑖𝑋𝑋𝑖𝑖𝑡 𝑋𝑋𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖= 𝑋𝑋𝑋𝑋𝑖𝑖𝑖𝑖(𝑡𝑡−1) + ෍𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑞𝑞∈{1,2} −𝐵𝐵𝑖𝑖𝑖𝑖−𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 , ∀𝑖𝑖, ∀𝑤𝑤, 𝑡𝑡> 1 (11) 𝑋𝑋𝑋𝑋𝑚𝑚𝑚𝑚𝑚𝑚= 𝑋𝑋𝑋𝑋𝑚𝑚𝑚𝑚(𝑡𝑡−1) + ෍𝑋𝑋𝑖𝑖𝑖𝑖(𝑡𝑡−1) 𝑞𝑞∈{1,2} −𝑢𝑢𝑢𝑢𝑢𝑢𝑖𝑖𝑖𝑖, ∀𝑖𝑖, ∀𝑤𝑤, 𝑡𝑡> 1 (12) 𝑆𝑆𝑆𝑆𝑆𝑆𝑚𝑚≤෍𝑋𝑋𝑋𝑋𝑚𝑚𝑚𝑚𝑚𝑚 𝑤𝑤∈𝑊𝑊 , ∀𝑚𝑚, ∀𝑡𝑡, (13) (11) (12) (13) and the quantity of produced finished goods type I in period t in both working shifts. A set of limitations (12) assures that there is a balance between raw materials, and (13) guarantees that the safety stock of raw materials in warehouses is satisfied. Constraints (11) ensures that the amount of finished product type 𝐼𝐼 in period 𝑡𝑡 in warehouse 𝑤𝑤 is equal to the amount of finished product type 𝐼𝐼 in period 𝑡𝑡−1 in warehouse w plus the quantity of produced finished goods type I in period t in both working shifts, less the amount of product type 𝐼𝐼 in period 𝑡𝑡 that is on backorder 134 www.ejtas.com EJTAS 2023 \| Volume 1 \| Number 5 134 2023 \| Volume 1 \| Number 5 2023 \| Volume 1 \| Number 5 EJTAS Production Constraint 𝑆𝑆𝑆𝑋𝑖𝑡 Production Constraint 𝑆𝑆𝑆𝑋𝑖𝑡 𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖≤෍𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑞𝑞∈{1,2} , ∀𝑖𝑖, ∀𝑡𝑡, (14) 𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖≤൬1 −𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖 𝛽𝛽𝑖𝑖 ൰∗෍𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑞𝑞∈{1,2} + 𝑋𝑋𝑋𝑋𝑖𝑖(𝑡𝑡−1), ∀𝑖𝑖, ∀𝑡𝑡, 𝑡𝑡> 1 (15) 𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖≤෍𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑞𝑞∈{1,2} , ∀𝑖𝑖, ∀𝑡𝑡, 𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖≤൬1 −𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖 𝛽𝛽𝑖𝑖 ൰∗෍𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑞𝑞∈{1,2} + 𝑋𝑋𝑋𝑋𝑖𝑖(𝑡𝑡−1), ∀𝑖𝑖, ∀𝑡𝑡, 𝑡𝑡> 1 (14) (15) of non-defected products plus the inventory of finished-product in previous period should be greater than or equal to demand of the finished- product in current period. Set of constraints (14), which is written for all product types and all periods of planning, guarantee the satisfaction of safety stock of finished-products in working shifts. Set of constraints (15) represents the total production Machine capacity Constraints 𝑀𝑀𝑋𝐼𝑀𝑚𝑚 ෍𝑀𝑀𝑀𝑀𝑖𝑖𝑖𝑖∗ 𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝐼𝐼 𝑖𝑖=1 ≤𝑀𝑀𝑚𝑚𝑚𝑚𝑞𝑞𝑞𝑞−𝑀𝑀𝑀𝑀𝑀𝑀𝑡𝑡∗𝑀𝑀𝑚𝑚𝑚𝑚𝑞𝑞𝑞𝑞, ∀𝑡𝑡, 𝑞𝑞= 1 (16) ෍𝑀𝑀𝑀𝑀𝑖𝑖𝑖𝑖∗ 𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝐼𝐼 𝑖𝑖=1 ≤𝑀𝑀𝑀𝑀𝑀𝑀∗𝑀𝑀𝑚𝑚𝑚𝑚𝑞𝑞𝑞𝑞−𝑀𝑀𝑀𝑀𝑀𝑀𝑡𝑡∗𝑀𝑀𝑀𝑀𝑀𝑀∗𝑀𝑀𝑚𝑚𝑚𝑚𝑞𝑞𝑞𝑞, ∀𝑡𝑡, 𝑞𝑞= 2 (17) 𝑀𝑀𝑀𝑀𝑖𝑖𝑖𝑖∗ 𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖𝐼𝑖≤𝑀𝑀𝑚𝑚𝑚𝑚𝑞𝑞𝑞𝑞−𝑀𝑀𝑀𝑀𝑀𝑀𝑡𝑡∗𝑀𝑀𝑚𝑚𝑚𝑚𝑞𝑞𝑞𝑞, ∀𝑡𝑡, 𝑞𝑞= 1 (16) 𝑀𝑀𝑋𝐼𝑀𝑀𝑀𝑀𝑚𝑚𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑚𝑚𝑡𝑞 (16) (17) Constraints (16) and (17) pledge that in regular time and overtime, the machine capacity is assured. ts (16) and (17) pledge that in regular overtime, the machine capacity is Warehouse Capacity Constraint 𝑋𝑋𝑊𝑊𝑐𝑐𝑊𝑖 ෍𝑋𝑋𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑊𝑊 𝑤𝑤=1 ≤෍𝑊𝑊ℎ𝑐𝑐𝑐𝑐𝑤𝑤𝑤𝑤𝑤𝑤 𝑊𝑊 𝑤𝑤=1 , ∀𝑖𝑖, ∀𝑡𝑡, (18) ෍෍𝑋𝑋𝑋𝑋𝑚𝑚𝑚𝑚𝑚𝑚 𝑊𝑊 𝑤𝑤=1 𝑀𝑀 𝑚𝑚=1. ≤෍෍𝑊𝑊ℎ𝑐𝑐𝑐𝑐𝑚𝑚𝑚𝑚𝑚𝑚 𝑀𝑀 𝑚𝑚=1 𝑊𝑊 𝑤𝑤=1 , ∀𝑡𝑡, (19) ෍𝑊𝑊ℎ𝑐𝑐𝑐𝑐𝑤𝑤𝑤𝑤𝑤𝑤 𝑊𝑊 𝑤𝑤=1 + ෍𝑊𝑊ℎ𝑐𝑐𝑐𝑐𝑚𝑚𝑚𝑚𝑚𝑚 𝑊𝑊 𝑤𝑤=1 ≤𝑀𝑀𝑆𝑆𝑆𝑆ℎ𝑚𝑚, ∀𝑖𝑖, ∀𝑡𝑡, (20) ෍𝑋𝑋𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑊𝑊 𝑤𝑤=1 ≤෍𝑊𝑊ℎ𝑐𝑐𝑐𝑐𝑤𝑤𝑤𝑤𝑤𝑤 𝑊𝑊 𝑤𝑤=1 , ∀𝑖𝑖, ∀𝑡𝑡, (18) ෍෍𝑋𝑋𝑋𝑋𝑚𝑚𝑚𝑚𝑚𝑚 𝑊𝑊 𝑤𝑤=1 𝑀𝑀 𝑚𝑚=1. ≤෍෍𝑊𝑊ℎ𝑐𝑐𝑐𝑐𝑚𝑚𝑚𝑚𝑚𝑚 𝑀𝑀 𝑚𝑚=1 𝑊𝑊 𝑤𝑤=1 , ∀𝑡𝑡, (19) ෍𝑊𝑊ℎ𝑐𝑐𝑐𝑐𝑤𝑤𝑤𝑤𝑤𝑤 𝑊𝑊 𝑤𝑤=1 + ෍𝑊𝑊ℎ𝑐𝑐𝑐𝑐𝑚𝑚𝑚𝑚𝑚𝑚 𝑊𝑊 𝑤𝑤=1 ≤𝑀𝑀𝑆𝑆𝑆𝑆ℎ𝑚𝑚, ∀𝑖𝑖, ∀𝑡𝑡, (20) (18) (19) (20) products an raw materials beyond its maximum warehouse available space. Backorder, Budget limit and Non-negativity Constraints There is backorder obeying the following; products an raw materials beyond its maximum warehouse available space. products an raw materials beyond its maximum warehouse available space. The first two constraints (18) and (19) gives the restrictions of actual inventories of finished products and raw materials. While (20) guarantees that each warehouse at each period will not be able to allow storage capacity of There is backorder obeying the following; ෍𝐵𝐵𝑖𝑖𝑖𝑖 𝑊𝑊 𝑤𝑤=1 ≤෍𝐴𝐴𝐴𝐴𝐴𝐴𝑖𝑖𝑖𝑖 𝑊𝑊 𝑤𝑤=1 ∗𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 ∀𝑖𝑖, 𝑡𝑡≠𝑇𝑇 𝐵𝐵𝑖𝑖𝑖𝑖= 0, ∀𝑖𝑖 ෍𝐵𝐵𝑖𝑖𝑖𝑖 𝑊𝑊 𝑤𝑤=1 ≤෍𝐴𝐴𝐴𝐴𝐴𝐴𝑖𝑖𝑖𝑖 𝑊𝑊 𝑤𝑤=1 ∗𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 ∀𝑖𝑖, 𝑡𝑡≠𝑇𝑇 (21) 𝐵𝐵𝑖𝑖𝑖𝑖= 0, ∀𝑖𝑖 (22) ( ) (22) (22) 135 2023 \| Volume 1 \| Number 5 www.ejtas.com EJTAS 𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 ≤෍𝐵𝐵𝐵𝐵𝐵𝐵𝑡𝑡 𝑇𝑇 𝑡𝑡=1 (23) 𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖, 𝑋𝑋𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖, 𝐵𝐵𝑖𝑖𝑖𝑖,𝑋𝑋𝑋𝑋𝑚𝑚𝑚𝑚𝑚𝑚, 𝑋𝑋𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖≥0, ∀𝑖𝑖, ∀𝑞𝑞, ∀𝑡𝑡, ∀𝑚𝑚, ∀𝑤𝑤 (24) 𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘, 𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘, 𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 ≥0, ∀𝑡𝑡, ∀𝑘𝑘, ∀𝑙𝑙 (25) 𝐷𝐷𝐷𝐷𝑜𝑃𝐷𝐷𝐷𝐷 (23) of Demand is 𝐷𝐷𝐷𝐷𝐷𝐷𝚤𝚤𝚤𝚤 ෫= (𝐷𝐷𝑜𝑜𝑃𝑃𝑖𝑖𝑖𝑖 1, 𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 2, 𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 3), in which 𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 2 is the most possible demand that certainly belongs to the set of available values (with a membership value of 1 after it is normalized), see figure 1 below. The lower bound value 𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 1 is the most pessimistic demand that has a small likelihood to belong to the set of available values (with a membership value of zero if normalized) and the upper bound value 𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 3 is the most optimistic demand with a small likelihood to belong to the set of available values (with a membership value of zero if normalized). 𝜇𝐷𝐷𝐷𝚤𝚤 Constraints (21) represent the backorder level at the end of period t cannot exceed the certain percent-age of the demand which determines the upper limit of shortage. While (22) assure that there is no possibility for backordering at the end of time horizon or last period. A restriction on the available budget for each planning period is shown using (23), which ensures that the Total Cost (i.e., Eq. Warehouse Capacity Constraint 𝑋𝑋𝑊𝑊𝑐𝑐𝑊𝑖 (1)) cannot go beyond the predetermined budget for the time horizon. (24) and (25) both present non-negativity requirements on decision variables. Let 𝜇𝜇(𝐷𝐷𝐷𝐷𝐷𝐷𝚤𝚤𝚤𝚤 ෫) represent the arbitrary measurement of fuzzy demand in view of the Decision-maker, i.e. membership function, that defines the degree of 𝑥𝑥 in the fuzzy space 𝐷𝐷𝐷𝐷𝐷𝐷𝚤𝚤𝚤𝚤 ෫ and figure 1 depicts the relationships of this function. Formation of Fuzzy Demand Fuzzy numbers like triangular and trapezoidal fuzzy numbers, can be used to represent demand in order to reflect this informational ambiguity. TFNs are used in this study to represent demand-related fuzzy data. Assuming the TFN Figure 1. A Triangular Distribution of the Fuzzy Demand Figure 1. A Triangular Distribution of the Fuzzy Demand As seen in Figure 1 the membership function of fuzzy demand may be expressed as follows: 𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝐷𝐷𝐷𝑖𝑖𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷 As seen in Figure 1 the membership function of fuzzy demand may be expressed as follows: 𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷 𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖≤𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 1 𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 1 ≤𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖≤𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 2 𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 2 ≤𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖≤𝐷𝐷𝑜𝑜𝑜𝑜𝑖𝑖𝑖𝑖 3 𝑂𝑂𝑂𝑂ℎ𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝜇𝜇(𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖) = ⎩⎪⎨ ⎪⎧ 0 (𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖−𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 1) (𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 2 −𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 1) ⁄ (𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 2 −𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖) (𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 3 −𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 2) ⁄ 1 (26) 1 136 www.ejtas.com EJTAS information, the constraint equations (11), (15) and (21) will be replaced with the following equations (27 to 29): 𝑖𝑤𝑡 Supposing the decision-maker desires that APP meets the market demand for product 𝑖𝑖 in period 𝑡𝑡 with a possibility level. Formation of Fuzzy Demand Using the fuzzy demand 𝑋𝑋𝑖𝑖𝑖𝑋𝑋𝑖𝑖𝑡𝑋𝑖𝑖𝑖𝐵𝑖𝑖𝐷𝐷𝐷𝚤𝚤 𝑖𝑖𝑖𝑖= 𝑋𝑋𝑋𝑋𝑖𝑖𝑖𝑖(𝑡𝑡−1) + ෍𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑞𝑞∈{1,2} −𝐵𝐵𝑖𝑖𝑖𝑖−𝐷𝐷𝐷𝐷𝐷𝐷𝚤𝚤𝚤𝚤 ෫ , ∀𝑖𝑖, ∀𝑤𝑤, 𝑡𝑡> 1 (27) 𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝛽𝑋𝑋𝑋𝑖𝑡𝑡 (27) 𝑋𝑋𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖= 𝑋𝑋𝑋𝑋𝑖𝑖𝑖𝑖(𝑡𝑡−1) + ෍𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑞𝑞∈{1,2} −𝐵𝐵𝑖𝑖𝑖𝑖−𝐷𝐷𝐷𝐷𝐷𝐷𝚤𝚤𝚤𝚤 , ∀𝑖𝑖, ∀𝑤𝑤, 𝑡𝑡> 1 (27) 𝐷𝐷𝐷𝐷𝐷𝐷𝚤𝚤𝚤𝚤 ෫≤൬1 −𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖 𝛽𝛽𝑖𝑖 ൰∗෍𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑞𝑞∈{1,2} + 𝑋𝑋𝑋𝑋𝑖𝑖(𝑡𝑡−1), ∀𝑖𝑖, ∀𝑡𝑡, 𝑡𝑡> 1 (28) ෍𝐵𝐵𝑖𝑖𝑖𝑖 𝑊𝑊 𝑤𝑤=1 ≤෍𝐴𝐴𝐴𝐴𝐴𝐴𝑖𝑖𝑖𝑖 𝑊𝑊 𝑤𝑤=1 ∗𝐷𝐷𝐷𝐷𝐷𝐷𝚤𝚤𝚤𝚤 ෫ ∀𝑖𝑖, 𝑡𝑡≠𝑇𝑇 (29) 𝐷𝐷𝐷𝐷𝐷𝐷𝚤𝚤𝚤𝚤 ෫≤൬1 −𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖 𝛽𝛽𝑖𝑖 ൰∗෍𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑞𝑞∈{1,2} + 𝑋𝑋𝑋𝑋𝑖𝑖(𝑡𝑡−1), ∀𝑖𝑖, ∀𝑡𝑡, 𝑡𝑡> 1 (28) ෍𝐵𝐵𝑖𝑖𝑖𝑖 𝑊𝑊 𝑤𝑤=1 ≤෍𝐴𝐴𝐴𝐴𝐴𝐴𝑖𝑖𝑖𝑖 𝑊𝑊 𝑤𝑤=1 ∗𝐷𝐷𝐷𝐷𝐷𝐷𝚤𝚤𝚤𝚤 ෫ ∀𝑖𝑖, 𝑡𝑡≠𝑇𝑇 (29) 𝐷𝐷𝐷𝐷𝐷𝐷𝚤𝚤𝚤𝚤 ෫≤൬1 −𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖 𝛽𝛽𝑖𝑖 ൰∗෍𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑞𝑞∈{1,2} + 𝑋𝑋𝑋𝑋𝑖𝑖(𝑡𝑡−1), ∀𝑖𝑖, ∀𝑡𝑡, 𝑡𝑡> 1 (28) 𝐵𝑊𝐴𝐴𝐴𝑊𝐷𝐷𝐷𝑖𝑡𝑇 (28) ෍𝐵𝐵𝑖𝑖𝑖𝑖 𝑊𝑊 𝑤𝑤=1 ≤෍𝐴𝐴𝐴𝐴𝐴𝐴𝑖𝑖𝑖𝑖 𝑊𝑊 𝑤𝑤=1 ∗𝐷𝐷𝐷𝐷𝐷𝐷𝚤𝚤𝚤𝚤 ෫ ∀𝑖𝑖, 𝑡𝑡≠𝑇𝑇 (29) (29) Based on the ranking method developed by im'enez(1996), all fuzzy (imprecise) demand constraints in the model are translated to their corresponding crisp constraints as follows: 𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷 Based on the ranking method developed by Jim'enez(1996), all fuzzy (imprecise) demand Based on the ranking method developed by Jim'enez(1996), all fuzzy (imprecise) demand constraints in the model are translated to their corresponding crisp constraints as follows: 𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷 𝑋𝑋𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖= 𝑋𝑋𝑋𝑋𝑖𝑖𝑖𝑖(𝑡𝑡−1) + ෍𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑞𝑞∈{1,2} −𝐵𝐵𝑖𝑖𝑖𝑖−ቆ𝛼𝛼𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 1 + 𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 2 2 + (1 −𝛼𝛼) 𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 2 + 𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 3 2 ቇ, ∀𝑖𝑖, 𝑤𝑤, 𝑡𝑡> 1 (30) ቆ𝛼𝛼𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 1 + 𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 2 2 + (1 −𝛼𝛼) 𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 2 + 𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 3 2 ቇ≤൬1 −𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖 𝛽𝛽𝑖𝑖 ൰∗෍𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑞𝑞∈{1,2} + 𝑋𝑋𝑋𝑋𝑖𝑖(𝑡𝑡−1), ∀𝑖𝑖, 𝑡𝑡, 𝑡𝑡> 1 (31) ෍𝐵𝐵𝑖𝑖𝑖𝑖 𝑊𝑊 𝑤𝑤=1 ≤෍𝐴𝐴𝐴𝐴𝐴𝐴𝑖𝑖𝑖𝑖 𝑊𝑊 𝑤𝑤=1 ∗ቆ𝛼𝛼𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 1 + 𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 2 2 + (1 −𝛼𝛼) 𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 2 + 𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 3 2 ቇ ∀𝑖𝑖, 𝑡𝑡≠𝑇𝑇 (32) (32) The feasibility degree of the constraints that has been assigned by the DM based on the risk that he accepts on the violation of constraints imposed by the gotten solution is given by various values of 𝛼𝛼. The feasibility degree of the constraints that has been assigned by the DM based on the risk that he accepts on the violation of constraints imposed by the gotten solution is given by various values of 𝛼𝛼. satisfy market demand. On a medium time horizon, the environmental coefficients and associated parameters are often uncertain. As a result, across the planning horizon, related operational expenses and labor are imprecise. When dealing with such ambiguous APP decision problems, assigning a set of precise values for the environmental coefficients and associated parameters is problematic. The Net Profit APP objective function (4) can be restated as: 𝑇𝑇𝐾 𝑍𝑍= ෍෍𝑆 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1𝐾 Possibilistic Programming on APP Problem with Imprecise Costs The Net-Profit APP decision problem that has already been looked at may be summarized as follows. Suppose that over a planning horizon of T, a corporation produces N various products to 𝑍𝑆𝑆𝑆𝐷𝐷𝐷𝑇𝐼𝑆𝑆𝑆𝐵𝑇𝐼𝑊𝑀 o po at o p oduces N va ous p oducts to 𝑍𝑍= ෍෍𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 −෍෍𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖𝐵𝐵𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 −෍෍෍𝐶𝐶𝐶𝐶𝐶𝐶 ෫𝑚𝑚𝑚𝑚𝑚𝑚𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝑀𝑀 𝑚𝑚=1 −൥ ෍෍𝐶𝐶𝐶𝐶𝐶𝐶 ෫𝑘𝑘𝑘𝑘𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍𝐶𝐶𝐶𝐶𝐶𝐶 ෫𝑘𝑘𝑘𝑘𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍𝐶𝐶𝐶𝐶𝐶𝐶 ෪𝑘𝑘𝑘𝑘𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍෍𝐶𝐶𝐶𝐶ℎ𝑃𝑃 ෫𝑖𝑖𝑖𝑖𝑖𝑖𝑋𝑋𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝐼𝐼 𝑖𝑖=1 + ෍෍෍𝐶𝐶𝐶𝐶𝐶𝐶 ෪𝑚𝑚𝑚𝑚𝑚𝑚𝑋𝑋𝑋𝑋𝑚𝑚𝑚𝑚𝑚𝑚 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝑀𝑀 𝑚𝑚=1 + ෍෍𝐶𝐶𝐶𝐶𝐶𝐶 ෪𝑖𝑖𝑖𝑖𝐵𝐵𝑙𝑙𝑙𝑙 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 + ෍෍෍𝐶𝐶𝐶𝐶𝐶𝐶 ෫𝑖𝑖𝑖𝑖𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑞𝑞∈{1,2} 𝐼𝐼 𝑖𝑖=1 ቏ (33) (33) 137 www.ejtas.com EJTAS 2023 \| Volume 1 \| Number 5 137 2023 \| Volume 1 \| Number 5 www.ejtas.com EJTAS where (𝑍𝑍𝑝𝑝, 𝑍𝑍𝑚𝑚, 𝑍𝑍𝑜𝑜) is the vector of the objective functions 𝑍𝑍𝑝𝑝, 𝑍𝑍𝑚𝑚 and 𝑍𝑍𝑜𝑜. It is important to make a minor change in order to maintain the possibility distribution's triangular shape (normal and convex). Instead of concurrently maximizing these three objectives, the new approach will maximize 𝑍𝑍𝑚𝑚, minimize (𝑍𝑍𝑚𝑚−𝑍𝑍𝑝𝑝) and maximize (𝑍𝑍𝑜𝑜−𝑍𝑍𝑚𝑚), where the first objective function 𝑍𝑍𝑚𝑚, is the basis of the last two objective functions, which are actually relative measures from it (see Figure 2). The three new objectives also support the earlier claim that doing so would shift the triangular possibility distribution towards the right. This work uses Wang and Liang's (2005) Possibility Linear Programing (PLP) technique to solve the APP problem with uncertainty. Fortunately, possibility distribution offers a useful substitute for dealing with underlying confusing phenomena when assessing environmental coefficients and associated factors (Zadeh, 1978; Inuiguchi and Sakawa, 1996; Hsu and Wang, 2001). This is by adopting the triangular Fuzzy number (TFN) to the APP problem under Fuzzy operational expenses. Approach to Resolving the Imprecise Objective Function In general, especially in large-scale problems, this is a very difficult task, and the use of the Fuzzy Set theory in GP models can overcome such problem, allowing decision makers to work with imprecise aspiration levels (Yaghoobi and Tamiz, 2007). In multiobjective programming, In fuzzifying the inequality signs; “ = ” “ ≤” and “ ≥”, each of the objectives. In general, especially in large-scale problems, this is a very difficult task, and the use of the Fuzzy Set theory in GP models can overcome such problem, allowing decision makers to work with imprecise aspiration levels (Yaghoobi and Tamiz, 2007). In multiobjective programming, In fuzzifying the inequality signs; “ = ” “ ≤” and “ ≥”, Zimmermann (1978) used the symbol “~”, they are to be understood as “essentially greater than or equal to” and “essentially less than or equal to”. if an imprecise aspiration level is introduced to each of the objective functions then these fuzzy objectives are termed as fuzzy goals. Let 𝑔𝑔𝑘𝑘 be the aspiration level assigned to the kth objective 𝑍𝑍𝑘𝑘(𝑥𝑥). Then the fuzzy goals are: 𝑍𝑘𝑥𝑔𝑘𝑍𝑘𝑥𝑍𝑥𝑔𝑍𝑥 Also, the fuzzy decision-making of Bellman and Zadeh (1970) and Zimmermann's fuzzy programming (1978) approach may be used to transform the auxiliary MOLP issue into an analogous single-goal LP problem. The three objective functions' Positive Ideal Solutions (PIS) and Negative Ideal Solutions (NIS) can be correspondingly described as follows. 𝑍𝑃𝑃𝑃𝑀𝑀𝑀𝑍𝑚𝑍𝑁𝑁𝑁𝑀𝑀𝑛𝑍𝑚 Also, the fuzzy decision-making of Bellman and Zadeh (1970) and Zimmermann's fuzzy programming (1978) approach may be used to transform the auxiliary MOLP issue into an analogous single-goal LP problem. The three objective functions' Positive Ideal Solutions (PIS) and Negative Ideal Solutions (NIS) can be correspondingly described as follows. 𝑍𝑃𝑃𝑃𝑀𝑀𝑀𝑍𝑚𝑍𝑁𝑁𝑁𝑀𝑀𝑛𝑍𝑚 Zimmermann (1978) used the symbol “~”, they are to be understood as “essentially greater than or equal to” and “essentially less than or equal to”. if an imprecise aspiration level is introduced to each of the objective functions then these fuzzy objectives are termed as fuzzy goals. Let 𝑔𝑔𝑘𝑘 be the aspiration level assigned to the kth objective 𝑍𝑍𝑘𝑘(𝑥𝑥). Approach to Resolving the Imprecise Objective Function Approach to Resolving the Imprecise Objective Function The imprecise objective function of the Net- Profit Possibility APP programing model in the preceding section has a triangular possibility distribution. Geometrically, this imprecise objective is fully defined by three corner points: (𝑍𝑍𝑝𝑝, 0), (𝑍𝑍𝑚𝑚, 1) and (𝑍𝑍𝑜𝑜,0). The imprecise objective can be maximized by pushing the three corner points towards the right. Because of the vertical coordinates of the critical points being fixed at either 1 or 0, the three horizontal coordinates are the only considerations. The new problem will be to solve; 𝑍𝑝𝑍𝑚𝑍𝑜 (34) This suggested approach equates to maximizing the most possible value of the imprecise profit (at the point of possibility degree = 1). At the same time, it has minimized the inferior side of the possibility distribution. This means minimizing the region (I), which in the perspective is similar to "the danger of receiving reduced profit." Also, this has increased "the chance of generating larger profit," which is similar to area (II) of the probability distribution. Similar to Figure 2, it would be preferred to have the possibility distribution of B against that of A. thus the auxiliary problem of equation (27) then result to three brand-new, precise objective functions as shown below; Maximize (𝑍𝑍𝑝𝑝, 𝑍𝑍𝑚𝑚, 𝑍𝑍𝑜𝑜) Maximize (𝑍𝑍𝑝𝑝, 𝑍𝑍𝑚𝑚, 𝑍𝑍𝑜𝑜) Maximize (𝑍𝑍𝑝𝑝, 𝑍𝑍𝑚𝑚, 𝑍𝑍𝑜𝑜) Figure 2. The Approach to Maximize the Net Profit Figure 2. Approach to Resolving the Imprecise Objective Function The Approach to Maximize the Net Profit www.ejtas.com EJTAS 2023 \| Volume 1 \| Number 5 138 138 2023 \| Volume 1 \| Number 5 www.ejtas.com 𝑀𝑀𝑀𝑀𝑀𝑀 𝑍𝑍1 = 𝑍𝑍𝑚𝑚 = ෍෍𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 −෍෍𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖𝐵𝐵𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 −෍෍෍𝐶𝐶𝐶𝐶𝑀𝑀𝑚𝑚𝑚𝑚𝑚𝑚 𝑚𝑚 𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝑀𝑀 𝑚𝑚=1 −൥ ෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑚𝑚𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑚𝑚𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑚𝑚𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍෍𝐶𝐶𝐶𝐶ℎ𝑃𝑃𝑖𝑖𝑖𝑖𝑖𝑖 𝑚𝑚𝑋𝑋𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝐼𝐼 𝑖𝑖=1 + ෍෍෍𝐶𝐶𝐶𝐶ℎ𝑅𝑅𝑖𝑖𝑖𝑖𝑖𝑖 𝑚𝑚𝑋𝑋𝑋𝑋𝑚𝑚𝑚𝑚𝑚𝑚 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝑀𝑀 𝑚𝑚=1 + ෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖 𝑚𝑚𝐵𝐵𝑙𝑙𝑙𝑙 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 + ෍෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖 𝑚𝑚𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑞𝑞∈{1,2} 𝐼𝐼 𝑖𝑖=1 ቏ (35) 𝑀𝑀𝑀𝑀𝑀𝑀 𝑍𝑍2 = (𝑍𝑍𝑚𝑚−𝑍𝑍𝑝𝑝) = ෍෍𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 −෍෍𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖𝐵𝐵𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 −෍෍෍൫𝐶𝐶𝐶𝐶𝐶𝐶𝑚𝑚𝑚𝑚𝑚𝑚 𝑚𝑚 −𝐶𝐶𝐶𝐶𝐶𝐶𝑚𝑚𝑚𝑚𝑚𝑚 𝑝𝑝 ൯𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝑀𝑀 𝑚𝑚=1 −൥ ෍෍൫𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑚𝑚−𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑝𝑝൯𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍൫𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑚𝑚−𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑝𝑝൯𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍൫𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑚𝑚−𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑝𝑝൯𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍෍൫𝐶𝐶𝐶𝐶ℎ𝑃𝑃𝑖𝑖𝑖𝑖𝑡𝑡 𝑚𝑚−𝐶𝐶𝐶𝐶ℎ𝑃𝑃𝑖𝑖𝑖𝑖𝑖𝑖 𝑝𝑝൯𝑋𝑋𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝐼𝐼 𝑖𝑖=1 + ෍෍෍൫𝐶𝐶𝐶𝐶ℎ𝑅𝑅𝑖𝑖𝑖𝑖𝑖𝑖 𝑚𝑚−𝐶𝐶𝐶𝐶ℎ𝑅𝑅𝑖𝑖𝑖𝑖𝑖𝑖 𝑝𝑝൯𝑋𝑋𝑋𝑋𝑚𝑚𝑚𝑚𝑚𝑚 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝑀𝑀 𝑚𝑚=1 + ෍෍൫𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖 𝑚𝑚−𝐶𝐶𝐶𝐶ℎ𝑅𝑅𝑖𝑖𝑖𝑖𝑖𝑖 𝑝𝑝൯𝐵𝐵𝑙𝑙𝑙𝑙 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 + ෍෍෍൫𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖 𝑚𝑚−𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖 𝑝𝑝൯𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑞𝑞∈{1,2} 𝐼𝐼 𝑖𝑖=1 ቏ (36) 𝑀𝑀𝑀𝑀𝑀𝑀 𝑍𝑍3 = (𝑍𝑍𝑜𝑜−𝑍𝑍𝑚𝑚) = ෍෍𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 −෍෍𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖𝐵𝐵𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 −෍෍෍(𝐶𝐶𝐶𝐶𝐶𝐶𝑚𝑚𝑚𝑚𝑚𝑚 𝑜𝑜 −𝐶𝐶𝐶𝐶𝐶𝐶𝑚𝑚𝑚𝑚𝑚𝑚 𝑚𝑚 )𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝑀𝑀 𝑚𝑚=1 −൥ ෍෍(𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑜𝑜−𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑚𝑚)𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍(𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑜𝑜−𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑚𝑚)𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍(𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑜𝑜−𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑚𝑚)𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍෍(𝐶𝐶𝐶𝐶ℎ𝑃𝑃𝑖𝑖𝑖𝑖𝑖𝑖 𝑜𝑜−𝐶𝐶𝐶𝐶ℎ𝑃𝑃𝑖𝑖𝑖𝑖𝑖𝑖 𝑚𝑚)𝑋𝑋𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝐼𝐼 𝑖𝑖=1 + ෍෍෍(𝐶𝐶𝐶𝐶ℎ𝑅𝑅𝑖𝑖𝑖𝑖𝑖𝑖 𝑜𝑜 −𝐶𝐶𝐶𝐶ℎ𝑅𝑅𝑖𝑖𝑖𝑖𝑖𝑖 𝑚𝑚)𝑋𝑋𝑋𝑋𝑚𝑚𝑚𝑚𝑚𝑚 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝑀𝑀 𝑚𝑚=1 + ෍෍(𝐶𝐶𝐶𝐶ℎ𝑅𝑅𝑖𝑖𝑖𝑖𝑖𝑖 𝑜𝑜−𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖 𝑚𝑚)𝐵𝐵𝑙𝑙𝑙𝑙 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 + ෍෍෍൫𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖 𝑜𝑜−𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖 𝑚𝑚൯𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑞𝑞∈{1,2} 𝐼𝐼 𝑖𝑖=1 ቏ (37) 𝑀𝑀𝑀𝑀𝑀𝑀 𝑍𝑍1 = 𝑍𝑍𝑚𝑚 = ෍෍𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 −෍෍𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖𝐵𝐵𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 −෍෍෍𝐶𝐶𝐶𝐶𝑀𝑀𝑚𝑚𝑚𝑚𝑚𝑚 𝑚𝑚 𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝑀𝑀 𝑚𝑚=1 −൥ ෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑚𝑚𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑚𝑚𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑚𝑚𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍෍𝐶𝐶𝐶𝐶ℎ𝑃𝑃𝑖𝑖𝑖𝑖𝑖𝑖 𝑚𝑚𝑋𝑋𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝐼𝐼 𝑖𝑖=1 + ෍෍෍𝐶𝐶𝐶𝐶ℎ𝑅𝑅𝑖𝑖𝑖𝑖𝑖𝑖 𝑚𝑚𝑋𝑋𝑋𝑋𝑚𝑚𝑚𝑚𝑚𝑚 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝑀𝑀 𝑚𝑚=1 + ෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖 𝑚𝑚𝐵𝐵𝑙𝑙𝑙𝑙 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 + ෍෍෍𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖 𝑚𝑚𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑞𝑞∈{1,2} 𝐼𝐼 𝑖𝑖=1 ቏ (35) 𝑀𝑀𝑀𝑍𝑍𝑚𝑍𝑝 (35) ෍෍෍𝐶𝐶𝑅 𝑖𝑖𝑖𝑖𝑖𝑖𝑚𝑋𝑋 𝑚𝑚𝑚𝑚𝑚𝑚𝑇 𝑡𝑡=1𝑊 𝑤𝑤=1𝑀 𝑚𝑚=1 ෍෍𝐶𝐶𝐶 𝑖𝑖𝑖𝑖𝑚𝐵 𝑙𝑙𝑙𝑙𝑇 𝑡𝑡=1𝐼 𝑖𝑖=1 ෍෍෍𝐶𝐶𝐶 𝑖𝑖𝑖𝑖𝑚𝑋 𝑖𝑖𝑖𝑖𝑖𝑖𝑇 𝑡𝑡=1 𝑞𝑞∈{1,2}𝐼 𝑖𝑖=1 ቏ 𝑀𝑀𝑀𝑀𝑀𝑀 𝑍𝑍2 = (𝑍𝑍𝑚𝑚−𝑍𝑍𝑝𝑝) = ෍෍𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 −෍෍𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖𝐵𝐵𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 −෍෍෍൫𝐶𝐶𝐶𝐶𝐶𝐶𝑚𝑚𝑚𝑚𝑚𝑚 𝑚𝑚 −𝐶𝐶𝐶𝐶𝐶𝐶𝑚𝑚𝑚𝑚𝑚𝑚 𝑝𝑝 ൯𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝑀𝑀 𝑚𝑚=1 −൥ ෍෍൫𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑚𝑚−𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑝𝑝൯𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍൫𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑚𝑚−𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑝𝑝൯𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍൫𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑚𝑚−𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑝𝑝൯𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍෍൫𝐶𝐶𝐶𝐶ℎ𝑃𝑃𝑖𝑖𝑖𝑖𝑡𝑡 𝑚𝑚−𝐶𝐶𝐶𝐶ℎ𝑃𝑃𝑖𝑖𝑖𝑖𝑖𝑖 𝑝𝑝൯𝑋𝑋𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝐼𝐼 𝑖𝑖=1 + ෍෍෍൫𝐶𝐶𝐶𝐶ℎ𝑅𝑅𝑖𝑖𝑖𝑖𝑖𝑖 𝑚𝑚−𝐶𝐶𝐶𝐶ℎ𝑅𝑅𝑖𝑖𝑖𝑖𝑖𝑖 𝑝𝑝൯𝑋𝑋𝑋𝑋𝑚𝑚𝑚𝑚𝑚𝑚 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝑀𝑀 𝑚𝑚=1 + ෍෍൫𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖 𝑚𝑚−𝐶𝐶𝐶𝐶ℎ𝑅𝑅𝑖𝑖𝑖𝑖𝑖𝑖 𝑝𝑝൯𝐵𝐵𝑙𝑙𝑙𝑙 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 + ෍෍෍൫𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖 𝑚𝑚−𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖 𝑝𝑝൯𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑞𝑞∈{1,2} 𝐼𝐼 𝑖𝑖=1 ቏ (36) 𝑀𝑀𝑀𝑀𝑀𝑀 𝑍𝑍3 = (𝑍𝑍𝑜𝑜−𝑍𝑍𝑚𝑚) = ෍෍𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 −෍෍𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖𝐵𝐵𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 −෍෍෍(𝐶𝐶𝐶𝐶𝐶𝐶𝑚𝑚𝑚𝑚𝑚𝑚 𝑜𝑜 −𝐶𝐶𝐶𝐶𝐶𝐶𝑚𝑚𝑚𝑚𝑚𝑚 𝑚𝑚 )𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝑀𝑀 𝑚𝑚=1 −൥ ෍෍(𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑜𝑜−𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑚𝑚)𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍(𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑜𝑜−𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑚𝑚)𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍(𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑜𝑜−𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑚𝑚)𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍෍(𝐶𝐶𝐶𝐶ℎ𝑃𝑃𝑖𝑖𝑖𝑖𝑖𝑖 𝑜𝑜−𝐶𝐶𝐶𝐶ℎ𝑃𝑃𝑖𝑖𝑖𝑖𝑖𝑖 𝑚𝑚)𝑋𝑋𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝐼𝐼 𝑖𝑖=1 + ෍෍෍(𝐶𝐶𝐶𝐶ℎ𝑅𝑅𝑖𝑖𝑖𝑖𝑖𝑖 𝑜𝑜 −𝐶𝐶𝐶𝐶ℎ𝑅𝑅𝑖𝑖𝑖𝑖𝑖𝑖 𝑚𝑚)𝑋𝑋𝑋𝑋𝑚𝑚𝑚𝑚𝑚𝑚 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝑀𝑀 𝑚𝑚=1 + ෍෍(𝐶𝐶𝐶𝐶ℎ𝑅𝑅𝑖𝑖𝑖𝑖𝑖𝑖 𝑜𝑜−𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖 𝑚𝑚)𝐵𝐵𝑙𝑙𝑙𝑙 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 + ෍෍෍൫𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖 𝑜𝑜−𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖 𝑚𝑚൯𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑞𝑞∈{1,2} 𝐼𝐼 𝑖𝑖=1 ቏ (37) 𝑀𝑀𝑀𝑀𝑀𝑀 𝑍𝑍2 = (𝑍𝑍𝑚𝑚−𝑍𝑍𝑝𝑝) = ෍෍𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 −෍෍𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖𝐵𝐵𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 −෍෍෍൫𝐶𝐶𝐶𝐶𝐶𝐶𝑚𝑚𝑚𝑚𝑚𝑚 𝑚𝑚 −𝐶𝐶𝐶𝐶𝐶𝐶𝑚𝑚𝑚𝑚𝑚𝑚 𝑝𝑝 ൯𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝑀𝑀 𝑚𝑚=1 −൥ ෍෍൫𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑚𝑚−𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑝𝑝൯𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍൫𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑚𝑚−𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑝𝑝൯𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍൫𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑚𝑚−𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑝𝑝൯𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍෍൫𝐶𝐶𝐶𝐶ℎ𝑃𝑃𝑖𝑖𝑖𝑖𝑡𝑡 𝑚𝑚−𝐶𝐶𝐶𝐶ℎ𝑃𝑃𝑖𝑖𝑖𝑖𝑖𝑖 𝑝𝑝൯𝑋𝑋𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝐼𝐼 𝑖𝑖=1 + ෍෍෍൫𝐶𝐶𝐶𝐶ℎ𝑅𝑅𝑖𝑖𝑖𝑖𝑖𝑖 𝑚𝑚−𝐶𝐶𝐶𝐶ℎ𝑅𝑅𝑖𝑖𝑖𝑖𝑖𝑖 𝑝𝑝൯𝑋𝑋𝑋𝑋𝑚𝑚𝑚𝑚𝑚𝑚 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝑀𝑀 𝑚𝑚=1 + ෍෍൫𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖 𝑚𝑚−𝐶𝐶𝐶𝐶ℎ𝑅𝑅𝑖𝑖𝑖𝑖𝑖𝑖 𝑝𝑝൯𝐵𝐵𝑙𝑙𝑙𝑙 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 + ෍෍෍൫𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖 𝑚𝑚−𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖 𝑝𝑝൯𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑞𝑞∈{1,2} 𝐼𝐼 𝑖𝑖=1 ቏ (36) (36) 𝑀𝑀𝑀𝑀𝑀𝑀 𝑍𝑍3 = (𝑍𝑍𝑜𝑜−𝑍𝑍𝑚𝑚) = ෍෍𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖𝐷𝐷𝐷𝐷𝐷𝐷𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 −෍෍𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖𝐵𝐵𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 −෍෍෍(𝐶𝐶𝐶𝐶𝐶𝐶𝑚𝑚𝑚𝑚𝑚𝑚 𝑜𝑜 −𝐶𝐶𝐶𝐶𝐶𝐶𝑚𝑚𝑚𝑚𝑚𝑚 𝑚𝑚 )𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝑀𝑀 𝑚𝑚=1 −൥ ෍෍(𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑜𝑜−𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑚𝑚)𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍(𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑜𝑜−𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑚𝑚)𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍(𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑜𝑜−𝐶𝐶𝐶𝐶𝐶𝐶𝑘𝑘𝑘𝑘 𝑚𝑚)𝑋𝑋𝑋𝑋𝑘𝑘𝑘𝑘 𝑇𝑇 𝑡𝑡=1 𝐾𝐾 𝑘𝑘=1 + ෍෍෍(𝐶𝐶𝐶𝐶ℎ𝑃𝑃𝑖𝑖𝑖𝑖𝑖𝑖 𝑜𝑜−𝐶𝐶𝐶𝐶ℎ𝑃𝑃𝑖𝑖𝑖𝑖𝑖𝑖 𝑚𝑚)𝑋𝑋𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝐼𝐼 𝑖𝑖=1 + ෍෍෍(𝐶𝐶𝐶𝐶ℎ𝑅𝑅𝑖𝑖𝑖𝑖𝑖𝑖 𝑜𝑜 −𝐶𝐶𝐶𝐶ℎ𝑅𝑅𝑖𝑖𝑖𝑖𝑖𝑖 𝑚𝑚)𝑋𝑋𝑋𝑋𝑚𝑚𝑚𝑚𝑚𝑚 𝑇𝑇 𝑡𝑡=1 𝑊𝑊 𝑤𝑤=1 𝑀𝑀 𝑚𝑚=1 + ෍෍(𝐶𝐶𝐶𝐶ℎ𝑅𝑅𝑖𝑖𝑖𝑖𝑖𝑖 𝑜𝑜−𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖 𝑚𝑚)𝐵𝐵𝑙𝑙𝑙𝑙 𝑇𝑇 𝑡𝑡=1 𝐼𝐼 𝑖𝑖=1 + ෍෍෍൫𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖 𝑜𝑜−𝐶𝐶𝐶𝐶𝐶𝐶𝑖𝑖𝑖𝑖 𝑚𝑚൯𝑋𝑋𝑖𝑖𝑖𝑖𝑖𝑖 𝑇𝑇 𝑡𝑡=1 𝑞𝑞∈{1,2} 𝐼𝐼 𝑖𝑖=1 ቏ (37) (37) each of the objectives. Approach to Resolving the Imprecise Objective Function Then the fuzzy goals are: 𝑍𝑘𝑥𝑔𝑘𝑍𝑘𝑥𝑍𝑥𝑔𝑍𝑥 𝑍𝑍1 𝑃𝑃𝑃𝑃𝑃𝑃= 𝑀𝑀𝑀𝑀𝑀𝑀𝑍𝑍𝑚𝑚; 𝑍𝑍1 𝑁𝑁𝑁𝑁𝑁𝑁= 𝑀𝑀𝑀𝑀𝑛𝑛𝑍𝑍𝑚𝑚 𝑍𝑍2 𝑃𝑃𝑃𝑃𝑃𝑃= 𝑀𝑀𝑀𝑀𝑀𝑀(𝑍𝑍𝑚𝑚−𝑍𝑍𝑝𝑝); 𝑍𝑍2 𝑁𝑁𝑁𝑁𝑁𝑁 = 𝑀𝑀𝑀𝑀𝑀𝑀(𝑍𝑍𝑚𝑚−𝑍𝑍𝑝𝑝)൫𝑣𝑣𝑗𝑗 ∗൯ 𝑍𝑍3 𝑃𝑃𝑃𝑃𝑃𝑃= 𝑀𝑀𝑀𝑀𝑀𝑀(𝑍𝑍𝑜𝑜−𝑍𝑍𝑚𝑚); 𝑍𝑍3 𝑁𝑁𝑁𝑁𝑁𝑁 = 𝑀𝑀𝑀𝑀𝑀𝑀(𝑍𝑍𝑜𝑜−𝑍𝑍𝑚𝑚) Fuzzy Multi-objective Goal Programing Development Fuzzy Multi-objective Goal Programing Development 𝑍𝑍𝑘𝑘(𝑥𝑥) ≥෩𝑔𝑔𝑘𝑘 [for maximizing 𝑍𝑍𝑘𝑘(𝑥𝑥)] and 𝑍𝑍𝑘𝑘(𝑥𝑥) ≤෩𝑔𝑔𝑘𝑘 [for minimizing 𝑍𝑍𝑘𝑘(𝑥𝑥)] 𝑍𝑍𝑘𝑘(𝑥𝑥) ≥෩𝑔𝑔𝑘𝑘 [for maximizing 𝑍𝑍𝑘𝑘(𝑥𝑥)] and 𝑍𝑍𝑘𝑘(𝑥𝑥) ≤෩𝑔𝑔𝑘𝑘 [for minimizing 𝑍𝑍𝑘𝑘(𝑥𝑥)] In classic models of GP, the decision maker has to specify a precise aspiration level (goal) for 139 EJTAS 2023 \| Volume 1 \| Number 5 EJTAS In solving the problem, a general form of FGP model is considered: 𝑥 In solving the problem, a general form of FGP model is considered: 𝑥 find 𝑥𝑥 to satisfy; subjet to 𝑍𝑍𝑘𝑘(𝑥𝑥) ≥෩𝑔𝑔𝑘𝑘 𝑍𝑍𝑘𝑘(𝑥𝑥) ≤෩𝑔𝑔𝑘𝑘 𝐴𝐴𝐴𝐴൭ ≤ = ≥ ൱𝑏𝑏 𝑋𝑋≥0 𝑘𝑘= 1 … 𝑛𝑛 𝑘𝑘= 𝑛𝑛+ 1 … 𝐽𝐽 (38) (38) For this paper, a FGP is employed in solving any of the APP models, like (4) –(25). Being able to use FGP approach with fuzzy goals, the aspiration levels should be calculated. Payoff table is used when the decision maker has no enough view point to determine the aspiration levels. Zimmermann (1978) used a Payoff table to develop an upper and lower limit that was used to formulate the membership functions of the fuzzy goals. 𝑋 kth fuzzy goal is approximately less than or equal to the aspiration level 𝑔𝑔𝑘𝑘, and 𝑍𝑍𝑘𝑘(𝑥𝑥) ≥෩𝑔𝑔𝑘𝑘 Means that the kth fuzzy goal is approximately greater than or equal to the aspiration level 𝑔𝑔𝑘𝑘 (Hannan, 1981). The fuzzy decision-making concept of Bellman and Zadeh (1970) can be used to solve the planned multi-objective APP problem (4)–(25). Linear membership functions as proposed by Zimmermann (1978) are used to represent the fuzzy goals of decision makers. In the general form (38), the purpose of FGP is to find compromise solution 𝑋𝑋 such that all fuzzy goals are satisfied. 𝑔𝑔𝑘𝑘 is the aspiration level for kth goal, 𝐴𝐴𝐴𝐴≤𝑏𝑏 are system constraints in vector notation. Approach to Resolving the Imprecise Objective Function 𝑍𝑍𝑘𝑘(𝑥𝑥) ≤෩𝑔𝑔𝑘𝑘 Means that the The corresponding linear membership function for each objective function is defined (see figure 3) by; The corresponding linear membership function for each objective function is defined (see figure 𝑍𝑍1(𝑥𝑥) ≤𝑍𝑍1 𝑃𝑃𝑃𝑃𝑃𝑃 , 𝜇𝜇(𝑍𝑍1(𝑥𝑥)) = ⎩ ⎪ ⎨ ⎪ ⎧ 1 𝑍𝑍1 −𝑍𝑍1 𝑁𝑁𝑁𝑁𝑁𝑁 𝑍𝑍1 𝑃𝑃𝑃𝑃𝑃𝑃−𝑍𝑍1 𝑁𝑁𝑁𝑁𝑁𝑁 0 𝜇𝜇(𝑍𝑍2(𝑥𝑥)) = ⎩ ⎪ ⎨ ⎪ ⎧ 1 𝑍𝑍2 𝑁𝑁𝑁𝑁𝑁𝑁−𝑍𝑍2 𝑍𝑍2 𝑁𝑁𝑁𝑁𝑁𝑁−𝑍𝑍2 𝑃𝑃𝑃𝑃𝑃𝑃 0 𝜇𝜇(𝑍𝑍1(𝑥𝑥)) = ⎩ ⎪ ⎨ ⎪ ⎧ 1 𝑍𝑍1 −𝑍𝑍1 𝑁𝑁𝑁𝑁𝑁𝑁 𝑍𝑍1 𝑃𝑃𝑃𝑃𝑃𝑃−𝑍𝑍1 𝑁𝑁𝑁𝑁𝑁𝑁 0 𝜇𝜇(𝑍𝑍2(𝑥𝑥)) = ⎩ ⎪ ⎨ ⎪ ⎧ 1 𝑍𝑍2 𝑁𝑁𝑁𝑁𝑁𝑁−𝑍𝑍2 𝑍𝑍2 𝑁𝑁𝑁𝑁𝑁𝑁−𝑍𝑍2 𝑃𝑃𝑃𝑃𝑃𝑃 0 𝑍𝑍1 𝑁𝑁𝑁𝑁𝑁𝑁≤𝑍𝑍1(𝑥𝑥) ≤𝑍𝑍1 𝑁𝑁𝑁𝑁𝑁𝑁 , (39) 𝑍𝑍1(𝑥𝑥) ≥𝑍𝑍1 𝑁𝑁𝑁𝑁𝑁𝑁 , 𝑍𝑍2(𝑥𝑥) ≤𝑍𝑍2 𝑃𝑃𝑃𝑃𝑃𝑃 , 𝑍𝑍𝑥𝑍 (39) 𝑍𝑍2 𝑃𝑃𝑃𝑃𝑃𝑃≤𝑍𝑍2(𝑥𝑥) ≤𝑍𝑍2 𝑁𝑁𝑁𝑁𝑁𝑁 , (40) 𝑍𝑍2(𝑥𝑥) ≥𝑍𝑍2 𝑁𝑁𝑁𝑁𝑁𝑁 , 𝑍𝑍2 𝑃𝑃𝑃𝑃𝑃𝑃≤𝑍𝑍2(𝑥𝑥) ≤𝑍𝑍2 𝑁𝑁𝑁𝑁𝑁𝑁 , (40) 𝑍𝑍2(𝑥𝑥) ≥𝑍𝑍2 𝑁𝑁𝑁𝑁𝑁𝑁 , (40) 0 and 𝜇𝜇(𝑍𝑍1(𝑥𝑥)) and 𝜇𝜇(𝑍𝑍3(𝑥𝑥)) are similar. and 𝜇𝜇(𝑍𝑍1(𝑥𝑥)) and 𝜇𝜇(𝑍𝑍3(𝑥𝑥)) are similar. and 𝜇𝜇(𝑍𝑍1(𝑥𝑥)) and 𝜇𝜇(𝑍𝑍3(𝑥𝑥)) are similar. www.ejtas.com EJTAS 2023 \| Volume 1 \| Number 5 140 140 2023 \| Volume 1 \| Number 5 2023 \| Volume 1 \| Number 5 Figure 3. Linear Membership form Figure 3. Linear Membership form cost for the Net-Profit APP problem (4)-(25) is formulate as follows: Lastly, the APP is solved following equivalent single-objective linear programming model. Hence, the associated FGP model with fuzzy 𝑥 find 𝑥𝑥 𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 𝜇𝜇(𝑍𝑍𝑘𝑘(𝑥𝑥)) to satisfy; 𝜇𝜇(𝑍𝑍1(𝑥𝑥)) = 𝑍𝑍1 −𝑍𝑍1 𝑁𝑁𝑁𝑁𝑁𝑁 𝑍𝑍1 𝑃𝑃𝑃𝑃𝑃𝑃−𝑍𝑍1 𝑁𝑁𝑁𝑁𝑁𝑁 𝜇𝜇(𝑍𝑍2(𝑥𝑥)) = 𝑍𝑍2 𝑁𝑁𝑁𝑁𝑁𝑁−𝑍𝑍2 𝑍𝑍2 𝑁𝑁𝑁𝑁𝑁𝑁−𝑍𝑍2 𝑃𝑃𝑃𝑃𝑃𝑃 𝜇𝜇(𝑍𝑍3(𝑥𝑥)) = 𝑍𝑍3 −𝑍𝑍3 𝑁𝑁𝑁𝑁𝑁𝑁 𝑍𝑍3 𝑃𝑃𝑃𝑃𝑃𝑃−𝑍𝑍3 𝑁𝑁𝑁𝑁𝑁𝑁 𝜇𝜇ቀ𝑍𝑍𝑗𝑗(𝑥𝑥)ቁ∈[0,1], 𝑗𝑗= 1,2,3 𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 (5 to 10, 12 to 14, 16 to 20, 22 to 25, 30 to 32) 𝑥𝑥𝑖𝑖≥0, 𝑖𝑖= 1 … . . 𝑛𝑛; 𝑗𝑗= 1,2,3 (41) find 𝑥𝑥 𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 𝜇𝜇(𝑍𝑍𝑘𝑘(𝑥𝑥)) to satisfy;𝜇𝑍𝑥𝑍𝑍𝑁𝑁𝑁𝑍𝑃𝑃𝑃𝑍𝑁𝑁𝑁 find 𝑥𝑥 𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 𝜇𝜇(𝑍𝑍𝑘𝑘(𝑥𝑥)) to satisfy;𝜇𝑍𝑥𝑍𝑍𝑁𝑁𝑁𝑍𝑃𝑃𝑃𝑍𝑁𝑁𝑁 𝜇𝜇(𝑍𝑍1(𝑥𝑥)) = 𝑍𝑍1 −𝑍𝑍1 𝑁𝑁𝑁𝑁𝑁𝑁 𝑍𝑍1 𝑃𝑃𝑃𝑃𝑃𝑃−𝑍𝑍1 𝑁𝑁𝑁𝑁𝑁𝑁 𝜇𝜇(𝑍𝑍2(𝑥𝑥)) = 𝑍𝑍2 𝑁𝑁𝑁𝑁𝑁𝑁−𝑍𝑍2 𝑍𝑍2 𝑁𝑁𝑁𝑁𝑁𝑁−𝑍𝑍2 𝑃𝑃𝑃𝑃𝑃𝑃 𝜇𝜇(𝑍𝑍3(𝑥𝑥)) = 𝑍𝑍3 −𝑍𝑍3 𝑁𝑁𝑁𝑁𝑁𝑁 𝑍𝑍3 𝑃𝑃𝑃𝑃𝑃𝑃−𝑍𝑍3 𝑁𝑁𝑁𝑁𝑁𝑁𝜇𝑍𝑗𝑥𝑗𝐶𝐶𝐶𝐶𝐶𝐶𝐶 (41) 𝜇𝜇ቀ𝑍𝑍𝑗𝑗(𝑥𝑥)ቁ∈[0,1], 𝑗𝑗 1,2,3 𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 (5 to 10, 12 to 14, 16 to 20, 22 to 25, 30 to 32) 𝑥𝑥𝑖𝑖≥0, 𝑖𝑖= 1 … . . 𝑛𝑛; 𝑗𝑗= 1,2,3 Model Implementation through the use of inventories, overtime, and backorders. Case description Related operating cost data Period 𝐶𝐶𝐶𝐶𝐶𝐶 ෫(₦ /unit) 𝐶𝐶𝐶𝐶𝐶𝐶 ෫(₦ /unit) 𝐶𝐶𝐶𝐶𝐶𝐶 ෫(₦ /unit) 𝐶𝐶𝐶𝐶𝐶𝐶 ෪(₦ /unit) 𝐶𝐶𝐶𝐶ℎ𝑃𝑃 ෫(₦ /unit) 𝐶𝐶𝐶𝐶ℎ𝑅𝑅 ෫(₦/unit) 𝐶𝐶𝐶𝐶𝐶𝐶 ෪(₦ /unit) 𝐶𝐶𝐶𝐶𝐶𝐶 ෫(₦ /unit) 1 45, 80, 140 35, 64, 100 10, 30, 55 15, 40, 60 0.5, 2, 4 800, 1400, 1600 1.5, 2, 4.5 5, 7, 11 2 45, 80, 135 35, 64, 100 10, 30, 55 15, 40, 60 0.5, 2, 4 850, 1400, 1600 1.5, 2, 4.5 5, 7, 10 3 48, 80, 140 35, 64, 100 10, 30, 55 15, 40, 60 0.5, 2, 4 800, 1400, 1500 1, 2, 4.5 5.5, 7, 10 4 47, 80, 140 35, 64, 100 10, 30, 55 15, 40, 60 0.5, 2, 4 850, 1400, 1500 1, 2, 4.5 5.5, 7, 11 5 47, 80, 145 35, 64, 100 10, 30, 55 15, 40, 60 0.5, 2, 4 850, 1400, 1500 1.5, 2, 4.5 5, 7, 11 6 47, 80, 140 35, 64, 100 10, 30, 55 15, 40, 60 0.5, 2, 4 800, 1400, 1600 1.5, 2, 4.5 5, 7, 10 𝑫𝑫𝑫𝑫𝑫𝑫𝟏𝑫𝑫𝑫𝟐𝑫𝑫𝑫𝟑 Table 4. Related operating cost data 𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝑃𝐶𝐶𝑅 Table 5. Product Demand Forecasting in six months 𝑫𝑫𝑫𝑫𝑫𝑫𝒊𝒊𝒊𝒊 ෫= (𝑫𝑫𝑫𝑫𝑫𝑫𝒊𝒊𝒊𝒊 𝟏𝟏, 𝑫𝑫𝑫𝑫𝑫𝑫𝒊𝒊𝒊𝒊 𝟐𝟐, 𝑫𝑫𝑫𝑫𝑫𝑫𝒊𝒊𝒊𝒊 𝟑𝟑) (unit/month) Period 𝑡𝑡 Produc t 𝑖𝑖 1 2 3 4 5 6 1 (320, 252, 290) (370, 295, 280) (520, 430, 440) (350, 260, 280) (380, 300, 300) (320, 270, 290) 2 (340, 255, 280) (290, 284, 270) (450, 430, 400) (300, 260, 305) (410, 300, 320) (330, 270, 260) Results and Discussion under the presumption that the DM provided the most likely value of the triangular distribution of each Fuzzy number as the precise value. LINGO 18.0 solver is used to solve the model. The objective values of the initial solutions using the model are 𝑀𝑀𝑀𝑀𝑀𝑀 𝑍𝑍1 = 2770238, 𝑀𝑀𝑀𝑀𝑀𝑀 𝑍𝑍2 = 1472820 and 𝑀𝑀𝑀𝑀𝑀𝑀 𝑍𝑍2 = 3135912 𝑍𝑃𝑃𝑃𝑍𝑁𝑁𝑁 Case description Related operating cost data Period 𝐶𝐶𝐶𝐶𝐶𝐶 ෫(₦ /unit) 𝐶𝐶𝐶𝐶𝐶𝐶 ෫(₦ /unit) 𝐶𝐶𝐶𝐶𝐶𝐶 ෫(₦ /unit) 𝐶𝐶𝐶𝐶𝐶𝐶 ෪(₦ /unit) 𝐶𝐶𝐶𝐶ℎ𝑃𝑃 ෫(₦ /unit) 𝐶𝐶𝐶𝐶ℎ𝑅𝑅 ෫(₦/unit) 𝐶𝐶𝐶𝐶𝐶𝐶 ෪(₦ /unit) 𝐶𝐶𝐶𝐶𝐶𝐶 ෫(₦ /unit) 1 45, 80, 140 35, 64, 100 10, 30, 55 15, 40, 60 0.5, 2, 4 800, 1400, 1600 1.5, 2, 4.5 5, 7, 11 2 45, 80, 135 35, 64, 100 10, 30, 55 15, 40, 60 0.5, 2, 4 850, 1400, 1600 1.5, 2, 4.5 5, 7, 10 3 48, 80, 140 35, 64, 100 10, 30, 55 15, 40, 60 0.5, 2, 4 800, 1400, 1500 1, 2, 4.5 5.5, 7, 10 4 47, 80, 140 35, 64, 100 10, 30, 55 15, 40, 60 0.5, 2, 4 850, 1400, 1500 1, 2, 4.5 5.5, 7, 11 5 47, 80, 145 35, 64, 100 10, 30, 55 15, 40, 60 0.5, 2, 4 850, 1400, 1500 1.5, 2, 4.5 5, 7, 11 6 47, 80, 140 35, 64, 100 10, 30, 55 15, 40, 60 0.5, 2, 4 800, 1400, 1600 1.5, 2, 4.5 5, 7, 10 Table 5. Product Demand Forecasting in six months 𝑫𝑫𝑫𝑫𝑫𝑫𝒊𝒊𝒊𝒊 ෫= (𝑫𝑫𝑫𝑫𝑫𝑫𝒊𝒊𝒊𝒊 𝟏𝟏, 𝑫𝑫𝑫𝑫𝑫𝑫𝒊𝒊𝒊𝒊 𝟐𝟐, 𝑫𝑫𝑫𝑫𝑫𝑫𝒊𝒊𝒊𝒊 𝟑𝟑) (unit/month) Period 𝑡𝑡 Produc t 𝑖𝑖 1 2 3 4 5 6 1 (320, 252, 290) (370, 295, 280) (520, 430, 440) (350, 260, 280) (380, 300, 300) (320, 270, 290) 2 (340, 255, 280) (290, 284, 270) (450, 430, 400) (300, 260, 305) (410, 300, 320) (330, 270, 260) Table 4. Case description Alternately, the DM can use a mathematical programming technique to create an aggregate production schedule for RPL factory. Based on company reports, the planning horizon spans for six months, May to October. The model includes two types of standard products. Production expenses for overtime are capped at 30% of production expenses for regular hours. Additionally, it is assumed that each product has no beginning inventory and no backorders at the last period. The inventory's maximum allowed storage area is 3000𝑚𝑚3. In a day, there are two working shifts. 8 hours are allotted for regular production per shift, while 3 hours allotted for The case study of Rich Pharmaceuticals Limited(RPL) was utilized to show how useful the suggested methodology is. RPL is one of the leading producers of pharmaceuticals in Nigeria. RPL's goods are mostly sold in Southern and Middle belt of Nigeria, some parts of West and East Africa, they have recently experienced strong demand. RPL must monitor financial data and assess performance if it is to expand its company. The company's net profit margin is one statistic they have to pay attention to. RPL's business APP approach is to consider a dynamic labor force level over the planning horizon, allowing for the flexible meeting of demand www.ejtas.com EJTAS 2023 \| Volume 1 \| Number 5 141 141 2023 \| Volume 1 \| Number 5 2023 \| Volume 1 \| Number 5 www.ejtas.com www.ejtas.com The purpose of the APP decision issue for the industrial instance that is addressed here is to develop a multiple fuzzy goals programming model for determining the optimal approach to adjust output rates, hiring and firing, inventory levels, overtime, and backorders in order to meet the targeted maximum profit using the Throughput accounting process. This APP choice is expected to reduce overall manufacturing costs, shorten the process, and increase sales and profit. overtime production. To produce these products, 10 types of raw materials are required. Repairs are done just in shift 2 (i.e., overtime) and the overall operating cost is as stated on Table 4 below. Table 5 gives the forecasted monthly demand for production, when demand for a certain period exceeds production capacity during regular hours and inventory levels are likewise insufficient to meet this demand, production is continued during overtime. Table 4. Results and Discussion Equation (33), in addition, may be used to create the full equivalent single-objective LP model for the RPL situation. Figure 4. The Approach to Maximize the Net Profit Figure 4. The Approach to Maximize the Net Profit Fifth Stage: Applying the FGP-APP gives the compromise solution as 𝑍𝑍1 = 2560411, 𝑍𝑍2 = 898850, 𝑍𝑍3 = 3049203. As will be determined by the triangular possibility distribution (see fig. 4), (₦1661561, ₦2560411, ₦5609614) is present in the improved profit as a result, and the overall degree of DM satisfaction is 0.873278. Results and Discussion The following is a description of the RPL case's solution process using the suggested APP-PP technique: First Stage: Create the PP model for the APP choice issue in accordance with Equations (4) to (25). Second Stage: Triangular possibility distributions are used to model the imprecise data as shown in Tables 4. Fourth Stage: The PIS and NIS of the three new 𝑍𝑃𝑃𝑃𝑍𝑁𝑁𝑁𝑍𝑃𝑃𝑃𝑍𝑁𝑁𝑁 Fourth Stage: The PIS and NIS of the three new objective functions (𝑍𝑍1 𝑃𝑃𝑃𝑃𝑃𝑃, 𝑍𝑍1 𝑁𝑁𝑁𝑁𝑁𝑁) = (2770238,1114430) (𝑍𝑍2 𝑃𝑃𝑃𝑃𝑃𝑃, 𝑍𝑍2 𝑁𝑁𝑁𝑁𝑁𝑁) = (1472820 ,2227640) and (𝑍𝑍3 𝑃𝑃𝑃𝑃𝑃𝑃, 𝑍𝑍3 𝑁𝑁𝑁𝑁𝑁𝑁) = (3135912,1658514). The fuzzy aspiration levels can be quantified using the linear and continuous membership function. According to Eq. (39) and (40), the relevant linear membership functions can be defined as shown below. (𝑍𝑍1 𝑃𝑃𝑃𝑃𝑃𝑃, 𝑍𝑍1 𝑁𝑁𝑁𝑁𝑁𝑁) = (𝑍𝑍2 𝑃𝑃𝑃𝑃𝑃𝑃, 𝑍𝑍2 𝑁𝑁𝑁𝑁𝑁𝑁) = (𝑍𝑍3 𝑃𝑃𝑃𝑃𝑃𝑃, 𝑍𝑍3 𝑁𝑁𝑁𝑁𝑁𝑁) = objective functions (𝑍𝑍1 𝑃𝑃𝑃𝑃𝑃𝑃, 𝑍𝑍1 𝑁𝑁𝑁𝑁𝑁𝑁) = (2770238,1114430) (𝑍𝑍2 𝑃𝑃𝑃𝑃𝑃𝑃, 𝑍𝑍2 𝑁𝑁𝑁𝑁𝑁𝑁) = (1472820 ,2227640) and (𝑍𝑍3 𝑃𝑃𝑃𝑃𝑃𝑃, 𝑍𝑍3 𝑁𝑁𝑁𝑁𝑁𝑁) = objective functions (𝑍𝑍1 𝑃𝑃𝑃𝑃𝑃𝑃, 𝑍𝑍1 𝑁𝑁𝑁𝑁𝑁𝑁) = (2770238,1114430) (𝑍𝑍2 𝑃𝑃𝑃𝑃𝑃𝑃, 𝑍𝑍2 𝑁𝑁𝑁𝑁𝑁𝑁) = (1472820 ,2227640) and (𝑍𝑍3 𝑃𝑃𝑃𝑃𝑃𝑃, 𝑍𝑍3 𝑁𝑁𝑁𝑁𝑁𝑁) = Third Stage: According to Equations (35) to (37) of the supplementary MOLP problem, create three new precise objective functions. In order to get the initial solutions for each of the objective functions, the original issue is solved using the standard single-objective LP method (3135912,1658514). The fuzzy aspiration levels can be quantified using the linear and continuous membership function. According to Eq. (39) and (40), the relevant linear membership functions can be defined as shown below. www.ejtas.com EJTAS 2023 \| Volume 1 \| Number 5 142 142 2023 \| Volume 1 \| Number 5 2023 \| Volume 1 \| Number 5 𝜇𝜇(𝑍𝑍1(𝑥𝑥)) = ⎩ ⎪ ⎨ ⎪ ⎧ 1 𝑍𝑍1(𝑥𝑥) ≥1114430 𝑍𝑍1(𝑥𝑥) −1114430 2770238 −1114430 1114430 ≤𝑍𝑍1(𝑥𝑥) ≤2770238 0 𝑍𝑍1(𝑥𝑥) ≤2770238 𝜇𝜇(𝑍𝑍2(𝑥𝑥)) = ⎩ ⎪ ⎨ ⎪ ⎧ 1 𝑍𝑍2(𝑥𝑥) ≤1472820 2227640 −𝑍𝑍2(𝑥𝑥) 2227640 −1472820 1472820 ≤𝑍𝑍2(𝑥𝑥) ≤2227640 0 𝑍𝑍2(𝑥𝑥) ≥2227640 1472820 2227640 1 𝜇𝜇(𝑍𝑍2(𝑥𝑥)) 0 𝑍𝑍2(𝑥𝑥) 𝜇𝜇(𝑍𝑍1(𝑥𝑥)) = ⎩ ⎪ ⎨ ⎪ ⎧ 1 𝑍𝑍1(𝑥𝑥) ≥1114430 𝑍𝑍1(𝑥𝑥) −1114430 2770238 −1114430 1114430 ≤𝑍𝑍1(𝑥𝑥) ≤2770238 0 𝑍𝑍1(𝑥𝑥) ≤2770238 𝜇𝜇(𝑍𝑍2(𝑥𝑥)) = ⎩ ⎪ ⎨ ⎪ ⎧ 1 𝑍𝑍2(𝑥𝑥) ≤1472820 2227640 −𝑍𝑍2(𝑥𝑥) 2227640 −1472820 1472820 ≤𝑍𝑍2(𝑥𝑥) ≤2227640 0 𝑍𝑍2(𝑥𝑥) ≥2227640 1472820 2227640 1 𝜇𝜇(𝑍𝑍2(𝑥𝑥)) 0 𝑍𝑍2(𝑥𝑥) 0 Equation (33), in addition, may be used to create the full equivalent single-objective LP model for the RPL situation. Additional Operational Data (Abridged) Workforce Production Z1 Z2 Z3 FGP Z1 Z2 Z3 FGP XL11 92.55738 92.78828 92.55738 92.78828 X111 246.8197 247.4354 246.8197 247.4354 XL12 102.8415 103.0981 102.8415 103.0981 X112 274.2441 274.9282 274.2441 274.9282 XL13 114.2684 114.5534 114.2684 114.5534 X113 304.7156 305.4758 304.7156 305.4758 XL14 120.0513 123.7031 121.95 99.43269 X114 320.1367 329.875 325.2 265.1538 XL15 120.0513 123.7031 121.95 110.4808 X115 320.1367 329.875 325.2 273.4154 XL16 120.0513 123.7031 121.95 96.08974 X116 320.1367 252.6713 325.2 248.3282 XL21 87.44262 87.21172 87.44262 87.21172 X121 74.0459 74.23062 74.0459 74.23062 XL22 97.15847 96.90192 97.15847 96.90192 X122 82.27322 82.47847 82.27322 82.47847 XL23 107.9539 107.6688 107.9539 107.6688 X123 91.41469 91.64274 91.41469 91.64274 XL24 119.9487 116.2969 118.05 119.632 X124 24.56328 98.9625 19.5 79.54615 XL25 119.9487 116.2969 118.05 129.5192 X125 41.66328 98.9625 36.6 88.38462 XL26 119.9487 116.2969 118.05 143.9103 X126 5.063279 98.9625 0 76.87179 XH11 0 282.7883 0 282.7883 X211 233.1803 232.5646 233.1803 232.5646 XH12 10.28415 290 10.28415 290 X212 259.0893 258.4051 259.0893 258.4051 XH13 11.42684 290 11.42684 290 X213 287.877 287.1168 287.877 287.1168 XH14 5.782905 290 7.681635 274.8793 X214 319.8633 310.125 314.8 228.2944 XH15 0 290 0 234.9509 X215 319.8633 310.125 314.8 270.4346 XH16 0 290 0 275.609 X216 319.8633 310.125 314.8 212.4718 XH21 0 277.2117 0 277.2117 X221 69.9541 69.76938 69.9541 69.76938 XH22 9.715847 290 9.715847 290 X222 77.72678 77.52153 77.72678 77.52153 XH23 10.79539 290 10.79539 10.76688 X223 86.36309 86.13504 86.36309 86.13504 XH24 11.99487 290 10.09614 290 X224 4.136721 93.0375 9.2 95.7056 XH25 0 290 0 290 X225 54.18672 93.0375 59.25 103.6154 XH26 0 290 0 14.39103 X226 7.736721 93.0375 12.8 115.1282 XF11 7.442624 290 7.442624 290 XF12 0 279.6902 0 279.6902 XF13 0 278.5447 0 278.5447 Table 6. Additional In aggregate production planning, it is crucial to understand the output table that results from the interaction of a dynamic labor and an uncertain demand. The dynamic workforce's influence on labor utilization and production capacity adds another level of complication. Finding patterns between labor variations and production output in response to various demand levels requires analyzing the Table 6 by the DM. It necessitates evaluating how the firm can fulfill erratic www.ejtas.com EJTAS 2023 \| Volume 1 \| Number 5 143 143 2023 \| Volume 1 \| Number 5 2023 \| Volume 1 \| Number 5 inventory control measures, allowing the company to optimize their production plans in the face of changing labor markets and workforce dynamics. demand while reducing overages or shortages by adjusting workforce numbers. Insights from Table 6 also be used to inform choices on resource allocation, hiring practices, and ational Data ged) Production Z1 Z2 Z3 FGP X111 246.8197 247.4354 246.8197 247.4354 X112 274.2441 274.9282 274.2441 274.9282 X113 304.7156 305.4758 304.7156 305.4758 X114 320.1367 329.875 325.2 265.1538 X115 320.1367 329.875 325.2 273.4154 X116 320.1367 252.6713 325.2 248.3282 X121 74.0459 74.23062 74.0459 74.23062 X122 82.27322 82.47847 82.27322 82.47847 X123 91.41469 91.64274 91.41469 91.64274 X124 24.56328 98.9625 19.5 79.54615 X125 41.66328 98.9625 36.6 88.38462 X126 5.063279 98.9625 0 76.87179 X211 233.1803 232.5646 233.1803 232.5646 X212 259.0893 258.4051 259.0893 258.4051 X213 287.877 287.1168 287.877 287.1168 X214 319.8633 310.125 314.8 228.2944 X215 319.8633 310.125 314.8 270.4346 X216 319.8633 310.125 314.8 212.4718 X221 69.9541 69.76938 69.9541 69.76938 X222 77.72678 77.52153 77.72678 77.52153 X223 86.36309 86.13504 86.36309 86.13504 X224 4.136721 93.0375 9.2 95.7056 X225 54.18672 93.0375 59.25 103.6154 X226 7.736721 93.0375 12.8 115.1282 Table 6. Additional Oper (Abrid Workforce Z1 Z2 Z3 FGP XL11 92.55738 92.78828 92.55738 92.78828 XL12 102.8415 103.0981 102.8415 103.0981 XL13 114.2684 114.5534 114.2684 114.5534 XL14 120.0513 123.7031 121.95 99.43269 XL15 120.0513 123.7031 121.95 110.4808 XL16 120.0513 123.7031 121.95 96.08974 XL21 87.44262 87.21172 87.44262 87.21172 XL22 97.15847 96.90192 97.15847 96.90192 XL23 107.9539 107.6688 107.9539 107.6688 XL24 119.9487 116.2969 118.05 119.632 XL25 119.9487 116.2969 118.05 129.5192 XL26 119.9487 116.2969 118.05 143.9103 XH11 0 282.7883 0 282.7883 XH12 10.28415 290 10.28415 290 XH13 11.42684 290 11.42684 290 XH14 5.782905 290 7.681635 274.8793 XH15 0 290 0 234.9509 XH16 0 290 0 275.609 XH21 0 277.2117 0 277.2117 XH22 9.715847 290 9.715847 290 XH23 10.79539 290 10.79539 10.76688 XH24 11.99487 290 10.09614 290 XH25 0 290 0 290 XH26 0 290 0 14.39103 XF11 7.442624 290 7.442624 290 XF12 0 279.6902 0 279.6902 XF13 0 278.5447 0 278.5447 XF14 0 280.8503 0 290 XF15 0 290 0 223.9028 www.ejtas.com EJTAS 2023 \| Volume 1 \| Number 5 144 144 2023 \| Volume 1 \| Number 5 2023 \| Volume 1 \| Number 5 XF16 0 290 0 290 XF21 12.55738 290 12.55738 290 XF22 0 280.3098 0 280.3098 XF23 0 279.2331 0 0 XF24 0 281.3719 0 278.0368 XF25 0 290 0 280.1128 XF26 0 290 0 0 helps businesses deal with the difficulties of varying demand, unpredictability in the supply chain, and cost considerations by concentrating on the overall picture of production over a specific time horizon. Conclusions In conclusion, the journal article "Possibilistic Aggregate Production Planning Considering Dynamic Workforce with Fuzzy Demand" clarifies the complicated world of production planning by fusing the complexity of dynamic workforce fluctuations and fuzzy demand forecasting. The study emphasizes the necessity of flexibility in resource allocation to successfully traverse the obstacles given by variable worker capabilities and uncertain demand. The paper provides insightful information on minimizing production risks and maximizing operational effectiveness by using a possibilistic approach. However, this study serves as a first step toward a deeper comprehension of this complex dynamic. Future research in this area may focus further on the creation of sophisticated prediction models that combine labor dynamics, demand volatility, and other contextual elements to improve the accuracy of decisions. Investigating the integration of cutting-edge technologies like artificial intelligence and machine learning might also result in ground- breaking approaches to agile production planning in the face of flexible demand scenarios and a mobile workforce. References Ballou, R.H. (2004) Business Logistics/Supply Chain Management: Planning, Organizing and Controlling the Supply chain. 5th Edition, Pearson/Prentice Hall Inc., New Jersey. Bellman, R.E. & Zadeh, L.A. (1970). Decision- making in a fuzzy environment. Management Science, 17(4), 141–164. Bradley, J. (2023). Warehousing: Warehousing Functions, Importance, and Benefits; Blue Cart. Retrieved from https://www.bluecart.com/blog/warehousing- functions-importance-benefits https://www.bluecart.com/blog/warehousing- functions-importance-benefits Dettmer, W.H. (1997). Goldratt’s theory of constraint: A systems approach to continuous improvement. Later Printing. Du, M., & Leung, S. Y. S. (2007). A fuzzy model for production-distribution planning in supply chain. European Journal of Operational Research, 178(3), 789-807. Effective production planning has become a key approach for firms to ensure efficient operations, satisfy consumer needs, and maximize resource usage in today's dynamic and competitive business environment. Aggregate Production Planning (APP), one of the many planning methods, stands out as a crucial strategy that enables companies to strike a careful balance between production levels and inventories while staying in line with market expectations. Aggregate production planning https://doi.org/10.22436/jmcs.002.01.08 https://www.semanticscholar.org/paper/Multi -period-%2C-Multi-product-%2C-Aggregate- Production-Hossain- Nahar/3b72a55d180334313ef4681614267751c bdfece0#paper-header Madanhire I, & Mbohwa C. (2015). Aggregate Production Planning Framework in a Multi-Product Factory. Proceedings of the 2015 International Conference on Industrial Engineering and Operations Management. Dubai, UAE. Hwang, C.L. & Yoon, K. (1981). Multiple Attribute Decision Making: Methods and Applications. Springer, Berlin. Mangan, J., Lalwani, C., Butcher, T. & Javadpour, R. (2011). Global Logistics and Supply Chain Management. Wiley. Jime´nez, M. (1996). Ranking fuzzy numbers through the comparison of its expected intervals. International Journal of Uncertainty, Fuzziness and Knowledge-Based Systems, 4(4), 379– 388. Nahmias, S. (2005). Production and Operations Analysis. Retrieved from https://www.researchgate.net/publication/243 773464_Production_and_Operations_Analysis https://doi.org/10.1142/S0218488596000226 1064. https://doi.org/10.1016/j.cie.2008.09.017 https://doi.org/10.1016/j.cie.2008.09.017 Guneri, A. F., & Olgun, H. (2018). A fuzzy multi-objective model for aggregate production planning with workforce allocation. Soft Computing, 22(15), 4959-4973. Leung, S. C., Wu, Y., & Lai, K. K. (2003). Multi- site aggregate production planning with multiple objectives: a goal programming approach, Production Planning and Control, 14(5), 425- 436. Hannan, E. L. (1981). Linear programming with multiple fuzzy goals. Fuzzy Sets and Systems, 6(3), 235-248. https://doi.org/10.1016/0165- 2 2 https://www.semanticscholar.org/paper/Multi -period-%2C-Multi-product-%2C-Aggregate- Production-Hossain- Nahar/3b72a55d180334313ef4681614267751c bdfece0#paper-header https://doi.org/10.22436/jmcs.002.01.08 Fung, R. Y., Tang, J., and Wang, Q. (2003). Multiproduct aggregate production planning with fuzzy demands and fuzzy capacities. IEEE Transactions on Systems, Man and Cybernetics, Part A: Systems and Humans, 33(3), 302-313. https://doi.org/10.1109/TSMCA.2003.820609 Guillermo G. (2001) Production Management; IEOR 4000: Lecture 5. Retrieved from 145 EJTAS 2023 \| Volume 1 \| Number 5 EJTAS http://www.columbia.edu/~gmg2/4000/pdf/l ect_05.pdf https://doi.org/10.1080/095372803100015426 Li, B., Wang, H., Yang, J., Guo, M., & Qi, C. (2013). A belief-rule-based inference method for aggregate production planning under uncertainty. International Journal of Production Research, 51(1), 83-105. https://doi org/10 1080/00207543 2011 65226 0114(81)90002-6 Hossain, M.M., Nahar, K., Reza, S., & Shaifullah, K.M. (2016). Multi-period, Multi-product, Aggregate Production Planning under demand uncertainty by considering Wastage Cost and Incentives. Retrieved from https://www.semanticscholar.org/paper/Multi -period-%2C-Multi-product-%2C-Aggregate- Production-Hossain- Nahar/3b72a55d180334313ef4681614267751c bdfece0#paper-header Hossain, M.M., Nahar, K., Reza, S., & Shaifullah, K.M. (2016). Multi-period, Multi-product, Aggregate Production Planning under demand uncertainty by considering Wastage Cost and Incentives. Retrieved from https://doi.org/10.1142/S0218488596000226 Narasimhan, R. & Talluri, S. (2009). Perspectives on Risk Management in Supply Chains. Journal of Operations Management, 27, 114-118. https://doi.org/10.1016/j.jom.2009.02.001 Jime´nez, M., Rodrı´guez, M.V., Arenas, M., & Bilbao, A. (2005). Linear programming with fuzzy parameters: An interactive method resolution. European Journal of Operational Research, 17(2007), 1599–1609 Phruksaphanrat, B., Ohsato, A. & Yenradee, P. (2006). A comment on the formulation of an aggregate production planning problem. The 2nd IEEE International Conference on Cybernetics and Intelligent Systems, 292-297. https://doi.org/10.1016/j.ejor.2005.10.002 Lai, Y.J., & Hwang, C.L. (1992). Fuzzy Mathematical Programming: Methods and Applications. Springer, Berlin. Pishvaee, M., Zanjirani F., Reza & Dullaert, W. (2010). A memetic algorithm for bi-objective integrated forward/reverse logistics network design. Computers & Operations Research, 37, 1100- 1112. Altomonte, L. (2023). Functions of a Warehouse. Safetyculture. Retrieved from Altomonte, L. (2023). Functions of a Warehouse. Safetyculture. Retrieved from https://safetyculture.com/topics/warehouse- management/functions-of-a-warehouse/ https://doi.org/10.1016/j.cor.2009.09.018 Leung, S. C. & Chan, S. S. (2009). A goal programming model for aggregate production planning with resource utilization constraint. Computers and Industrial Engineering, 56(3), 1053- Sakall, U.S., Baykoç, O.F., & Birgoren, B. (2010). A possibilistic aggregate production planning model for brass casting industry. Production 146 EJTAS 2023 \| Volume 1 \| Number 5 www.ejtas.com EJTAS Planning andControl, 21(3), 319-338. https://doi.org/10.1080/09537280903449438 based on MINMAX approach. European Journal of Operational Research, 177, 1580–1590. https://doi.org/10.1016/j.ejor.2005.10.022 Planning h //d Silver, E., Pyke, D. & Peterson, R. (1998). Inventory Management and Production Scheduling. Retrieved from https://www.researchgate.net/publication/229 124356_Inventory_Management_and_Producti on_Scheduling Silver, E., Pyke, D. & Peterson, R. (1998). Inventory Management and Production Scheduling. Retrieved from Yen, B. P. C., & Liu, C. (2009). A fuzzy aggregate production planning model for make-to-order industry. International Journal of Production Research, 47(10), 2647-2668. on_Scheduling https://doi.org/10.1109/IEEM.2007.4419464 Zadeh, L.A. (1965). Fuzzy Sets. Information Control, 8, 338-353. http://dx.doi.org/10.1016/S0019- 9958(65)90241-X Zadeh, L.A. (1965). Fuzzy Sets. Information Control, 8, 338-353. Sunderesh, S. H. (2022). Facilities Design. 5th Edition. CRC Press. Taleizadeh, A. A., & Pentico, D. W. (2012). Fuzzy multi-objective linear programming approaches to aggregate production planning problems. Computers & Industrial Engr, 62(2), 368-381. Zadeh, L.A. (1978). Fuzzy sets as a basis for a theory of possibility. Fuzzy Sets and Systems, 1, 3– 28. https://doi.org/10.1016/S0165- 0114(99)80004 9 0114(99)80004-9 Wang, R.C. & Fang, H.H. (2001) Aggregate production planning with multiple objectives in a fuzzy environment. European Journal of Operational Research, 133, 521–536. https://doi.org/10.1016/S0377- 2217(00)00196-X Zhang, R., Zhang, L., Xiao, Y., & Kaku, I. (2012). The activity-based aggregate production planning with capacity expansion in manufacturing systems. Computers and Industrial Engineering, 62(2), 491-503. https://doi.org/10.1016/j.cie.2011.10.016 Wang, R.C., & Liang, T.F. (2004). Application of fuzzy multiobjective linear programming to aggregate production planning. Computers and Industrial Engineering, 46(1), 17-41. https://doi.org/10.1016/j.cie.2003.09.009 Zimmermann, H.J. (1991). Fuzzy Set Theory and Its Applications, (2nd rev .ed). Boston: Kulwer,. Zimmermann, H.J. (1976). Description and optimization of fuzzy systems. International Journal of General Systems, 2, 209–215. https://doi.org/10.1080/03081077608547470 Wang, R.-C. & Liang, T.-F. (2005). Applying possibilistic linear programming to aggregate production planning. International Journal of Production Economics, 98, 328-341. https://doi.org/10.1016/j.ijpe.2004.09.011 Zimmermann, H.-J. (1978). Fuzzy programming and linear programming with several objective functions. Fuzzy Sets and Systems, 1, 45–56. https://doi.org/10.1016/0165-0114(78)90031- Woeppel, M.J. (2001). Manufacturer’s guide to implementing the theory of constraints. St. Lucie Press, New York. Yaghoobi, M.A., & Tamiz, M. (2007). A method for solving fuzzy goal programming problems www.ejtas.com EJTAS 2023 \| Volume 1 \| Number 5 147 147 2023 \| Volume 1 \| Number 5 2023 \| Volume 1 \| Number 5
https://openalex.org/W4362470031	https://hal.science/hal-04055988/file/Maitre%20et%20al.%202023.pdf	English	null	Myelin in Alzheimer’s disease: culprit or bystander?	Acta neuropathologica communications	2,023	cc-by	19,303	Myelin in Alzheimer’s disease: culprit or bystander? Michel Maitre, Hélène Jeltsch-David, Nwife Getrude Okechukwu, Christian Klein, Christine Patte-Mensah, Ayikoe-Guy Mensah-Nyagan To cite this version: Michel Maitre, Hélène Jeltsch-David, Nwife Getrude Okechukwu, Christian Klein, Christine Patte- Mensah, et al.. Myelin in Alzheimer’s disease: culprit or bystander?. Acta Neuropathologica Com- munications, 2023, 11, pp.56. 10.1186/s40478-023-01554-5. hal-04055988 Distributed under a Creative Commons Attribution 4.0 International License © 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://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. REVIEW Open Access HAL Id: hal-04055988 https://hal.science/hal-04055988v1 Submitted on 3 Apr 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 (2023) 11:56 Acta Neuropathologica Communications Maitre et al. Acta Neuropathologica Communications https://doi.org/10.1186/s40478-023-01554-5 Abstract Alzheimer’s disease (AD) is a neurodegenerative disorder with neuronal and synaptic losses due to the accumulation of toxic amyloid β (Αβ) peptide oligomers, plaques, and tangles containing tau (tubulin-associated unit) protein. While familial AD is caused by specific mutations, the sporadic disease is more common and appears to result from a complex chronic brain neuroinflammation with mitochondriopathies, inducing free radicals’ accumulation. In aged brain, mutations in DNA and several unfolded proteins participate in a chronic amyloidosis response with a toxic effect on myelin sheath and axons, leading to cognitive deficits and dementia. Αβ peptides are the most frequent form of toxic amyloid oligomers. Accumulations of misfolded proteins during several years alters different metabolic mechanisms, induce chronic inflammatory and immune responses with toxic consequences on neuronal cells. Myelin composition and architecture may appear to be an early target for the toxic activity of Aβ peptides and others hydrophobic misfolded proteins. In this work, we describe the possible role of early myelin alterations in the genesis of neuronal alterations and the onset of symptomatology. We propose that some pathophysiological and clinical forms of the disease may arise from structural and metabolic disorders in the processes of myelination/demyelination of brain regions where the accumulation of non-functional toxic proteins is important. In these forms, the primacy of the deleterious role of amyloid peptides would be a matter of questioning and the initiating role of neuropathology would be primarily the fact of dysmyelination. Keywords Myelin, Early biomarker, Alzheimer’s disease, Oligodendrocytes, Aβ peptides Introduction Sporadic AD typically occurs after the age of 65 and is the most common cause of dementia in older people. We consider here the disease under its main pathophysi ological definition that classically consists of extracellular amyloid plaques and intracellular neurofibrillary tangles [69, 70]. These abnormalities lead to a cascade of events eventually conducting to cognitive disorders and demen tia. There are, however, a significant number of diverse clinical presentations with ages of onset and evolution in the disease that suggest non-univocal pathophysiological mechanisms [61]. It seems that some cases of sporadic AD involve changes in the constitution and architecture of myelin, and this early in the life of the future patient. Correspondence: Michel Maitre maitre@unistra.fr 1Biopathologie de la Myéline, Neuroprotection et Stratégies Thérapeutiques, Fédération de Médecine Translationnelle de Strasbourg (FMTS), INSERM U1119, Université de Strasbourg, Bâtiment CRBS de la Faculté de Médecine, 1 rue Eugène Boeckel, Strasbourg 67000, France 2Biotechnologie et signalisation cellulaire, UMR 7242 CNRS, Université de Strasbourg, 300 Boulevard Sébastien Brant CS 10413, Illkirch cedex 67412, France Myelin in Alzheimer’s disease: culprit or bystander? Michel Maitre1 , Hélène Jeltsch-David1,2 , Nwife Getrude Okechukwu1 , Christian Klein1 , Christine Patte-Mensah1 and Ayikoe-Guy Mensah-Nyagan1 In general, extracellular abnormalities of the amyloid cascade predominate, followed most often by other Tau mediated biological mechanisms at the intracellular level, accompanied by inflammatory, neuroimmune and neu rochemical disorders that can put dysmyelination at the forefront of neurodegenerative disorders. y g [ ] AD involves progressive neurodegeneration with neu ronal losses leading to cognitive, memory, emotional, behavioral disorders, and a progressive dependence [210]. Long considered to primarily affect the grey mat ter, many studies have described early lesions of the white matter in patients with nascent and moderate intensity [55]. The main molecular alterations of the disease are considered to contain essentially a pathology of the pro duction/degradation and an intra-brain accumulation of amyloid β (Aβ) peptides producing deposits in the form of hydrophobic plates of aggregated toxic peptides (senile plaques) [69, 159]. Another proteinopathy usually accom panies the previous protein in the form of hyperphos phorylated tau proteins and deposits of neurofibrillary tangles. These toxic proteins maintain chronic inflam mation and oxidative stress accompanied by neuronal and synaptic losses at the origin of the symptomatology [31]. This paper reviews the fundamental importance of myelin and correct CNS myelination for its develop ment, functional adaptations, and permanent reshuf fling. During aging in the human patient, sporadic AD Myelin consists of a multilayered membrane wrapped around the axons of most central nervous system (CNS) neurons. This membrane is produced by expansions of specialized glial cells of the brain, “the mature oligo dendrocytes”, derived from oligodendrocytes progenitor cells (OPCs) [21]. This cell line constitutes the precursor cells for the constitution of the myelin sheath through a well-defined program of proliferation, migration, and dif ferentiation to lead to the myelination of neuronal axons [100]. Among the properties of myelin, the best known is the saltatory conduction of nerve impulses, which gives it more speed and efficiency. Furthermore, it is now well acknowledged that oligodendrocytes’ expansions display a trophic, plastic, and metabolic influence on the axons they envelop (Fig. 1) [130, 139]. Myelin is constantly reshaping and its alteration in degenerative phenomena such as Alzheimer’s disease (AD) may be a fundamental Fig. 1 Oligodendrocytes are derived from the differentiation of oligodendrocyte precursor cells (OPCs) and are the main cell for remyelin ation. (modified from [175]). The differentiated oligodendrocytes of OPCs migrate to different axons via positive chemotactism [216]. © 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://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. Maitre et al. Acta Neuropathologica Communications (2023) 11:56 Maitre et al. Acta Neuropathologica Communications (2023) 11:56 Page 2 of 18 (2023) 11:56 Maitre et al. Acta Neuropathologica Communications element for the genesis of pathophysiological and clinical disorders observed in the early stages of the disease [22]. AD involves progressive neurodegeneration with neu ronal losses leading to cognitive, memory, emotional, behavioral disorders, and a progressive dependence [210]. Long considered to primarily affect the grey mat ter, many studies have described early lesions of the white matter in patients with nascent and moderate intensity [55]. The main molecular alterations of the disease are considered to contain essentially a pathology of the pro duction/degradation and an intra-brain accumulation of amyloid β (Aβ) peptides producing deposits in the form of hydrophobic plates of aggregated toxic peptides (senile plaques) [69, 159]. Another proteinopathy usually accom panies the previous protein in the form of hyperphos phorylated tau proteins and deposits of neurofibrillary tangles. These toxic proteins maintain chronic inflam mation and oxidative stress accompanied by neuronal and synaptic losses at the origin of the symptomatology [31]. This paper reviews the fundamental importance of myelin and correct CNS myelination for its develop ment, functional adaptations, and permanent reshuf fling. During aging in the human patient, sporadic AD element for the genesis of pathophysiological and clinical disorders observed in the early stages of the disease [22]. A variety of growth and trophic factors regulate the development of oligodendrocytes and their temporal and geographical attractions [17]. Many of these factors are pro duced by both neurons and astrocytes, regulating the proliferation, survival, or degeneration of OPCs. The neuroregulin, which activates Erb-tyrosines kinases receptors, promotes the survival and proliferation of oligodendrocytes. The activation of the Notch 1 cascade inhibits the differentiation of oligo dendrocytes, and an integrin/contactin complex coordinates signals from the extracellular matrix and the axonal surface to regulate oligodendrocyte sur vival and myelination. This also depends closely on the electrical activity propagated in the axons. OPCs express functional adenosine receptors, activated by action potential [149, 183]. Adenosine acts as a powerful transmitter between glia and neurons to inhibit the proliferation of OPCs, stimulate their dif ferentiation and stimulate myelin production. The LIF (leukemia inhibitory factor) is heavily involved in oligodendrocyte development kinetics and in the overall myelination process [133, 198]. Abbreviations: CNTF, ciliary neurotrophic factor; FGF, fibroblast growth factor; IGF, insulin-like growth factor; LIF, leuke mia inhibitory factor; NCAM, neural cell adhesion molecule; NT-3, neurotrophin 3; OPCs, oligodendrocyte precursor cells; PDGF-A, platelet-derived growth factor-A tes are derived from the differentiation of oligodendrocyte precursor cells (OPCs) and are the main cell for remyel Fig. 1 Oligodendrocytes are derived from the differentiation of oligodendrocyte precursor cells (OPCs) and are the main cell for remyelin ation. (modified from [175]). The differentiated oligodendrocytes of OPCs migrate to different axons via positive chemotactism [216]. A variety of growth and trophic factors regulate the development of oligodendrocytes and their temporal and geographical attractions [17]. Many of these factors are pro duced by both neurons and astrocytes, regulating the proliferation, survival, or degeneration of OPCs. The neuroregulin, which activates Erb-tyrosines kinases receptors, promotes the survival and proliferation of oligodendrocytes. The activation of the Notch 1 cascade inhibits the differentiation of oligo dendrocytes, and an integrin/contactin complex coordinates signals from the extracellular matrix and the axonal surface to regulate oligodendrocyte sur vival and myelination. This also depends closely on the electrical activity propagated in the axons. OPCs express functional adenosine receptors, activated by action potential [149, 183]. Adenosine acts as a powerful transmitter between glia and neurons to inhibit the proliferation of OPCs, stimulate their dif ferentiation and stimulate myelin production. The LIF (leukemia inhibitory factor) is heavily involved in oligodendrocyte development kinetics and in the overall myelination process [133, 198]. Abbreviations: CNTF, ciliary neurotrophic factor; FGF, fibroblast growth factor; IGF, insulin-like growth factor; LIF, leuke mia inhibitory factor; NCAM, neural cell adhesion molecule; NT-3, neurotrophin 3; OPCs, oligodendrocyte precursor cells; PDGF-A, platelet-derived growth factor-A Fig. 1 Oligodendrocytes are derived from the differentiation of oligodendrocyte precursor cells (OPCs) and are the main cell for remyelin ation. (modified from [175]). The differentiated oligodendrocytes of OPCs migrate to different axons via positive chemotactism [216]. A variety of growth and trophic factors regulate the development of oligodendrocytes and their temporal and geographical attractions [17]. Many of these factors are pro duced by both neurons and astrocytes, regulating the proliferation, survival, or degeneration of OPCs. The neuroregulin, which activates Erb-tyrosines kinases receptors, promotes the survival and proliferation of oligodendrocytes. The activation of the Notch 1 cascade inhibits the differentiation of oligo dendrocytes, and an integrin/contactin complex coordinates signals from the extracellular matrix and the axonal surface to regulate oligodendrocyte sur vival and myelination. This also depends closely on the electrical activity propagated in the axons. OPCs express functional adenosine receptors, activated by action potential [149, 183]. Adenosine acts as a powerful transmitter between glia and neurons to inhibit the proliferation of OPCs, stimulate their dif ferentiation and stimulate myelin production. The LIF (leukemia inhibitory factor) is heavily involved in oligodendrocyte development kinetics and in the overall myelination process [133, 198]. Abbreviations: CNTF, ciliary neurotrophic factor; FGF, fibroblast growth factor; IGF, insulin-like growth factor; LIF, leuke mia inhibitory factor; NCAM, neural cell adhesion molecule; NT-3, neurotrophin 3; OPCs, oligodendrocyte precursor cells; PDGF-A, platelet-derived growth factor-A Maitre et al. Acta Neuropathologica Communications (2023) 11:56 Maitre et al. Acta Neuropathologica Communications (2023) 11:56 Maitre et al. Acta Neuropathologica Communications (2023) 11:56 Page 3 of 18 (2023) 11:56 Maitre et al. Acta Neuropathologica Communications Interestingly, normal adults at the cognitive level show microstructural changes in myelin architecture when carrying homozygous alleles ε4 that is a major marker for late AD [128, 145, 146]. has plurifocal impairments that induces various clinical presentations depending on the intensity of inflamma tory and immune reactions, and ischemic, mitochondrial, and free radical disorders. In many cases, the hypothesis of an alteration of the amyloid cascade Aβ as a primitive mechanistic etiology is questionable and multiple pro teinopathies can be implicated, which depend on somatic mosaicism, transcriptional and translational alterations. Multiple causes, multiple targets AD as an heterogeneous diseaseh The amyloid hypothesis as the essential cause of neuronal loss and brain atrophy is a matter of discussion mainly because removal or reduction of amyloid plaques by immunological treatments display no significant effect on clinical symptoms of AD [39, 140]. However, before aggregation, the soluble oligomeric forms of Aβ peptides possess strong toxic properties against myelin integrity and neuronal survival [160, 188]. White matter lesions are commonly found in magnetic resonance imaging (MRI) scans of elderly people and are associated with cognitive decline [128]. Whether or not a primary role of Aβ peptides is fundamental in these lesions is a matter of debate. Increase in the concentration of Aβ peptides in brain has also been described because of head trauma or cerebral ischemia [178, 220]. This increase in the con centration of Aβ peptides is additional evidence for the existence of various forms of Alzheimer’s like diseases with various clinical pictures and various mechanisms of neurodegenerative processes in term of pathophysi ological evolution and biological markers [43, 214]. In addition to Aβ peptides accumulation and toxicity, hyperphosphorylation of tau proteins, which disturbs microtubules assembly and axonal transport, could have some impact on the trophic effect on the myelin envelop [209]. These basic alterations are also supplemented with several others biological modifications of many molecu lar pathways and functions namely in the domain of energy metabolism and cholesterol transports [19]. Cho lesterol is fundamental for oligodendrocytes survival and for synthetis of myelin, this compounds represent a large proportion of the human brain and abnormalities in cholesterol metabolism are present and associated with brain age and in Alzheimer’s disease [12, 105]. Regarding cholesterol delivery to axons and synapses, the ε4 allele of the apolipoprotein E (APOE) gene is the less effective factor for cholesterol transport compared to ε3 and ε2. Accumulating evidence supports a multi-factor for the origin of many forms of sporadic AD. Multi-organ altera tions could initiate or worsen neurodegeneration [8, 193, 208]. Developing heart failure promoting hypoxia, intes tinal and hepatic disorders altering brain metabolism through the microbiome, ischemic symptoms due to vas cular deposits and chronic inflammation, could also con tribute to the decrease in neuronal survival [114, 197]. Sporadic AD is a multifactorial disease Sporadic AD is a multifactorial disease While familial AD has essentially genetic causes expressed in amyloid precursor protein (APP) and pre senilin leading early in life to specific proteinopathies and inducing neurodegenerative pathologies [63], these mechanisms are less consistent in the sporadic AD of elderly subjects. In these patients displaying accumula tions of cerebral amyloid peptides, the question arises as to whether this accumulation is the cause or the con sequence of other factors inducing neurodegeneration. Over the years, many mutations are present in brain neurons, generating multiple proteinopathies after tran scriptional, translational, or post-translational errors. Some of these are of exogenous origin and enter the CNS due to the pathological porosity of the blood-brain bar rier (BBB). The accumulation of these abnormal proteins generates inflammatory and immune responses that lasts for many years. Such accumulation is increased by mito chondriopathies, and the genesis of free radicals related to the disruption of oxidative phosphorylation and the depreciation of energy metabolism. The most deleterious and widespread proteinopathy is that affecting the regu lated proteolysis of the APP, giving rise to toxic peptides interfering with many neuronal functions and leading to synaptic and neuronal losses, as well as inducing pro found cognitive and behavioral functional abnormalities. These peptides exhibit amyloid properties and accumu late over time into hydrophobic plaques, which can be detected by PET scan ligands or post-mortem histology. While familial AD has essentially genetic causes expressed in amyloid precursor protein (APP) and pre senilin leading early in life to specific proteinopathies and inducing neurodegenerative pathologies [63], these mechanisms are less consistent in the sporadic AD of elderly subjects. In these patients displaying accumula tions of cerebral amyloid peptides, the question arises as to whether this accumulation is the cause or the con sequence of other factors inducing neurodegeneration. In many cases, myelin and its integrity appear to be a preferential and early target in multiple forms of AD, and oligodendrocytes represent a cell population highly sen sitive to Aβ and other proteinopathies. These misfolded proteins result in multiple dysmetabolism that accentuate and modify the course and clinical forms of the disease. Myelin has morphological alterations in the early stages of AD Change of the lipid composition of myelin over time [127] Oligodendrocytes-derived myelin accounts for about 40% of CNS lipids, consisting of 50% phospholipids, 40% glycolipids, 10% cholesterol and cholesterol esters, and polyunsaturated long-chain fatty acids (Fig. 3) [82] . At the hepatic level, functional alterations (cirrhosis, hepatitis) could aggravate the elimination of deleterious proteins including Aβ [201]. Modifications of bile acids synthesized by the altered liver tissue exhibit impaired neuroprotective functions. Frequently, the accumulation of mutations in the mitochondrial genome accelerates pathological phenomena at the level of the tricarboxylic cycle or the respiratory chain and increase ROS produc tion [73]. Metabolic disorders of the periphery of the body often affect brain metabolism via abnormal perme ability of BBB and the presence of abnormal metabolites in the cerebrospinal fluid (CSF). This mainly concerns certain intermediates of amino acids metabolism, par ticularly regarding the catabolites of tryptophan degrada tion [167]. This essential amino acid is the precursor not Synthesized by oligodendrocytes, cholesterol comes almost exclusively from ketone bodies as precursors. This lipid has structural functions at the level of the myelin by regulating the fluidity and permeability of this mem brane around the axons, and it regulates the speed of myelination according to its uptake by the membrane in formation. The typical lipids of myelin are essen tially galactosyl ceramides and sulfatides. They stabilize and organize myelin in direct association with the basic protein.i Changes in the configuration of myelin are observed with age but are more accentuated in AD. Not all regions of the brain are affected in the same way. In general, the volume of white matter decreases over time and the phe nomena of demyelination/remyelination accentuated by pathology leads to the decrease in the size of axons and the reduction in the size of the internodal distances. These structural changes induce functional consequences for conduction rates and vulnerability to traumatic, isch emic, dysmetabolic conditions and toxic factors such as oligomers Aβ peptides. These processes are commonly encountered as factors favoring Alzheimer’s genesis and pathology. Fig. 2 Dysregulation in multiple biochemical pathways underlie the pathogenesis of AD. Metabolomic approaches conducted from the blood or CSF of AD patients compared to controls highlighted ab normalities in the energy metabolism of patients. A diabetic-type pathol ogy is often evoked with a decrease in insulin sensitivity. Metabolic disorders and AD Many metabolic alterations have often been encountered in AD with varying severities. Most of these alterations concern or affect brain energy metabolism, these phe nomena being aggravated by cerebral hypoperfusion and blood sugar abnormalities [10]. Carbohydrate metabo lism and dysfunctions in intestinal absorption phenom ena, nutritional abnormalities and deficiencies, resistance to glucose utilization via decreased insulin sensitivities combined with fatty acid metabolism disorders, induce energy deficiencies deleterious to neuronal functioning Maitre et al. Acta Neuropathologica Communications (2023) 11:56 (2023) 11:56 Page 4 of 18 (2023) 11:56 Maitre et al. Acta Neuropathologica Communications only of melatonin and serotonin, but also of the interme diates of the kynurenine cycle, some of which possess neuroprotective or neurotoxic properties or interfere with the elimination of amyloid peptides from the brain [116]. and survival [85]. The slowing down of the tricarboxylic cycle generates the accumulation of acetyl-CoA coming from the increased fatty acids degradation and the syn thesis of ketone bodies that could have a positive role on neuronal survival. White matter degeneration in AD could be in part due to the accelerated degradation of lipids in this context of decreased energetic metabolism coming from reduced glucose utilization (Fig. 2) [141, 206]. Myelin has morphological alterations in the early stages of AD In addition to disorders in glycolysis and the respiratory chain, abnormalities involving accumulations of ketone bodies resulting from the metabolism of acetyl- CoA residues, a product of the accelerated degradation of fatty acids by β-oxidation, have been described. The bioavailability and metabolism of several amino acids could also be affected, especially concerning tryp tophan degraded in the kynurenine cycle and resulting in the formation of neuroprotective (kynurenic acid) or neurotoxic (quinolinic acid) com pounds. Abbreviations: AD, Alzheimer’s disease; CSF, cerebrospinal fluid; NADH, nicotinamide-adenine-dinucleotide; ROS, reactive oxygen species; TCA, tricarboxylic acid; βOHD, beta-hydroxybutyrate A step for the conversion of mild cognitive impairment (MCI) into dementia? Studies of myelin sheath’s conformation in AD were mostly conducted by electron microscopy and MRI both in animals and humans. The 5XFAD mouse is a trans genic model that expresses three different mutations in the APP and two in presinilin 1 (PS1). In this mouse, amyloid deposits can be detected with synaptic losses at an age as early as 1.5 months [142] and myelin abnor malities can be seen even earlier accompanying the first alterations in spatial memory occurring around the age of 1 month [67]. Several studies conducted in humans suggest that myelin disorders strongly contribute to the onset of AD symptoms. Neuroimaging shows myelina tion defects in several brain regions, but especially and firstly in the hippocampus and corpus callosum [55, 137, 147, 202]. Conformation abnormalities accompanied by Fig. 2 Dysregulation in multiple biochemical pathways underlie the pathogenesis of AD. Metabolomic approaches conducted from Fig. 2 Dysregulation in multiple biochemical pathways underlie the pathogenesis of AD. Metabolomic approaches conducted from the blood or CSF of AD patients compared to controls highlighted ab normalities in the energy metabolism of patients. A diabetic-type pathol ogy is often evoked with a decrease in insulin sensitivity. In addition to disorders in glycolysis and the respiratory chain, abnormalities involving accumulations of ketone bodies resulting from the metabolism of acetyl- CoA residues, a product of the accelerated degradation of fatty acids by β-oxidation, have been described. The bioavailability and metabolism of several amino acids could also be affected, especially concerning tryp tophan degraded in the kynurenine cycle and resulting in the formation of neuroprotective (kynurenic acid) or neurotoxic (quinolinic acid) com pounds. Abbreviations: AD, Alzheimer’s disease; CSF, cerebrospinal fluid; NADH, nicotinamide-adenine-dinucleotide; ROS, reactive oxygen species; TCA, tricarboxylic acid; βOHD, beta-hydroxybutyrate (2023) 11:56 Page 5 of 18 Maitre et al. Acta Neuropathologica Communications Fig. 3 Myelin composition and organization. The myelin wrapping around most of the CNS axons includes a large majority of complex lipids and 15–30% of specific proteins. Lipids are essentially made up of cholesterol, galactocerebrosides and phospholipids. This envelope is constantly reshuffled in time and space from the oligodendrocytes that make up the bulk of the glial cells of the CNS. Chronic inflammatory and autoimmune reactions, muta tions in certain constituent proteins, attacks by free radicals or ischemic, and metabolic problems related to aging alter the myelin sheath that releases its constituents into the CSF and the bloodstream. A step for the conversion of mild cognitive impairment (MCI) into dementia? Abbreviations: AD, Alzheimer’ disease; CNP, C-type natriuretic peptide; CNS, central nervous system; CSF, cerebrospinal fluid; MAG, myelin-associated glycoprotein; MBP, myelin basic protein; MOG, myelin oligodendrocyte glycoprotein; PLP, proteolipid protein thinning of the myelin sheath are frequently encountered even before the onset of axonal lesions, which may indi cate premises for demyelination. In the pre-clinical stages of the disease, MRI shows altered longitudinal and trans verse relaxation times and increased myelin hydration degrees [18]. In general, abnormalities in the structure and formation of the cerebral white matter have been identified in many presentations of Alzheimer’s disease that can be warning signs for a disease in progress [156]. Variations in T1w/T2w ratios in patients with risk factors (close family history, APOE4 phenotypes) were identified compared to control individuals. In addition, individu als at risk had an association with altered patterns of resting-state functional connectivity (rs-FC) [52]. These abnormalities support the idea of significant alterations in myelin developing with age and constituting signals of vulnerability [53]. Interestingly, some studies have shown a relationship between structural abnormalities of myelin in ADs in the pre-clinical period and peptide concentra tions of Aβ1–42 in patients’ CSF [34, 38]. S d f l fi h h b are related to axonal lesions and inflammatory disorders, BBB permeability abnormalities and multiple dissemi nated micro-hemorrhagic structures [88, 102]. thinning of the myelin sheath are frequently encountered even before the onset of axonal lesions, which may indi cate premises for demyelination. In the pre-clinical stages of the disease, MRI shows altered longitudinal and trans verse relaxation times and increased myelin hydration degrees [18]. In general, abnormalities in the structure and formation of the cerebral white matter have been identified in many presentations of Alzheimer’s disease that can be warning signs for a disease in progress [156]. Variations in T1w/T2w ratios in patients with risk factors (close family history, APOE4 phenotypes) were identified compared to control individuals. In addition, individu als at risk had an association with altered patterns of resting-state functional connectivity (rs-FC) [52]. These abnormalities support the idea of significant alterations in myelin developing with age and constituting signals of vulnerability [53]. Interestingly, some studies have shown a relationship between structural abnormalities of myelin in ADs in the pre-clinical period and peptide concentra tions of Aβ1–42 in patients’ CSF [34, 38].i A step for the conversion of mild cognitive impairment (MCI) into dementia? In AD, myelin is one of the first bulwarks for the anatomical and functional integrity of the axons it sur rounds and undergoes early toxic action of misfolded extracellular toxic proteins or peptides. Abbreviations: AD, Alzheimer’ disease; CNP, C-type natriuretic peptide; CNS, central nervous system; CSF, cerebrospinal fluid; MAG, myelin-associated glycoprotein; MBP, myelin basic protein; MOG, myelin oligodendrocyte glycoprotein; PLP, proteolipid protein Fig. 3 Myelin composition and organization. The myelin wrapping around most of the CNS axons includes a large majority of complex lipids and 15–30% of specific proteins. Lipids are essentially made up of cholesterol, galactocerebrosides and phospholipids. This envelope is constantly reshuffled in time and space from the oligodendrocytes that make up the bulk of the glial cells of the CNS. Chronic inflammatory and autoimmune reactions, muta tions in certain constituent proteins, attacks by free radicals or ischemic, and metabolic problems related to aging alter the myelin sheath that releases its constituents into the CSF and the bloodstream. In AD, myelin is one of the first bulwarks for the anatomical and functional integrity of the axons it sur rounds and undergoes early toxic action of misfolded extracellular toxic proteins or peptides. Abbreviations: AD, Alzheimer’ disease; CNP, C-type natriuretic peptide; CNS, central nervous system; CSF, cerebrospinal fluid; MAG, myelin-associated glycoprotein; MBP, myelin basic protein; MOG, myelin oligodendrocyte glycoprotein; PLP, proteolipid protein Fig. 3 Myelin composition and organization. The myelin wrapping around most of the CNS axons includes a large majority of complex lipids and 15–30% of specific proteins. Lipids are essentially made up of cholesterol, galactocerebrosides and phospholipids. This envelope is constantly reshuffled in time and space from the oligodendrocytes that make up the bulk of the glial cells of the CNS. Chronic inflammatory and autoimmune reactions, muta tions in certain constituent proteins, attacks by free radicals or ischemic, and metabolic problems related to aging alter the myelin sheath that releases its constituents into the CSF and the bloodstream. In AD, myelin is one of the first bulwarks for the anatomical and functional integrity of the axons it sur rounds and undergoes early toxic action of misfolded extracellular toxic proteins or peptides. Potential involvements of epigenetic mechanisms in myelin reshufflei Oligodendrocytes and their progenitors are directly involved in membrane and metabolic interac tions with neurons during the different phases of destruc tion and regeneration of the myelin sheath, driven by the dynamic and fluctuating expression of many transcrip tion factors [179]. The activity of the nervous system is intimately linked to the epigenetic regulation of the activ ity of these factors and to the neo-expression of certain genes involved in the functional dynamics of the produc tion/destruction of myelin [158]. Correct myelination is essential for the proper development and evolution of neuronal connections and the adaptation of brain func tion to the environment. It constantly reshapes neuron/ oligodendrocytes interactions following many factors such as learning, social relationships, emotional stimuli (emotions, anxiety) [166, 217]. These stimuli can induce epigenetic modifications that alter the physiology and functionality of precursors and oligodendrocytes [165]. membrane remodeling [37]. Histologically, BIN1 is mostly expressed at the Ranvier nodes. BACE1 (beta-site amyloid precursor protein cleav ing enzyme 1) codes for a transmembrane β secretase expressed in several cell types including oligodendro cytes. It cleaves APP giving birth to amyloid peptides, but also neuroregulin 1, which modulates the myelination and differentiation of oligodendrocytes [50, 195]. Many β-secretase inhibitors have effects on myelin abnormali ties caused by AD. Finally, several other genes that are also expressed in oligodendrocytes (PICALM, NME8, PSEN, for example) possess a special responsibility as genetic factors associated with the development of AD [123]. LINGO 1 (leucine rich repeat and Immunoglobulin-like domain-containing protein 1) codes for a transmembrane protein primarily expressed in the cortex, hippocampus, thalamus, and amygdala. The protein acts primarily as a negative regulator of myelination and its inhibition may have potential applications for the treatment of myelin damage in neurodegenerative diseases [204]. As such, anti-LINGO 1 antibodies promote the action of oligo dendrocytes and the repair of myelin disease [212].h The biochemical markers of the white matter indicating the evolution of late AD are of many natures and depend on the stage of the disease. Since myelin is mainly com posed of complex lipids synthesized by oligodendrocytes, reduced levels of galactosyl ceramide (cerebroside) and sulfatide can be found in both the grey and white matters of AD brains [89]. These compounds are the most specific lipids of myelin, decreasing in parallel with the severity of the disease and altering long before fibrillary deposits of tau protein [28, 87]. Potential involvements of epigenetic mechanisms in myelin reshufflei GWAS (genome-wide association study) has identified about 40 loci associated with AD in the European popu lation and in these respective loci, several genes involved directly in the causative mechanism of the disease have been described (APOE, CR1, BIN1, TREM2, CLU SORL1, ADAM10, ABCA7, CD33, SP11, PIRLA). It remains to identify the functions of many genes in the identified loci. Many risk genes are involved in the innate immune response and neuroinflammation. The CD33 and TREM2 microglia receptors, implicated in microglial pathology, represent new targets for the development of therapeu tic tools. It is possible in many cases that the activation of innate immunity, like that encountered in other myelin pathologies, associated with long-term inflammatory mechanisms, is responsible for subtle alterations of myelin during the incubation period of the disease [5, 65]. Studies of cortical stratification in the human brain provide important knowledge on the level of degenera tion, in addition to the information given by the level of volumetric atrophy [147, 155]. MRI studies show hyper- densities in the white matter with volume increase con sistent with the abnormalities of amyloid peptides and tau proteins in CSF. At the histological level, it appears that these stratification disorders are mainly due to alter ations in myelin architecture in which iron ions could play an important role [194]. Vascularization and oxygen supply in injured hyper-dense regions are decreased and Over 20 AD risk loci falling mainly in noncoding regions of the genome have been identified by genome- wide association studies, explaining the complexity of the disease at the genetic level [81, 98, 126]. The regulation of gene expression by microRNA is a promising issue for the diagnostic and treatment of several kind of MCI and AD at the beginning of the symptoms, as well as to discriminate with other myelin pathologies like multiple Maitre et al. Acta Neuropathologica Communications (2023) 11:56 (2023) 11:56 (2023) 11:56 Maitre et al. Acta Neuropathologica Communications Page 6 of 18 sclerosis [103, 226]. In such diseases, the dynamics of the myelination/demyelination/remyelination balance is continuously evolving under normal conditions of plas ticity of the nervous system, but also under pathological conditions, where this balance is affected [47]. In this respect, the process of myelin degeneration are particu larly concerned both in multiple sclerosis and in the early phases of AD. Potential involvements of epigenetic mechanisms in myelin reshufflei Cholesterol concentrations, another majority lipid compound of myelin sheaths, is known to decrease with the onset of cerebral atrophy [44]. Changes in the epigenome have a role in the manifes tations of AD [13]. Social isolation impacts the inten sity of neuronal activity and reduces the importance of myelination [6, 139]. Modifications in the acetylation and methylation of histones were detected, as well as in DNA [131]. These adaptations participate in the regulation of genes involved in the processes of myelination/demyelin ation and in the pathophysiology of certain neurodegen erative diseases where these processes play a central role (AD, but also multiple sclerosis) [30]. This last pathology of myelin includes analogies with those existing in some phenotypic form of AD and may be the consequence of a combined alteration of genetic and epigenetic factors, the latter involving DNA methylations, histone modifica tions, chromatin remodeling and modified regulation of non-coding RNA [16]. Myelin proteins are also involved in relatively early stages of the disease (Braak stage I and II), in which alter ations of oligodendrocytes and myelin are noted even before the onset of clinically detectable cognitive disor ders [55]. The level of most myelin proteins is likewise decreased in more advanced stages of AD (Braak stages V and VI). Lowered concentrations of basic myelin pro tein (MBP), proteolipid (PLP) and 2’-3’ cyclic nucleotide phosphodiesterase (CNPase) are observed, specifically in several regions of the cerebral cortex.i Oligodendrocyte’s dysfunction: A major risk factor in AD and a process in the onset of the disease? Before the appearance of amyloid and tau pathology, many forms of AD showed a breakdown of myelin due to the vulnerability of oligodendrocytes under this neuro degenerative pathology. In many cases, the loss of myelin sheaths appears to be the initiating step in the earliest stages of the disease. Extensive evidence has indicated that the breakdown of myelin is associated with AD since the vulnerability of oligodendrocytes under Alzheimer’s pathology easily induces the myelin breakdown and the loss of the myelin sheath. Aging itself is already an important factor of myelin alterations and multiple cellular partners are involved in this process. Brain MRI often reveals signs including several hyper signal outbreaks in T2-weighted images (T2WI) with chronic cerebral hypoperfusion often associated with carotid stenosis [112]. These alterations appear more massive at the stage of MCI both in animal models and in human pathology than in established AD. In myelin abnormalities, association of oligodendrocytes’ losses with axonal alterations are commonly encoun tered in post-mortem patients [137]. The accumulation of Aβ peptides is considered a princeps factor in the neu rodegenerative process even before the appearance of aggregates in the form of amyloid plaques [23, 75]. The response of oligodendrocytes to the presence of amyloid peptides or plaques has been the subject of several stud ies. During aging, the spontaneous involution of these cells is important, and their disappearance is close to 25% from the age of 50 years, this phenomenon being potenti ated by the presence of the APOE ε4 allele in the genomic baggage of the individual [107, 132, 134]. Furthermore, the importance of increase in the expression of myelinat ing genes in oligodendrocytes from Alzheimer’s patients is related to the severity of the disease [64, 79]. In trans genic animals over-expressing the APP, the myelin sheath has an increased thickness and a modified architecture [54, 67, 211]. Myelination is directly related to the intensity of neural activity, which affects the electrical properties of axons. The toxicity of Aβ peptides proteinopathy affects imme diately the whole myelin-axon, which forms a couple with multiple functional and metabolic relationships [181]. p p The toxicity of proteinopathies, which causes degen erations in AD, mainly concerns oligomeric Aβ peptides and hyperphosphorylated tau proteins [2, 41, 82]. Senile plaques are rarely seen in the hydrophobic white mat ter and does not lend itself to the aggregation of toxic oligomers. Brain markers of myelin in Alzheimer’s patients Looking for myelin components in biological fluids At the genetic markers level, several genes associated with the corpus of the oligodendrocyte ecosystem have been described as risk factors in late-onset AD [124]. In genomic association studies, the BIN1 (bridging inte grator 1) gene is considered to be significantly involved in late AD behind the APOE gene [169]. It is mainly expressed in mature oligodendrocytes and white mat ter in rodents and humans, where it regulates mem brane dynamics in the phenomena of endocytosis and In the field of protein markers present in patients’ CSF, there is a wide heterogeneity and variability, which confirm the impression that sporadic AD may be the consequence of multiple and varied alterations in many metabolic circuits. This reinforces the idea that the pathophysiological mechanisms leading to late AD are multifactorial and reveal a disease of great complexity [205]. Many cognitive pathologies with MCI are often accompanied in a non-specific way by the presence of Maitre et al. Acta Neuropathologica Communications (2023) 11:56 Page 7 of 18 (2023) 11:56 Page 7 of 18 (2023) 11:56 Maitre et al. Acta Neuropathologica Communications inflammatory markers and proteins associated with the complement cascade in the CSF or blood of patients. neuroregulin 1 and brain-derived neurotrophic factors (BDNF), whose release depends on neuronal activity [77, 199]. Many studies have been conducted to explore early oligodendrocyte alterations in AD in association with changes in myelination and early symptoms of the disease. Most commonly, oligodendrocyte differentiation abnormalities are associated with disruption of oxidative stress phenomena associated with excitotoxicity, medi ated by glutamatergic metabotropic receptors in large amounts in oligodendrocyte precursors [22, 137]. Other factors, such as high iron ion levels and disorders in the glutathione cycle, would accentuate the presence of free radicals, without forgetting the mitochondrial chain disorders induced by the toxicity of Aβ peptides [190]. Mitochondria pathologies are at the forefront of axonal survival for functional and metabolic exchanges with the myelin envelope [66, 219]. Oligodendrocyte’s dysfunction: A major risk factor in AD and a process in the onset of the disease? These specific depopula tions probably precede the disorganization of the neural connectome that precedes the appearance of AD, and a contemporary demyelination around the outbreaks of amyloid peptide deposits [94]. These dysfunctions are strongly associated with abnormalities in lipoprotein metabolism given that the amyloid oligodendrocytes actively participate in the synthesis of cholesterol consti tuting synaptic contacts [120]. They secrete apolipopro teins E and J, which are severe risk factors depending on the alleles involved in the onset of AD [85]. Interestingly, the production of new oligodendrocytes seems funda mental for motor learning in mice [111]. as disorders in the cascades of the complement and clot ting factors testify to changes in the immune response at the periphery [143]. Cleavage fragments of abnormal proteins, numerous glycated proteins and a large popula tion of phosphoproteins contribute to microglial activa tion in the brain and alteration of many resident proteins [24]. This include myelin constituent proteins that are presented as new antigens to the immune system. Studies have shown significant accumulation of autoantibodies in the serum of patients with AD, especially directed against myelin proteins [62, 115]. IgG and IgM immunoglobulins directed against the MOG, MBP, MAG and PLP proteins are frequently present in the CSF and circulating blood [152]. This strongly suggests the involvement of the immune system in myelin alterations observed in many AD patients and in some animal models of the disease. p Since the discovery of mutations in APP, PSEN1 and PSEN2 genes, which induce familial ADs, the hypothesis of the amyloid cascade at the origin of the pathophysiol ogy of AD remains the preferred mechanism of this type of neurodegeneration [136]. The problem is that spo radic AD does not usually present this type of mutation, although similar pathologies of Aβ peptides and tau pro teins are encountered in familial and late forms of the dis ease [106]. The main hypothesis remains those long-term abnormalities in Aβ peptide metabolism are the starting point of tau dysfunction and a series of toxic phenom ena inducing neuronal and cognitive losses. It appears that during aging, multiple mutations accumulate in the nuclear and mitochondrial DNA of neural cells that add up to the increased loss of editing and quality control of translated proteins [76, 108]. Oligodendrocyte’s dysfunction: A major risk factor in AD and a process in the onset of the disease? As a result, it is mainly these latter that exert toxicity on myelin. The therapeutic strategies currently developed for plaque removal seem not to display huge impact on clinical symptomatology [196]. In fact, the degree to which the therapeutic strategies for plaque removal have clinical effects remains an open question, as the reason why such therapies are not working well. For more than twenty years, therapeutic research against AD has focused on reducing the accumulation of patho logical amyloid peptides and the substances studied have made it possible to achieve this goal without significant improvement in cognitive impairment in patients. The amyloid hypothesis revised in many cases is questioned, sometimes in favor of primitive alterations of the myelin envelope [61]. Oligodendrocytes are very active cells from a metabolic point of view, especially during the process of myelination or remyelination. A cellular respi ratory abnormality that may be related to hypo-vascular ization or ischemia may be the source of a myelination disorder [137]. Vascular pathologies affecting the white matter are common in the elderly or with symptomato logically occurring early AD [78, 109]. The human brain is largely myelinated, which may partly explain its vulner ability to neurodegeneration [192]. Originating mostly from the ventricular and subven tricular regions of the brain, OPCs are present in the brain, even in the adult stage [36, 164, 187, 218]. OPCs control the angiogenesis of the white matter, its vascu larization/oxygenation, and the myelination of axons according to spatial-temporal parameters, which con tribute to their stability, functions, and integrity [96, 153]. Intimate exchanges between neurons regulate ionic homeostasis, making of real synaptic connections and activity of OPCs via numerous neurotransmitters [58, 68, 172]. These cells are also the target for several mitogens produced by neurons and trophic factors like Oligodendrocytes are widely represented in many areas of the human CNS, especially in the neocortex, where they account for about 75% of glial cells [45]. They Maitre et al. Acta Neuropathologica Communications (2023) 11:56 (2023) 11:56 Maitre et al. Acta Neuropathologica Communications Page 8 of 18 Page 8 of 18 are considered very fragile, and their density decreases sharply in the brain of the elderly person from 50 years of age [223]. Various methods of labeling these cells have shown a severe loss of oligodendrocytes in several regions of the hippocampus, not correlated with the den sity of amyloid β deposits [40]. Oligodendrocyte’s dysfunction: A major risk factor in AD and a process in the onset of the disease? It is estimated that in the normal individual, about 20% of the proteins synthesized by ribosomes have structural and folding abnormalities and must be eliminated by the proteasome, lysosome, or resident proteases of the cell membrane [97, 157, 180]. Overloading these mechanisms leads to chronic neuro inflammation, microglial and macrophagic activation, and immune responses against abnormal non-functional proteins over the long-term [122]. Among the oligomeric peptides that accumulate in the brain and display signifi cant cellular toxicity are Aβ peptides and particularly the peptide Aβ1–42. This accumulation most often comes from a drop in the clearance to the vascular compart ment and the CSF. During the development of AD, including in the early stages characterized by mild, worsening memory disor ders (MCI), numerous studies have been performed to characterize the changes observable by MRI techniques in the structure and architecture of myelin. Schemati cally, the results obtained showed very early the exis tence of a reduction in cerebral myelin levels, with losses of oligodendrocytes and axons, microglial activations accompanied by dilated perivascular regions in the white matter. These studies are essentially based on the con trasts between the aqueous contents of the intra- and extra-cellular spaces at the periventricular level, com paring MCI patients and control persons. It seems that progressive ischemia with vascular and energy losses associated with the toxicity of certain proteinopathies (especially amyloidosis Aβ) alters the myelin structure very early and hinders proliferation and oligodendrocytic re-myelination [88, 137, 148]. Adaptative immunity to myelin components in AD An auto-immune process for degeneration? Many results support the existence of mutual interac tions between immune processes (innate and acquired) and neurodegenerative events, especially those occurring during the incubation of AD [33, 51, 144].l Neuroinflammation phenomena are considered to pre- exist for a very long time in the brain before the onset of cellular stigmas of neurodegeneration and clinical symp tomatology. Chronic inflammation, microglia activation and lymphocytic infiltration are thought to be the result of intracerebral accumulation of misfolded proteins and/ or multiple exogenous attacks of various infectious agents during the individual’s lifetime [42]. Amyloid peptides and hyperphosphorylated tau are particularly involved in the inflammatory reaction and progressive onset of auto immunity [207]. Changes in circulating cytokines as well The rupture of the myelin envelope appears to be an early phenomenon in the pathophysiology of AD [38, 150, 202]. In humans, the vulnerability of myelin materi alizes on MRI through morphological changes, thinning and hydration swelling [49, 229]. At the same time, there are elevations of tau, phosphotau, soluble APPβ (sAPPβ) peptides and Aβ1–42 peptides. The latter peptide has a high toxicity to myelin in oligodendrocyte cultures and in animal models of familial AD (e.g. 5XFAD mice), where Maitre et al. Acta Neuropathologica Communications (2023) 11:56 (2023) 11:56 Page 9 of 18 Maitre et al. Acta Neuropathologica Communications andymes modifications inducing inflammatory and immune responses. morphological alterations of myelin are the first patho logical stigmas to appear in animals at 1 month of age [67]. The mechanisms of toxicity of peptides Aβ are still the subject of speculation; it seems that the oligomeric Aβ peptides are the main culprits of this toxicity [75]. Several cellular receptors (glutamates, ephrin’s, adrener gic, cholinergic, and immunoglobulins) bind oligomeric Aβ peptides and could mediate the toxicity of amyloid oligomers during the years of incubation of the disease [171, 215]. Several evidence from studies of the population of abnormal proteins in the CSF showed that abnormal pro teins in CSF represent a picture close to that of abnor mal proteins in the brain [11]. This methodology can provide information on the biochemical and metabolic changes that occur in the CNS of patients with neuro degeneration. CSF amyloid peptides and tau proteins are used for the diagnosis and evolution of AD [154]. Adaptative immunity to myelin components in AD An auto-immune process for degeneration? Aβ1– 42 peptides correlation has been described with several CSF proteins belonging to the endocannabinoid and the somatostatin systems [71] with the latter regulating the proteolytic degradation of the amyloid peptide. The pres ence of other proteins has been linked to the degradation of the myelin [170].h Multi-proteinopathies are associated with aging brains During lifetime, mutations accumulate in the post- mitotic cells of neurons due to non-replication of DNA, but also within mitochondrial DNA [86, 95]. This results in harmful mitochondriopathies for neuronal survival, as well as increased production of misfolded and non-func tional proteins. Deficiencies in quality control and pro tein structure editing also contribute to the intra-brain accumulation of protease-resistant hydrophobic deposits with intrinsic toxicity [1, 151]. These chronic accumula tions lead to long-term inflammatory and phagocytic reactions, as well as immune responses accompanied by infiltration of immunocompetent cells. Gradually, an amyloid reaction develops, in which peptides Aβ par ticipates largely because of their cerebral accumula tion. The elimination of these peptides from the brain is largely conditioned by the effectiveness of the enzymes that degrade them and allow their clearance [222]. The peptide Aβ1–42 is particularly toxic to myelin sheaths, axonal and synaptic endings that finally degenerate [75, 137]. In several transgenic models of familial forms of Alzheimer’s, the first stigmas of the disease result in morphological abnormalities of myelin sheaths, in the form of edema and thinning of the envelope surround ing myelinated axons. At the same time, disturbances in animal behavior appear manifesting as reduced anxiety manifestations and reduction of memory and spatial rec ognition [56, 57]. The quality control processes of in vivo newly formed proteins and the elimination of abnormal proteins are phenomena with growing alterations with age [92, 138]. This results in the cellular and extra-cellular accumula tion of an increasing number of non-functional proteins that tend to form hydrophobic aggregates [117]. At the brain level, these toxic aggregates induce significant cel lular and functional losses that are the basis of many neu rodegenerative diseases [14]. In addition to the amyloid peptides and tau protein that are the canonical proteins of early AD and whose toxic deposits in brain tissue are the basis of mechanistic theories of neurodegeneration, it has been shown that a wide range of protein aggregates from other sources exist in the brain of elderly patients displaying a cognitive impairment or at first stages of AD [162]. Adaptative immunity to myelin components in AD An auto-immune process for degeneration? Among the proteins significantly altered compared to controls, many are found in the biochemical cascade of glycolysis that primarily feeds cellular energy and whose intensity decreases with age, even faster in patients with AD [135]. Other strategic proteins form larger insoluble aggregates depending on symptomatologic impairment. These include glucose 6 phosphate isomerase creatine kinase B, certain forms of adenylate cyclase and calcium/ calmodulin protein kinase 2. This list is not exhaustive but reflects the importance of metabolic and functional disorders that develop over time in the brains of patients with mild cognitive impairments that worsen in AD [91]. It ld b sp l t d th t th l ti f is Depending on the individual, brain aging does not occur unequivocally but depends on multiple factors related to specific genes and environmental situations (Fig. 4) [176]. The accumulation of mutations in the nuclear DNA of post-mitotic cells and mitochondrial DNA induce deleterious mitochondriopathies [121, 186], promote the production of abnormal proteins, impair respiratory and energy functions, and amplify cellular and oxidative stress [91]. Toxicity of abnormal oligomers seems to be the result of their misfolded nature, which exposes hydrophobic residues leading to aggregation and abnormal interactions with a large range of cellular com ponents [4]. Membranes like myelin constituted mainly by complex hydrophobic lipids could be an important target for amyloid oligomers for direct interactions It could be speculated that the accumulation of mis folded proteins during old age in multiple regions of the brain alters mitochondrial and metabolic functions, saturates the processes of cleaning and elimination of senescent cells, and slows down the neurogenesis that persists in the older brain [20, 80]. Inflammatory vasculi tis, hypoxia, and oxidative stress due to the accumulation of non-functional deleterious proteins are considered the primary factors in myelin envelope impairment. The decrease in electrical and metabolic activity of axons con tributes to the decrease in myelin density that surrounds (2023) 11:56 Page 10 of 18 Maitre et al. Acta Neuropathologica Communications Fig. 4 Multiple Alzheimer’s disease etiologies and many cellular partners. Deleterious proteinopathies (in the first-place amyloid peptides) are to be integrated into the complex cellular environment of the brain. These multiple cellular elements participate in progressive multi-focal neuro-axonal degeneration leading to the irreversible symptomatology of AD. This is expressed when the toxic peptide removal systems are overwhelmed, which ap pears only after a long incubation period. Adaptative immunity to myelin components in AD An auto-immune process for degeneration? Altered neurons express phases of hypo- and hyperexcitability with deficits in axonal transport and synaptic activity that affects myelination/remyelination activity and oligodendrocyte trophism. These are very vulnerable cells whose density decreases sharply with age. There seems to be a link between the intensity of neuronal involvement and the extent of demyelination. This is strongly accentuated in AD in which remyelination processes seem deficient. The activation of astrocytes participates in the elimination of deficient neurons and synapses. They actively participate in the elimination of abnormal proteins and inflammation processes, in the same way that the activation of microglia facilitates the phagocy tosis of cellular debris. In the same way, these cells participate in the activation of the innate immune responses, the activation of the complement and the secretion of inflammatory cytokines. Abbreviations: AD, Alzheimer’s disease; ROS, reactive oxygen species Microbiome and myelin dysregulation in the neurodegenerative brain them leading to its gradual dislocation. The toxicity of Aβ peptides has been demonstrated in vitro against neurons, endothelial cells, astrocytes, vascular smooth muscle cells and oligodendrocytes [224]. Aβ peptides cytotoxic ity might involve the susceptibility of oligodendrocytes to oxidative stress because of its low content of reduced glutathione and high concentration of iron [190]. The Aβ peptides activation of the neutral sphingomyelinase- ceramide pathway has been reported to induce oligo dendrocyte death [101]. In addition, inhibition of neutral sphingomyelinase 2 in these cells reduces their ceramide content and favor the myelination process by improving the quality of myelin structure [221]. A role for the gut-brain axis and for hepatic metabolism Some arguments favor a view of AD as a disease that is not limited to the CNS alone but reflects multi-organ dysfunctions that contribute to or influence brain neuro degeneration [200]. Multiple proteinopathies, including the Aβ cascade, may come from peripheral organs that no longer metabolize abnormal proteins properly and allow their dissemination through a permeable BBB. Abnor mal communication between various compartments of amyloid proteins can contribute to altering brain disease. Chronic peripheral metabolic abnormalities are sus pected to participate or to worse neurodegeneration. In this regard, intestinal metabolism is often questioned.h The population of microorganisms of the gut micro biota constitutes a true symbiotic organ that has a great inter-individual heterogeneity due to many intrinsic and Maitre et al. Acta Neuropathologica Communications (2023) 11:56 Maitre et al. Acta Neuropathologica Communications (2023) 11:56 (2023) 11:56 Page 11 of 18 Maitre et al. Acta Neuropathologica Communications Page 11 of 18 The problems of pathological cerebral aging are prob ably the result either of the evolution of protein targets at the central level, or the modification of a peripheral immune response [60, 182], the two mechanisms can combine over time to lead to an autoimmune altera tion colonizing the CNS and involving, in the first place, the components of myelin. Many environmental factors can promote this chronic process by perpetuating the homeostasis of the intestinal flora and at the origin of certain metabolic and cytotoxic disorders [15, 90]. This primarily affect oligodendrocytes, which are fragile cells of the CNS, and which adapt their functions through out the life of the individual. Many factors contribute to the activity of oligodendrocytes, intrinsic and environ mental factors that modify the status of the epigenome [165]. Microbiome and myelin dysregulation in the neurodegenerative brain Among these factors, the composition and activity of the microbiome plays a reweighting role and interferes with the spatio-temporal character of myelination in the brain. In general, the relationships between the intesti nal sphere and the brain are of primary importance for myelination. This sphere includes not only the intestinal epithelium, but also hepatic metabolism, sympathetic and parasympathetic nerve activity, endocrine, and cyto kine secretions and metabolites of microbial origin [46]. The microbiota has an important role in the regulation of myelin plasticity as the existence of hyper-myelinated extrinsic factors dependent on genetic, medication (e.g. antibiotics), physical and hormonal activity, and infec tious factors [185]. The composition of the microbiome changes with age and the reactivity of the immune sys tem [129]. An active exchange via the bloodstream and intestinal innervation between the microbiome and the nervous system exist, whose influence is important dur ing the neurogenesis, the molecular organization of the connectome, and the variations of CNS myelination. These phenomena are especially different during periods of brain development or during aging [228]. In various transgenic models of familial AD, distur bances in the composition and diversity of the intestinal microbiome compared to healthy animals are observed [174]. In humans, it has also been described qualitative and quantitative changes in the population of intestinal bacteria in patients with cognitive disorders associated with cerebral amyloidosis [59, 118]. These disturbances can be the source of chronic neuroinflammation target ing several organs including the brain and a decrease in the immune response inducing neurodegeneration over the long term [26, 74]. These phenomena are increased by the leaky permeability of the BBB as a function of age, allowing the passage at the cerebral level of many toxics present at the periphery (Fig. 5) [213]. Fig. 5 Multiple communication system that includes neural, immune, endocrine, and metabolic pathways lead to degeneration. Continuous fluctuation of the microbiota due to the environment constantly influences the inflammatory, immune, and metabolic responses of the CNS [110]. With age, the permeability of intestinal and BBB is often impaired [84, 161]. The gut microbiota metabolizes and release many growths, metabolic and inflam matory factors which could penetrate the brain via the circulating blood. These substances contribute to increase the inflammatory, immune, and oxida tive phenomena that exist in the elderly brain due to the accumulation over time of many abnormal proteins due to their hydrophobic conformation. Conclusion and perspectives To conclude, myelin damage and its several possible outcomes (Table 1) is one of the early lesions observed in many clinical forms of AD. Even though many differ ences exist in the presentations and structural alterations between multiple sclerosis and AD, neurodegenerative alterations between both pathologies have common etiol ogies and mechanisms [113]: long-standing inflammatory disorders, some autoimmune reactions, cognitive impair ments, and mitochondrial alterations [104, 184]. Amy loid disorders are not absent from the pathophysiology of multiple sclerosis and Aβ peptides levels are generally lower in the CSF of patients with multiple sclerosis who have cognitive impairment [93]. The accumulation of APP in the brain of these patients appears parallel to the worsening of symptomatology and dynamic processes of demyelination/remyelination [25]. These parallels remain hypotheses at present, but there are indications that some mechanistic similarities exist. axons has been demonstrated in germ-free mice or treated chronically with antibiotics [72]. This abnormal ity could be a consequence of neuronal hyperactivity in certain regions of the brain of these mice, such as the amygdala or the prefrontal cortex. The development of myelinating oligodendrocytes is controlled by a set of transcription factors (Sox 10 and Myrf for example) that drive the steps of myelination and re-myelination [7]. The anomalies of these phenomena alter certain brain func tions, those concerning cognitive functions. Restoring a normal microbiome in germ-free mice greatly improves their social and executive performance [173, 189, 191]. Current mechanistic hypothesis favors long-term dys functions in the proteolysis of APP and in the accumula tion of hydrophobic Aβ peptides with multiple toxicities. These lead to inflammatory, oxidative, and immune reac tions leading to massive cellular apoptosis accompanied by post-translational modifications on target proteins inducing profound functional alterations in brain cells activities. It seems possible that a multiplicity of muta tions and epigenetic alterations of neuronal genomes, associated with intrinsic or extrinsic predisposing fac tors, generate metabolic and inflammatory alterations over the long term, inducing a multiplicity of phenotypic and clinical presentations involving secondarily multiple deleterious proteinopathies, including amyloidosis of Aβ types. It is now recognized that disorders intestinal physiol ogy can influence the risk of Alzheimer’s and its rate of progression. Deposits of aggregate of Aβ peptides at the intestinal level have been detected in AD patients [74], but most of the results involving the intestinal sphere and the progression of AD have been obtained in animal models. Conclusion and perspectives The ratios between Firmicutes and Bacteroidetes are considered strategic in the composition of the human intestinal microbiota [168]. The fecal microbiota is the product of a very complex and diverse ecosystem, and its composition can modify the accumulation of intes tinal APP in the early phases of AD [29, 119]. In trans genic APP/PS1 animals, an increase in Aβ peptides levels have been observed in the CNS in relation to changes in the intestinal flora, accompanied by disorders of spatial memory [225]. Oligodendrocytes and myelin sheaths may be the first to be affected by these deleterious depos its. A parallel can be observed between the myelin altera tions observed in AD and during normal aging in the elderly. In the latter case, the installation of progressive ischemia could be the cause of this demyelination [137, Acknowledgements h k g This work was supported by recurring grants from the Institut National de la Santé et de la Recherche Médicale (INSERM, France, grant #U1119) and Université de Strasbourg, France (grant #UMR_S1119). Authors’ contributioni The first draft of the manuscript was written by Michel Maître and Hélène Jeltsch-David, who performed the literature search and data analysis. Idea for the article: Michel Maitre and Ayikoe-Guy Mensah-Nyagan. Table and pictures design and editing: Christian Klein, Michel Maitre and Hélène Jeltsch- David. All authors commented on previous versions of the manuscript and have approved the manuscript for submission; the content of which is not submitted or published elsewhere. All authors read and approved the final manuscript. Microbiome and myelin dysregulation in the neurodegenerative brain Acta Neuropathologica Communications Table 1 Examples of different outcomes of myelin damages Myelin modifications References Demyelination (as evidenced, for example, by decreased myelin water fraction) [18, 48, 83, 94, 137, 177] Rupture of the myelin envelope [38, 150, 202] Myelin reshuffle [16, 47, 165, 179] Defect in myelin biosynthesis (loss of ceramide synthase 2 activity) [32] Down-regulation of myelination network [3] Morphological abnormalities of myelin sheaths, in the form of edema and thinning of the envelope surround ing myelinated axons [49, 137, 229] Myelin degeneration -> driving cognitive and motor impairment [27, 153] Changes of myelin organization (q-Space myelin map imaging) [148] Myelin instability [35, 203] Myelin damage in cortical gray matter (Western blot quantification of MBP and dMBP) [227] Decrease of myelin density (multi-echo T2 relaxation time technique) [99] Table 1 Examples of different outcomes of myelin damages Myelin modifications References Demyelination (as evidenced, for example, by decreased myelin water fraction) [18, 48, 83, 94, 137, 177] Rupture of the myelin envelope [38, 150, 202] Myelin reshuffle [16, 47, 165, 179] Defect in myelin biosynthesis (loss of ceramide synthase 2 activity) [32] Down-regulation of myelination network [3] Morphological abnormalities of myelin sheaths, in the form of edema and thinning of the envelope surround ing myelinated axons [49, 137, 229] Myelin degeneration -> driving cognitive and motor impairment [27, 153] Changes of myelin organization (q-Space myelin map imaging) [148] Myelin instability [35, 203] Myelin damage in cortical gray matter (Western blot quantification of MBP and dMBP) [227] Decrease of myelin density (multi-echo T2 relaxation time technique) [99] 177]. The lesions often appear disseminated with a predi lection for intracortical axons of small diameters that are myelinated late during development. Myelin dystrophies lead to axonal alterations and neuronal death with differ ent rate in individuals [163]. Microbiome and myelin dysregulation in the neurodegenerative brain The very likely origin of these malformed proteins is found in the accumulation during senescence of many mutations in post-mitotic cells that are neurons [9, 76]. In addition, the role of epigenetic dysregulation of gene expression induced by aging or abnormal environmental stimulation is also considered to be an important factor in neurodegeneration and cognitive alterations [125]. Abbreviations: BBB, blood-brain barrier; CNS, central nervous system g. 5 Multiple communication system that includes neural, immune, endocrine, and metabolic pathways lead to deg cation system that includes neural, immune, endocrine, and metabolic pathways lead to degeneration. Continuous ll Fig. 5 Multiple communication system that includes neural, immune, endocrine, and metabol 5 Multiple communication system that includes neural, i Fig. 5 Multiple communication system that includes neural, immune, endocrine, and metabolic pathways lead to degeneration. Continuous fluctuation of the microbiota due to the environment constantly influences the inflammatory, immune, and metabolic responses of the CNS [110]. With age, the permeability of intestinal and BBB is often impaired [84, 161]. The gut microbiota metabolizes and release many growths, metabolic and inflam matory factors which could penetrate the brain via the circulating blood. These substances contribute to increase the inflammatory, immune, and oxida tive phenomena that exist in the elderly brain due to the accumulation over time of many abnormal proteins due to their hydrophobic conformation. The very likely origin of these malformed proteins is found in the accumulation during senescence of many mutations in post-mitotic cells that are neurons [9, 76]. In addition, the role of epigenetic dysregulation of gene expression induced by aging or abnormal environmental stimulation is also considered to be an important factor in neurodegeneration and cognitive alterations [125]. Abbreviations: BBB, blood-brain barrier; CNS, central nervous system (2023) 11:56 Page 12 of 18 Maitre et al. Acta Neuropathologica Communications (2023) 11:56 Maitre et al. References 1. Adav SS, Sze SK (2016) Insight of brain degenerative protein modifications in the pathology of neurodegeneration and dementia by proteomic profiling. Mol Brain 9:92. https://doi.org/10.1186/s13041-016-0272-9 1. Adav SS, Sze SK (2016) Insight of brain degenerative protein modifications in the pathology of neurodegeneration and dementia by proteomic profiling. Mol Brain 9:92. https://doi.org/10.1186/s13041-016-0272-9 p g 21. Butt AM, Papanikolaou M, Rivera A (2019) Physiology of Oligodendroglia. Adv Exp Med Biol 1175:117–128. https://doi.org/10.1007/978-981-13-9913-8_5 21. Butt AM, Papanikolaou M, Rivera A (2019) Physiology of Oligodendroglia. Adv Exp Med Biol 1175:117–128. https://doi.org/10.1007/978-981-13-9913-8_5 2. Allegri RF (2020) Moving from neurodegenerative dementias, to cognitive proteinopathies, replacing “where” by “what”…. Dement Neuropsychol 14:237–242. https://doi.org/10.1590/1980-57642020dn14-030005 22. Butt AM, De La Rocha IC, Rivera A (2019) Oligodendroglial cells in Alzheimer’s Disease. Adv Exp Med Biol 1175:325–333. https://doi. org/10.1007/978-981-13-9913-8_12 22. Butt AM, De La Rocha IC, Rivera A (2019) Oligodendroglial cells in Alzheimer’s Disease. Adv Exp Med Biol 1175:325–333. https://doi. org/10.1007/978-981-13-9913-8_12 3. Allen M, Wang X, Burgess JD, Watzlawik J, Serie DJ, Younkin CS, Nguyen T, Malphrus KG, Lincoln S, Carrasquillo MM, Ho C, Chakrabarty P, Strickland S, Murray ME, Swarup V, Geschwind DH, Seyfried NT, Dammer EB, Lah JJ, Levey AI, Golde TE, Funk C, Li H, Price ND, Petersen RC, Graff-Radford NR, Younkin SG, Dickson DW, Crook JR, Asmann YW, Ertekin-Taner N (2018) Conserved brain myelination networks are altered in Alzheimer’s and other neurodegen erative diseases. Alzheimers Dement J Alzheimers Assoc 14:352–366. https:// doi.org/10.1016/j.jalz.2017.09.012 23. Cai Z, Xiao M (2016) Oligodendrocytes and Alzheimer’s disease. Int J Neurosci 126:97–104. https://doi.org/10.3109/00207454.2015.1025778 23. Cai Z, Xiao M (2016) Oligodendrocytes and Alzheimer’s disease. Int J Neurosci 126:97–104. https://doi.org/10.3109/00207454.2015.1025778 24. Cao W, Zheng H (2018) Peripheral immune system in aging and Alzheimer’s disease. Mol Neurodegener 13:51. https://doi.org/10.1186/ s13024-018-0284-2 24. Cao W, Zheng H (2018) Peripheral immune system in aging and Alzheimer’s disease. Mol Neurodegener 13:51. https://doi.org/10.1186/ s13024-018-0284-2 25. Chandra A (2015) Role of amyloid from a multiple sclerosis perspective: a literature review. Neuroimmunomodulation 22:343–346. https://doi. org/10.1159/000375309 25. Chandra A (2015) Role of amyloid from a multiple sclerosis perspective: a literature review. Neuroimmunomodulation 22:343–346. https://doi. org/10.1159/000375309 g j j 4. Almeida ZL, Brito RMM (2020) Structure and aggregation mecha nisms in Amyloids. Mol Basel Switz 25:1195. https://doi.org/10.3390/ molecules25051195 26. Financial interests 18. Bouhrara M, Reiter DA, Bergeron CM, Zukley LM, Ferrucci L, Resnick SM, Spen cer RG (2018) Evidence of demyelination in mild cognitive impairment and dementia using a direct and specific magnetic resonance imaging measure of myelin content. Alzheimers Dement J Alzheimers Assoc 14:998–1004. https://doi.org/10.1016/j.jalz.2018.03.007 Received: 3 February 2023 / Accepted: 20 March 2023 g j j 19. Braak H, Del Tredici K (2015) The preclinical phase of the pathological process underlying sporadic Alzheimer’s disease. Brain J Neurol 138:2814–2833. https://doi.org/10.1093/brain/awv236 g j j 19. Braak H, Del Tredici K (2015) The preclinical phase of the pathological process underlying sporadic Alzheimer’s disease. Brain J Neurol 138:2814–2833. https://doi.org/10.1093/brain/awv236 20. Briston T, Hicks AR (2018) Mitochondrial dysfunction and neurodegenerative proteinopathies: mechanisms and prospects for therapeutic intervention. Biochem Soc Trans 46:829–842. https://doi.org/10.1042/BST20180025 Data Availability Not applicable. 13. Basavarajappa BS, Subbanna S (2021) Histone methylation regulation in neurode generative Disorders. Int J Mol Sci 22:4654. https://doi.org/10.3390/ijms22094654 13. Basavarajappa BS, Subbanna S (2021) Histone methylation regulation in neurode generative Disorders. Int J Mol Sci 22:4654. https://doi.org/10.3390/ijms22094654 14. Bayer TA (2015) Proteinopathies, a core concept for understanding and ultimately treating degenerative disorders? Eur Neuropsychopharmacol J Eur Coll Neuropsychopharmacol 25:713–724. https://doi.org/10.1016/j. euroneuro.2013.03.007 Consent for publication Not applicable. 16. Berry K, Wang J, Lu QR (2020) Epigenetic regulation of oligodendrocyte myelination in developmental disorders and neurodegenerative dis eases. F1000Research 9:F1000 Faculty Rev-105. https://doi.org/10.12688/ f1000research.20904.1 16. Berry K, Wang J, Lu QR (2020) Epigenetic regulation of oligodendrocyte myelination in developmental disorders and neurodegenerative dis eases. F1000Research 9:F1000 Faculty Rev-105. https://doi.org/10.12688/ f1000research.20904.1 17. Bonetto G, Kamen Y, Evans KA, Káradóttir RT (2020) Unraveling myelin plastic ity. Front Cell Neurosci 14:156. https://doi.org/10.3389/fncel.2020.00156 17. Bonetto G, Kamen Y, Evans KA, Káradóttir RT (2020) Unraveling myelin plastic ity. Front Cell Neurosci 14:156. https://doi.org/10.3389/fncel.2020.00156 References Chen Y, Fang L, Chen S, Zhou H, Fan Y, Lin L, Li J, Xu J, Chen Y, Ma Y, Chen Y (2020) Gut microbiome alterations precede cerebral amyloidosis and Microglial Pathology in a mouse model of Alzheimer’s Disease. BioMed Res Int 2020:8456596. https://doi.org/10.1155/2020/8456596 5. Andrews SJ, Fulton-Howard B, Goate A (2020) Interpretation of risk loci from genome-wide association studies of Alzheimer’s disease. Lancet Neurol 19:326–335. https://doi.org/10.1016/S1474-4422(19)30435-1 27. Chen J-F, Liu K, Hu B, Li R-R, Xin W, Chen H, Wang F, Chen L, Li R-X, Ren S-Y, Xiao L, Chan JR, Mei F (2021) Enhancing myelin renewal reverses cognitive dysfunction in a murine model of Alzheimer’s disease. Neuron 109:2292– 2307e5. https://doi.org/10.1016/j.neuron.2021.05.012 6. Antontseva E, Bondar N, Reshetnikov V, Merkulova T (2020) The Effects of chronic stress on Brain Myelination in humans and in various Rodent Models. Neuroscience 441:226–238. https://doi.org/10.1016/j. neuroscience.2020.06.013 p g j 28. Chew H, Solomon VA, Fonteh AN (2020) Involvement of lipids in Alzheimer’s Disease Pathology and potential therapies. Front Physiol 11:598. https://doi. org/10.3389/fphys.2020.00598 28. Chew H, Solomon VA, Fonteh AN (2020) Involvement of lipids in Alzheimer’s Disease Pathology and potential therapies. Front Physiol 11:598. https://doi. org/10.3389/fphys.2020.00598 7. Aprato J, Sock E, Weider M, Elsesser O, Fröb F, Wegner M (2020) Myrf guides target gene selection of transcription factor Sox10 during oligodendroglial develop ment. Nucleic Acids Res 48:1254–1270. https://doi.org/10.1093/nar/gkz1158 7. Aprato J, Sock E, Weider M, Elsesser O, Fröb F, Wegner M (2020) Myrf guides target gene selection of transcription factor Sox10 during oligodendroglial develop ment. Nucleic Acids Res 48:1254–1270. https://doi.org/10.1093/nar/gkz1158 29. Chi H, Cao W, Zhang M, Su D, Yang H, Li Z, Li C, She X, Wang K, Gao X, Ma K, Zheng P, Li X, Cui B (2021) Environmental noise stress disturbs commensal microbiota homeostasis and induces oxi-inflammmation and AD-like neuro pathology through epithelial barrier disruption in the EOAD mouse model. J Neuroinflammation 18:9. https://doi.org/10.1186/s12974-020-02053-3 g g 8. Ashraf GM, Greig NH, Khan TA, Hassan I, Tabrez S, Shakil S, Sheikh IA, Zaidi SK, Akram M, Jabir NR, Firoz CK, Naeem A, Alhazza IM, Damanhouri GA, Kamal MA (2014) Protein misfolding and aggregation in Alzheimer’s disease and type 2 diabetes mellitus. CNS Neurol Disord Drug Targets 13:1280–1293. https://doi.org/10.2174/1871527313666140917095514 8. Funding g Funding provided by the University of Strasbourg and by the INSERM. Maitre et al. Acta Neuropathologica Communications (2023) 11:56 Page 13 of 18 (2023) 11:56 Page 13 of 18 Maitre et al. Acta Neuropathologica Communications Declarations 15. Berer K, Mues M, Koutrolos M, Rasbi ZA, Boziki M, Johner C, Wekerle H, Krish namoorthy G (2011) Commensal microbiota and myelin autoantigen cooper ate to trigger autoimmune demyelination. Nature 479:538–541. https://doi. org/10.1038/nature10554 15. Berer K, Mues M, Koutrolos M, Rasbi ZA, Boziki M, Johner C, Wekerle H, Krish namoorthy G (2011) Commensal microbiota and myelin autoantigen cooper ate to trigger autoimmune demyelination. Nature 479:538–541. https://doi. org/10.1038/nature10554 References Gouw AA, Seewann A, Vrenken H, van der Flier WM, Rozemuller JM, Barkhof F, Scheltens P, Geurts JJG (2008) Heterogeneity of white matter hyperintensities in Alzheimer’s disease: post-mortem quantitative MRI and neuropathology. Brain J Neurol 131:3286–3298. https://doi.org/10.1093/brain/awn265 45. Edwards LJ, Kirilina E, Mohammadi S, Weiskopf N (2018) Microstructural imaging of human neocortex in vivo. NeuroImage 182:184–206. https://doi. org/10.1016/j.neuroimage.2018.02.055 46. Eiser AR, Fulop T (2020) Extra-cranial factors in the development of Alzheimer’s disease. Brain Res 1748:147076. https://doi.org/10.1016/j. brainres.2020.147076 46. Eiser AR, Fulop T (2020) Extra-cranial factors in the development of Alzheimer’s disease. Brain Res 1748:147076. https://doi.org/10.1016/j. brainres.2020.147076 65. Griciuc A, Tanzi RE (2021) The role of innate immune genes in Alzheim er’s disease. Curr Opin Neurol 34:228–236. https://doi.org/10.1097/ WCO.0000000000000911 47. Ettle B, Schlachetzki JCM, Winkler J (2016) Oligodendroglia and myelin in neurodegenerative diseases: more than just bystanders? Mol Neurobiol 53:3046–3062. https://doi.org/10.1007/s12035-015-9205-3 66. Griggs RB, Yermakov LM, Susuki K (2017) Formation and disruption of func tional domains in myelinated CNS axons. Neurosci Res 116:77–87. https://doi. org/10.1016/j.neures.2016.09.010 48. Faizy TD, Thaler C, Broocks G, Flottmann F, Leischner H, Kniep H, Nawabi J, Schön G, Stellmann J-P, Kemmling A, Reddy R, Heit JJ, Fiehler J, Kumar D, Hanning U (2020) The myelin Water Fraction serves as a marker for age-related myelin altera tions in the cerebral White Matter – A Multiparametric MRI Aging Study. Front Neurosci 14:136. https://doi.org/10.3389/fnins.2020.00136 67. Gu L, Wu D, Tang X, Qi X, Li X, Bai F, Chen X, Ren Q, Zhang Z (2018) Myelin changes at the early stage of 5XFAD mice. Brain Res Bull 137:285–293. https:// doi.org/10.1016/j.brainresbull.2017.12.013 68. Hamilton NB, Clarke LE, Arancibia-Carcamo IL, Kougioumtzidou E, Matthey M, Káradóttir R, Whiteley L, Bergersen LH, Richardson WD, Attwell D (2017) Endog enous GABA controls oligodendrocyte lineage cell number, myelination, and CNS internode length. Glia 65:309–321. https://doi.org/10.1002/glia.23093 g 49. Falangola MF, Nie X, Ward R, McKinnon ET, Dhiman S, Nietert PJ, Helpern JA, Jensen JH (2020) Diffusion MRI detects early brain microstructure abnormali ties in 2-month-old 3×Tg-AD mice. NMR Biomed 33:e4346. https://doi. org/10.1002/nbm.4346 69. Hardy J, Allsop D (1991) Amyloid deposition as the central event in the aetiol ogy of Alzheimer’s disease. Trends Pharmacol Sci 12:383–388. https://doi. org/10.1016/0165-6147(91)90609-v 50. Farah MH, Pan BH, Hoffman PN, Ferraris D, Tsukamoto T, Nguyen T, Wong PC, Price DL, Slusher BS, Griffin JW (2011) Reduced BACE1 activity enhances clearance of myelin debris and regeneration of axons in the injured periph eral nervous system. References Decourt B, Boumelhem F, Pope ED, Shi J, Mari Z, Sabbagh MN (2021) Critical Appraisal of amyloid lowering agents in AD. Curr Neurol Neurosci Rep 21:39. https://doi.org/10.1007/s11910-021-01125-y p g j 39. Decourt B, Boumelhem F, Pope ED, Shi J, Mari Z, Sabbagh MN (2021) Critical Appraisal of amyloid lowering agents in AD. Curr Neurol Neurosci Rep 21:39. https://doi.org/10.1007/s11910-021-01125-y 40. DeFlitch L, Gonzalez-Fernandez E, Crawley I, Kang SH (2022) Age and Alzheimer’s Disease-Related oligodendrocyte changes in hippocampal subregions. Front Cell Neurosci 16:847097. https://doi.org/10.3389/fncel.2022.847097 60. Friedland RP, McMillan JD, Kurlawala Z (2020) What are the Molecular Mechanisms by which functional bacterial amyloids influence amyloid Beta deposition and neuroinflammation in neurodegenerative Disorders? Int J Mol Sci 21:1652. https://doi.org/10.3390/ijms21051652 40. DeFlitch L, Gonzalez-Fernandez E, Crawley I, Kang SH (2022) Age and Alzheimer’s Disease-Related oligodendrocyte changes in hippocampal subregions. Front Cell Neurosci 16:847097. https://doi.org/10.3389/fncel.2022.847097 41. DeTure MA, Dickson DW (2019) The neuropathological diagnosis of Alzheimer’s disease. Mol Neurodegener 14:32. https://doi.org/10.1186/ s13024-019-0333-5 61. Frisoni GB, Altomare D, Thal DR, Ribaldi F, van der Kant R, Ossenkoppele R, Blennow K, Cummings J, van Duijn C, Nilsson PM, Dietrich P-Y, Scheltens P, Dubois B (2022) The probabilistic model of Alzheimer disease: the amyloid hypothesis revised. Nat Rev Neurosci 23:53–66. https://doi.org/10.1038/ s41583-021-00533-w 42. Di Domenico F, Pupo G, Giraldo E, Lloret A, Badia M-C, Schinina ME, Giorgi A, Butterfield DA, Vina J, Perluigi M (2016) Autoantibodies Profile in matching CSF and serum from AD and aMCI patients: potential pathogenic role and link to oxidative damage. Curr Alzheimer Res 13:112–122. https://doi.org/10.2 174/1567205013666151218131424 62. Ge W, Cheng Y, Zhang S, Ye S, Chen J, Wang R, Li C (2000) Variation of myelin basic protein and its antibody in serum in senile dementia patients. Chin Med Sci J Chung-Kuo Hsueh Ko Hsueh Tsa Chih 15:28 43. Di Fede G, Giaccone G, Tagliavini F (2013) Hereditary and sporadic beta-amyloidoses. Front Biosci Landmark Ed 18:1202–1226. https://doi. org/10.2741/4173 63. Goate A, Chartier-Harlin MC, Mullan M, Brown J, Crawford F, Fidani L, Giuffra L, Haynes A, Irving N, James L (1991) Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer’s disease. Nature 349:704–706. https://doi.org/10.1038/349704a0 44. Di Paolo G, Kim T-W (2011) Linking lipids to Alzheimer’s disease: cholesterol and beyond. Nat Rev Neurosci 12:284–296. https://doi.org/10.1038/nrn3012 64. References Ashraf GM, Greig NH, Khan TA, Hassan I, Tabrez S, Shakil S, Sheikh IA, Zaidi SK, Akram M, Jabir NR, Firoz CK, Naeem A, Alhazza IM, Damanhouri GA, Kamal MA (2014) Protein misfolding and aggregation in Alzheimer’s disease and type 2 diabetes mellitus. CNS Neurol Disord Drug Targets 13:1280–1293. https://doi.org/10.2174/1871527313666140917095514 30. Chomyk AM, Volsko C, Tripathi A, Deckard SA, Trapp BD, Fox RJ, Dutta R (2017) DNA methylation in demyelinated multiple sclerosis hippocampus. Sci Rep 7:8696. https://doi.org/10.1038/s41598-017-08623-5 9. Atwood CS, Bowen RL (2015) A unified hypothesis of early- and late-onset Alzheimer’s Disease Pathogenesis. J Alzheimers Dis JAD 47:33–47. https://doi. org/10.3233/JAD-143210 31. Ciccocioppo F, Bologna G, Ercolino E, Pierdomenico L, Simeone P, Lanuti P, Pieragostino D, Del Boccio P, Marchisio M, Miscia S (2020) Neurodegenera tive diseases as proteinopathies-driven immune disorders. Neural Regen Res 15:850–856. https://doi.org/10.4103/1673-5374.268971 g 10. Austin BP, Nair VA, Meier TB, Xu G, Rowley HA, Carlsson CM, Johnson SC, Prab hakaran V (2011) Effects of hypoperfusion in Alzheimer’s disease. J Alzheimers Dis JAD 26(Suppl 3):123–133. https://doi.org/10.3233/JAD-2011-0010 10. Austin BP, Nair VA, Meier TB, Xu G, Rowley HA, Carlsson CM, Johnson SC, Prab hakaran V (2011) Effects of hypoperfusion in Alzheimer’s disease. J Alzheimers Dis JAD 26(Suppl 3):123–133. https://doi.org/10.3233/JAD-2011-0010 32. Couttas TA, Kain N, Suchowerska AK, Quek L-E, Turner N, Fath T, Garner B, Don AS (2016) Loss of ceramide synthase 2 activity, necessary for myelin biosynthesis, precedes tau pathology in the cortical pathogenesis of Alzheimer’s disease. Neurobiol Aging 43:89–100. https://doi.org/10.1016/j. neurobiolaging.2016.03.027 11. Bader JM, Geyer PE, Müller JB, Strauss MT, Koch M, Leypoldt F, Koertvelyessy P, Bittner D, Schipke CG, Incesoy EI, Peters O, Deigendesch N, Simons M, Jensen MK, Zetterberg H, Mann M (2020) Proteome profiling in cerebrospinal fluid reveals novel biomarkers of Alzheimer’s disease. Mol Syst Biol 16:e9356. https://doi.org/10.15252/msb.20199356 33. Das R, Chinnathambi S (2019) Microglial priming of antigen presentation and adaptive stimulation in Alzheimer’s disease. Cell Mol Life Sci CMLS 76:3681–3694. https://doi.org/10.1007/s00018-019-03132-2 12. Bartzokis G (2004) Age-related myelin breakdown: a developmental model of cognitive decline and Alzheimer’s disease. Neurobiol Aging 25:5–18 author reply 49–62. https://doi.org/10.1016/j.neurobiolaging.2003.03.001 Page 14 of 18 Maitre et al. Acta Neuropathologica Communications (2023) 11:56 (2023) 11:56 53. Fernandez-Alvarez M, Atienza M, Cantero JL (2022) Effects of non-modifiable risk factors of Alzheimer’s disease on intracortical myelin content. Alzheimers Res Ther 14:202. https://doi.org/10.1186/s13195-022-01152-y 34. References Dayon L, Núñez Galindo A, Wojcik J, Cominetti O, Corthésy J, Oikonomidi A, Henry H, Kussmann M, Migliavacca E, Severin I, Bowman GL, Popp J (2018) Alzheimer disease pathology and the cerebrospinal fluid proteome. Alzheim ers Res Ther 10:66. https://doi.org/10.1186/s13195-018-0397-4 54. Ferreira S, Pitman KA, Wang S, Summers BS, Bye N, Young KM, Cullen CL (2020) Amyloidosis is associated with thicker myelin and increased oligoden drogenesis in the adult mouse brain. J Neurosci Res 98:1905–1932. https:// doi.org/10.1002/jnr.24672 35. de Faria O, Pivonkova H, Varga B, Timmler S, Evans KA, Káradóttir RT (2021) Periods of synchronized myelin changes shape brain function and plasticity. Nat Neurosci 24:1508–1521. https://doi.org/10.1038/s41593-021-00917-2 36. de la Fuente AG, Queiroz RML, Ghosh T, McMurran CE, Cubillos JF, Bergles DE, Fitzgerald DC, Jones CA, Lilley KS, Glover CP, Franklin RJM (2020) Changes in the Oligodendrocyte Progenitor Cell Proteome with Ageing. Mol Cell Proteomics MCP 19:1281–1302. https://doi.org/10.1074/mcp.RA120.002102 55. Ferrer I, Andrés-Benito P (2020) White matter alterations in Alzheimer’s disease without concomitant pathologies. Neuropathol Appl Neurobiol 46:654–672. https://doi.org/10.1111/nan.12618 56. Filley CM, Fields RD (2016) White matter and cognition: making the connec tion. J Neurophysiol 116:2093–2104. https://doi.org/10.1152/jn.00221.2016 37. De Rossi P, Buggia-Prévot V, Clayton BLL, Vasquez JB, van Sanford C, Andrew RJ, Lesnick R, Botté A, Deyts C, Salem S, Rao E, Rice RC, Parent A, Kar S, Popko B, Pytel P, Estus S, Thinakaran G (2016) Predominant expression of Alzheimer’s disease- associated BIN1 in mature oligodendrocytes and localization to white matter tracts. Mol Neurodegener 11:59. https://doi.org/10.1186/s13024-016-0124-1 57. Filley CM (2021) Cognitive dysfunction in White Matter Disorders: New Perspectives in Treatment and Recovery. J Neuropsychiatry Clin Neurosci 33:349–355. https://doi.org/10.1176/appi.neuropsych.21030080 58. Fontenas L, Welsh TG, Piller M, Coughenour P, Gandhi AV, Prober DA, Kucenas S (2019) The Neuromodulator Adenosine regulates Oligodendrocyte Migra tion at Motor Exit Point transition zones. Cell Rep 27:115–128e5. https://doi. org/10.1016/j.celrep.2019.03.013 38. Dean DC, Hurley SA, Kecskemeti SR, O’Grady JP, Canda C, Davenport-Sis NJ, Carlsson CM, Zetterberg H, Blennow K, Asthana S, Sager MA, Johnson SC, Alexander AL, Bendlin BB (2017) Association of amyloid Pathology with myelin alteration in preclinical Alzheimer Disease. JAMA Neurol 74:41–49. https://doi.org/10.1001/jamaneurol.2016.3232 59. Fotiadis P, Reijmer YD, Van Veluw SJ, Martinez-Ramirez S, Karahanoglu FI, Gok cal E, Schwab KM, Alzheimer’s Disease Neuroimaging Initiative study group, Goldstein JN, Rosand J, Viswanathan A, Greenberg SM, Gurol ME (2020) White matter atrophy in cerebral amyloid angiopathy. Neurology 95:e554–e562. https://doi.org/10.1212/WNL.0000000000010017 p g j 39. References Kövari E, Gold G, Herrmann FR, Canuto A, Hof PR, Bouras C, Giannakopoulos P (2007) Cortical microinfarcts and demyelination affect cognition in cases at high risk for dementia. Neurology 68:927–931. https://doi.org/10.1212/01. wnl.0000257094.10655.9a g g p g j g g 76. Hou Y, Song H, Croteau DL, Akbari M, Bohr VA (2017) Genome instability in Alzheimer disease. Mech Ageing Dev 161:83–94. https://doi.org/10.1016/j. mad.2016.04.005 77. Hu J-G, Wu X-J, Feng Y-F, Xi G-M, Wang Z-H, Zhou J-S, Lü H-Z (2012) PDGF-AA and bFGF mediate B104CM-induced proliferation of oligodendrocyte precursor cells. Int J Mol Med 30:1113–1118. https://doi.org/10.3892/ijmm.2012.1110 95. Krishnan KJ, Ratnaike TE, De Gruyter HLM, Jaros E, Turnbull DM (2012) Mito chondrial DNA deletions cause the biochemical defect observed in Alzheim er’s disease. Neurobiol Aging 33:2210–2214. https://doi.org/10.1016/j. neurobiolaging.2011.08.009 78. Ihara M, Polvikoski TM, Hall R, Slade JY, Perry RH, Oakley AE, Englund E, O’Brien JT, Ince PG, Kalaria RN (2010) Quantification of myelin loss in frontal lobe white matter in vascular dementia, Alzheimer’s disease, and dementia with Lewy bodies. Acta Neuropathol (Berl) 119:579–589. https://doi.org/10.1007/ s00401-009-0635-8 g g 96. Kuhn S, Gritti L, Crooks D, Dombrowski Y (2019) Oligodendrocytes in develop ment, myelin Generation and Beyond. Cells 8:1424. https://doi.org/10.3390/ cells8111424 97. Kundu D, Prerna K, Chaurasia R, Bharty MK, Dubey VK (2020) Advances in protein misfolding, amyloidosis and its correlation with human diseases. 3 Biotech 10:193. https://doi.org/10.1007/s13205-020-2166-x 79. Iordanishvili E, Schall M, Loução R, Zimmermann M, Kotetishvili K, Shah NJ, Oros-Peusquens A-M (2019) Quantitative MRI of cerebral white matter hyperintensities: a new approach towards understanding the underly ing pathology. NeuroImage 202:116077. https://doi.org/10.1016/j. neuroimage.2019.116077 98. Kunkle BW, Grenier-Boley B, Sims R, Bis JC, Damotte V, Naj AC, Boland A, Vron skaya M, van der Lee SJ et al (2019) Alzheimer Disease Genetics Consortium (ADGC), European Alzheimer’s Disease Initiative (EADI), Cohorts for Heart and Aging Research in Genomic Epidemiology Consortium (CHARGE), Genetic and Environmental Risk in AD/Defining Genetic, Polygenic and Environ mental Risk for Alzheimer’s Disease Consortium (GERAD/PERADES), Genetic meta-analysis of diagnosed Alzheimer’s disease identifies new risk loci and implicates Aβ, tau, immunity and lipid processing. Nat Genet 51:414–430. doi: https://doi.org/10.1038/s41588-019-0358-2 80. Irwin RW, Wang JM, Chen S, Brinton RD (2011) Neuroregenerative mecha nisms of allopregnanolone in Alzheimer’s disease. Front Endocrinol 2:117. https://doi.org/10.3389/fendo.2011.00117 81. References J Neurosci Off J Soc Neurosci 31:5744–5754. https://doi. org/10.1523/JNEUROSCI.6810-10.2011 70. Hardy J, Selkoe DJ (2002) The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 297:353–356. https://doi.org/10.1126/science.1072994 51. Feng Y-S, Tan Z-X, Wu L-Y, Dong F, Zhang F (2020) The involvement of NLRP3 inflammasome in the treatment of Alzheimer’s disease. Ageing Res Rev 64:101192. https://doi.org/10.1016/j.arr.2020.101192 71. Heilig M, Sjögren M, Blennow K, Ekman R, Wallin A (1995) Cerebrospinal fluid neuropeptides in Alzheimer’s disease and vascular dementia. Biol Psychiatry 38:210–216. https://doi.org/10.1016/0006-3223(94)00239-Y 72. Hoban AE, Stilling RM, Ryan FJ, Shanahan F, Dinan TG, Claesson MJ, Clarke G, Cryan JF (2016) Regulation of prefrontal cortex myelination by the micro biota. Transl Psychiatry 6:e774. https://doi.org/10.1038/tp.2016.42 52. Fernandez-Alvarez M, Atienza M, Zallo F, Matute C, Capetillo-Zarate E, Cantero JL (2022) Linking plasma amyloid Beta and neurofilament light chain to intracortical myelin content in cognitively normal older adults. Front Aging Neurosci 14:896848. https://doi.org/10.3389/fnagi.2022.896848 y y p g p 73. Holubiec MI, Gellert M, Hanschmann EM (2022) Redox signaling and metabo lism in Alzheimer’s disease. Front Aging Neurosci 14:1003721. https://doi. org/10.3389/fnagi.2022.1003721 73. Holubiec MI, Gellert M, Hanschmann EM (2022) Redox signaling and metabo lism in Alzheimer’s disease. Front Aging Neurosci 14:1003721. https://doi. org/10.3389/fnagi.2022.1003721 Page 15 of 18 Maitre et al. Acta Neuropathologica Communications (2023) 11:56 92. Kim D-K, Kim TH, Lee S-J (2016) Mechanisms of aging-related proteinopathies in Caenorhabditis elegans. Exp Mol Med 48:e263. https://doi.org/10.1038/ emm.2016.109 74. Honarpisheh P, Reynolds CR, Blasco Conesa MP, Moruno Manchon JF, Putluri N, Bhattacharjee MB, Urayama A, McCullough LD, Ganesh BP (2020) Dys regulated gut homeostasis observed prior to the Accumulation of the Brain Amyloid-β in Tg2576 mice. Int J Mol Sci 21:1711. https://doi.org/10.3390/ ijms21051711 93. Kobylarek D, Iwanowski P, Masolak D, Limphaibool N, Chou JT-T, Kozubski W (2020) A case report: co-occurrence of cerebral amyloid angiopathy and mul tiple sclerosis. Mult Scler Relat Disord 46:102517. https://doi.org/10.1016/j. msard.2020.102517 75. Horiuchi M, Maezawa I, Itoh A, Wakayama K, Jin L-W, Itoh T, Decarli C (2012) Amyloid β1–42 oligomer inhibits myelin sheet formation in vitro. Neurobiol Aging 33:499–509. https://doi.org/10.1016/j.neurobiolaging.2010.05.007 75. Horiuchi M, Maezawa I, Itoh A, Wakayama K, Jin L-W, Itoh T, Decarli C (2012) Amyloid β1–42 oligomer inhibits myelin sheet formation in vitro. Neurobiol Aging 33:499–509. https://doi.org/10.1016/j.neurobiolaging.2010.05.007 76. Hou Y, Song H, Croteau DL, Akbari M, Bohr VA (2017) Genome instability in Alzheimer disease. Mech Ageing Dev 161:83–94. https://doi.org/10.1016/j. mad.2016.04.005 94. References Jansen IE, Savage JE, Watanabe K, Bryois J, Williams DM, Steinberg S, Sealock J, Karlsson IK, Hägg S, Athanasiu L, Voyle N, Proitsi P, Witoelar A, Stringer S, Aarsland D, Almdahl IS, Andersen F, Bergh S, Bettella F, Bjornsson S, Brækhus A, Bråthen G, de Leeuw C, Desikan RS, Djurovic S, Dumitrescu L, Fladby T, Hohman TJ, Jonsson PV, Kiddle SJ, Rongve A, Saltvedt I, Sando SB, Selbæk G, Shoai M, Skene NG, Snaedal J, Stordal E, Ulstein ID, Wang Y, White LR, Hardy J, Hjerling-Leffler J, Sullivan PF, van der Flier WM, Dobson R, Davis LK, Stefansson H, Stefansson K, Pedersen NL, Ripke S, Andreassen OA, Posthuma D (2019) Genome-wide meta-analysis identifies new loci and functional pathways influencing Alzheimer’s disease risk. Nat Genet 51:404–413. https://doi. org/10.1038/s41588-018-0311-9 99. Laule C, Leung E, Lis DKB, Traboulsee AL, Paty DW, MacKay AL, Moore GRW (2006) Myelin water imaging in multiple sclerosis: quantitative correlations with histopathology. Mult Scler Houndmills Basingstoke Engl 12:747–753. https://doi.org/10.1177/1352458506070928 100. Lebel C, Deoni S (2018) The development of brain white matter microstructure. NeuroImage 182:207–218. https://doi.org/10.1016/j. neuroimage.2017.12.097 101. Lee J-T, Xu J, Lee J-M, Ku G, Han X, Yang D-I, Chen S, Hsu CY (2004) Amyloid- beta peptide induces oligodendrocyte death by activating the neutral sphingomyelinase-ceramide pathway. J Cell Biol 164:123–131. https://doi. org/10.1083/jcb.200307017 82. Jellinger KA (2020) Neuropathological assessment of the Alzheimer spec trum. J Neural Transm Vienna Austria 1996 127:1229–1256. doi: https://doi. org/10.1007/s00702-020-02232-9 83. Jean Harry G, Toews AD (1998) Myelination, dysmyelination, and demyelin ation. Handb Dev Neurotoxicology. Handb Dev Neurotoxicology 87–115. https://doi.org/10.1016/B978-012648860-9.50007-8 102. Lee S, Viqar F, Zimmerman ME, Narkhede A, Tosto G, Benzinger TLS, Marcus DS, Fagan AM, Goate A et al (2016) Dominantly Inherited Alzheimer Network White matter hyperintensities are a core feature of Alzheimer’s disease: Evidence from the dominantly inherited Alzheimer network. Ann Neurol 79:929–939. doi: https://doi.org/10.1002/ana.24647 84. Jin L, Pan Y, Tran NLL, Polychronopoulos LN, Warrier A, Brouwer KLR, Nicolazzo JA (2020) Intestinal permeability and oral absorption of selected drugs are reduced in a mouse model of familial Alzheimer’s Disease. Mol Pharm 17:1527–1537. https://doi.org/10.1021/acs.molpharmaceut.9b01227 103. Leidinger P, Backes C, Deutscher S, Schmitt K, Mueller SC, Frese K, Haas J, Ruprecht K, Paul F, Stähler C, Lang CJG, Meder B, Bartfai T, Meese E, Keller A (2013) A blood based 12-miRNA signature of Alzheimer disease patients. Genome Biol 14:R78. https://doi.org/10.1186/gb-2013-14-7-r78 85. Johnson LA (2020) APOE and metabolic dysfunction in Alzheimer’s disease. Int Rev Neurobiol 154:131–151. References https://doi.org/10.1016/bs.irn.2020.02.002 86. Kaeser GE, Chun J (2020) Mosaic somatic gene recombination as a potentially unifying hypothesis for Alzheimer’s Disease. Front Genet 11:390. https://doi. org/10.3389/fgene.2020.00390 104. Leszek J, Barreto GE, Gąsiorowski K, Koutsouraki E, Ávila-Rodrigues M, Aliev G (2016) Inflammatory mechanisms and oxidative stress as key factors respon sible for progression of neurodegeneration: role of Brain Innate Immune System. CNS Neurol Disord Drug Targets 15:329–336. https://doi.org/10.2174/ 1871527315666160202125914 g g 87. Kao Y-C, Ho P-C, Tu Y-K, Jou I-M, Tsai K-J (2020) Lipids and Alzheimer’s Disease. Int J Mol Sci 21:1505. https://doi.org/10.3390/ijms21041505 105. Li D, Zhang J, Liu Q (2022) Brain cell type-specific cholesterol metabolism and implications for learning and memory. Trends Neurosci 45:401–414. https:// doi.org/10.1016/j.tins.2022.01.002 88. Kavroulakis E, Simos PG, Kalaitzakis G, Maris TG, Karageorgou D, Zaganas I, Panagiotakis S, Basta M, Vgontzas A, Papadaki E (2018) Myelin content changes in probable Alzheimer’s disease and mild cognitive impairment: Associations with age and severity of neuropsychiatric impairment. J Magn Reson Imaging JMRI 47:1359–1372. https://doi.org/10.1002/jmri.25849 106. Lin X, Kapoor A, Gu Y, Chow MJ, Peng J, Zhao K, Tang D (2020) Contributions of DNA damage to Alzheimer’s Disease. Int J Mol Sci 21:1666. https://doi. org/10.3390/ijms21051666 89. Kaya I, Jennische E, Lange S, Tarik Baykal A, Malmberg P, Fletcher JS (2020) Brain region-specific amyloid plaque-associated myelin lipid loss, APOE deposition and disruption of the myelin sheath in familial Alzheimer’s disease mice. J Neurochem 154:84–98. https://doi.org/10.1111/jnc.14999 107. Liu C-C, Liu C-C, Kanekiyo T, Xu H, Bu G (2013) Apolipoprotein E and Alzheimer disease: risk, mechanisms and therapy. Nat Rev Neurol 9:106–118. https://doi.org/10.1038/nrneurol.2012.263 108. Lodato MA, Walsh CA (2019) Genome aging: somatic mutation in the brain links age-related decline with disease and nominates pathogenic mecha nisms. Hum Mol Genet 28:R197–R206. https://doi.org/10.1093/hmg/ddz191 90. Keogh CE, Kim DHJ, Pusceddu MM, Knotts TA, Rabasa G, Sladek JA, Hsieh MT, Honeycutt M, Brust-Mascher I, Barboza M, Gareau MG (2021) Myelin as a regulator of development of the microbiota-gut-brain axis. Brain Behav Immun 91:437–450. https://doi.org/10.1016/j.bbi.2020.11.001 109. Liu H, Yang Y, Xia Y, Zhu W, Leak RK, Wei Z, Wang J, Hu X (2017) Aging of cerebral white matter. Ageing Res Rev 34:64–76. https://doi.org/10.1016/j.arr.2016.11.006 91. Kepchia D, Huang L, Dargusch R, Rissman RA, Shokhirev MN, Fischer W, Schubert D (2020) Diverse proteins aggregate in mild cognitive impair ment and Alzheimer’s disease brain. Alzheimers Res Ther 12:75. https://doi. org/10.1186/s13195-020-00641-2 110. References Masters SL, O’Neill LAJ (2011) Disease-associated amyloid and misfolded protein aggregates activate the inflammasome. Trends Mol Med 17:276–282. https://doi.org/10.1016/j.molmed.2011.01.005 142. Oakley H, Cole SL, Logan S, Maus E, Shao P, Craft J, Guillozet-Bongaarts A, Ohno M, Disterhoft J, Van Eldik L, Berry R, Vassar R (2006) Intraneuronal beta- amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer’s disease mutations: potential factors in amyloid plaque formation. J Neurosci Off J Soc Neurosci 26:10129–10140. https://doi. org/10.1523/JNEUROSCI.1202-06.2006 123. Mathys H, Davila-Velderrain J, Peng Z, Gao F, Mohammadi S, Young JZ, Menon M, He L, Abdurrob F, Jiang X, Martorell AJ, Ransohoff RM, Hafler BP, Bennett DA, Kellis M, Tsai L-H (2019) Single-cell transcriptomic analysis of Alzheimer’s disease. Nature 570:332–337. https://doi.org/10.1038/s41586-019-1195-2 124. McKenzie AT, Moyon S, Wang M, Katsyv I, Song W-M, Zhou X, Dammer EB, Duong DM, Aaker J, Zhao Y, Beckmann N et al (2017) Multiscale network modeling of oligodendrocytes reveals molecular components of myelin dysregulation in Alzheimer’s disease. Mol Neurodegener 12:82. https://doi. org/10.1186/s13024-017-0219-3 g 143. Ogunmokun G, Dewanjee S, Chakraborty P, Valupadas C, Chaudhary A, Kolli V, Anand U, Vallamkondu J, Goel P, Paluru HPR, Gill KD, Reddy PH, De Feo V, Kandimalla R (2021) The potential role of cytokines and growth factors in the pathogenesis of Alzheimer’s Disease. Cells 10:2790. https://doi.org/10.3390/ cells10102790 g 125. Mohd Murshid N, Aminullah Lubis F, Makpol S (2022) Epigenetic changes and its intervention in Age-Related neurodegenerative Diseases. Cell Mol Neurobiol 42:577–595. https://doi.org/10.1007/s10571-020-00979-z 144. Olsen I, Singhrao SK (2016) Inflammasome involvement in Alzheimer’s Disease. J Alzheimers Dis JAD 54:45–53. https://doi.org/10.3233/JAD-160197 126. Morabito S, Miyoshi E, Michael N, Shahin S, Martini AC, Head E, Silva J, Leavy K, Perez-Rosendahl M, Swarup V (2021) Single-nucleus chromatin accessibil ity and transcriptomic characterization of Alzheimer’s disease. Nat Genet 53:1143–1155. https://doi.org/10.1038/s41588-021-00894-z 145. Operto G, Cacciaglia R, Grau-Rivera O, Falcon C, Brugulat-Serrat A, Ródenas P, Ramos R, Morán S, Esteller M, Bargalló N, Molinuevo JL, Gispert JD, ALFA Study (2018) White matter microstructure is altered in cognitively normal middle-aged APOE-ε4 homozygotes. Alzheimers Res Ther 10:48. https://doi. org/10.1186/s13195-018-0375-x 127. Morell P, Quarles RH (1999) Myelin Formation, Structure and Biochemistry. In: Basic Neurochemistry, 6th edition, 6th edition. Raven Press, New York, pp 70–93 146. References Liu P, Jia X-Z, Chen Y, Yu Y, Zhang K, Lin Y-J, Wang B-H, Peng G-P (2021) Gut microbiota interacts with intrinsic brain activity of patients with amnestic (2023) 11:56 Page 16 of 18 Page 16 of 18 Maitre et al. Acta Neuropathologica Communications mild cognitive impairment. CNS Neurosci Ther 27:163–173. https://doi. org/10.1111/cns.13451 mild cognitive impairment. CNS Neurosci Ther 27:163–173. https://doi. org/10.1111/cns.13451 131. Moyon S, Frawley R, Marechal D, Huang D, Marshall-Phelps KLH, Kegel L, Bøstrand SMK, Sadowski B, Jiang Y-H, Lyons DA, Möbius W, Casaccia P (2021) TET1-mediated DNA hydroxymethylation regulates adult remyelination in mice. Nat Commun 12:3359. https://doi.org/10.1038/s41467-021-23735-3 g 111. Long P, Corfas G (2014) Neuroscience. To learn is to myelinate. Science 346:298–299. https://doi.org/10.1126/science.1261127 g 132. Mueller SG, Weiner MW (2009) Selective effect of age, apo e4, and Alzheim er’s disease on hippocampal subfields. Hippocampus 19:558–564. https://doi. org/10.1002/hipo.20614 112. Lorenzini L, Fernandez M, Baldassarro VA, Bighinati A, Giuliani A, Calzà L, Giardino L (2020) White Matter and Neuroprotection in Alzheimer’s demen tia. Mol Basel Switz 25:503. https://doi.org/10.3390/molecules25030503 113. Luczynski P, Laule C, Hsiung G-YR, Moore GRW, Tremlett H (2019) Coexistence of multiple sclerosis and Alzheimer’s disease: a review. Mult Scler Relat Disord 27:232–238. https://doi.org/10.1016/j.msard.2018.10.109 133. Murphy M, Dutton R, Koblar S, Cheema S, Bartlett P (1997) Cytokines which signal through the LIF receptor and their actions in the nervous system. Prog Neurobiol 52:355–378. https://doi.org/10.1016/s0301-0082(97)00020-8 134. Najm R, Jones EA, Huang Y (2019) Apolipoprotein E4, inhibitory network dysfunction, and Alzheimer’s disease. Mol Neurodegener 14:24. https://doi. org/10.1186/s13024-019-0324-6 114. Maarouf CL, Walker JE, Sue LI, Dugger BN, Beach TG, Serrano GE (2018) Impaired hepatic amyloid-beta degradation in Alzheimer’s disease. PLoS ONE 13:e0203659. https://doi.org/10.1371/journal.pone.0203659 135. Nalbandian A, Llewellyn KJ, Gomez A, Walker N, Su H, Dunnigan A, Chwa M, Vesa J, Kenney MC, Kimonis VE (2015) In vitro studies in VCP-associated multisystem proteinopathy suggest altered mitochondrial bioenergetics. Mitochondrion 22:1–8. https://doi.org/10.1016/j.mito.2015.02.004 115. Maetzler W, Berg D, Synofzik M, Brockmann K, Godau J, Melms A, Gasser T, Hörnig S, Langkamp M (2011) Autoantibodies against amyloid and glial- derived antigens are increased in serum and cerebrospinal fluid of Lewy body-associated dementias. J Alzheimers Dis JAD 26:171–179. https://doi. org/10.3233/JAD-2011-110221 g j 136. Nardini E, Hogan R, Flamier A, Bernier G (2021) Alzheimer’s disease: a tale of two diseases? Neural Regen Res 16:1958–1964. https://doi. org/10.4103/1673-5374.308070 g 116. References Maitre M, Klein C, Patte-Mensah C, Mensah-Nyagan G (2020) Tryptophan metabolites modify brain Aβ peptide degradation: a role in Alzheimer’s dis ease? Prog Neurobiol 190. https://doi.org/10.1016/j.pneurobio.2020.101800 137. Nasrabady SE, Rizvi B, Goldman JE, Brickman AM (2018) White matter changes in Alzheimer’s disease: a focus on myelin and oligodendrocytes. Acta Neuropathol Commun 6:22. https://doi.org/10.1186/s40478-018-0515-3 117. Malampati S, Song J-X, Chun-Kit Tong B, Nalluri A, Yang C-B, Wang Z, Gopalkrish nashetty Sreenivasmurthy S, Zhu Z, Liu J, Su C, Krishnamoorthi S, Iyaswamy A, Cheung K-H, Lu J-H, Li AM (2020) Targeting Aggrephagy for the treatment of Alzheimer’s Disease. Cells 9:311. https://doi.org/10.3390/cells9020311 138. Noor A, Zafar S, Zerr I (2021) Neurodegenerative Proteinopathies in the Proteoform Spectrum-Tools and Challenges. Int J Mol Sci 22:1085. https://doi. org/10.3390/ijms22031085 118. Malek-Ahmadi M, Perez SE, Chen K, Mufson EJ (2020) Braak Stage, cerebral amyloid Angiopathy, and Cognitive decline in early Alzheimer’s Disease. J Alzheimers Dis JAD 74:189–197. https://doi.org/10.3233/JAD-191151 139. Nickel M, Gu C (2018) Regulation of Central Nervous System Myelina tion in higher brain functions. Neural Plast 2018:6436453. https://doi. org/10.1155/2018/6436453 119. Manocha GD, Floden AM, Miller NM, Smith AJ, Nagamoto-Combs K, Saito T, Saido TC, Combs CK (2019) Temporal progression of Alzheimer’s disease in brains and intestines of transgenic mice. Neurobiol Aging 81:166–176. https://doi.org/10.1016/j.neurobiolaging.2019.05.025 140. Nicoll JAR, Buckland GR, Harrison CH, Page A, Harris S, Love S, Neal JW, Holmes C, Boche D (2019) Persistent neuropathological effects 14 years following amyloid-β immunization in Alzheimer’s disease. Brain J Neurol 142:2113–2126. https://doi.org/10.1093/brain/awz142 120. Marangon D, Boccazzi M, Lecca D, Fumagalli M (2020) Regulation of oligo dendrocyte functions: targeting lipid metabolism and extracellular matrix for myelin repair. J Clin Med 9:470. https://doi.org/10.3390/jcm9020470 141. Novotny BC, Fernandez MV, Wang C, Budde JP, Bergmann K, Eteleeb AM, Bradley J, Webster C, Ebl C, Norton J, Gentsch J, Dube U, Wang F, Morris JC, Bateman RJ, Perrin RJ, McDade E, Xiong C, Chhatwal J, Dominantly Inherited Alzheimer Network (DIAN) Study Group, Alzheimer’s Disease Neuroimaging Initiative, and the Alzheimer’s Disease Metabolomics Consortium (ADMC), Goate A, Farlow M, Schofield P, Chui H, Karch CM, Cruchaga C, Benitez BA, Harari O (2022) Metabolomic and lipidomic signatures in autosomal dominant and late-onset Alzheimer’s disease brains. Alzheimers Dement J Alzheimers Assoc. doi: https://doi.org/10.1002/alz.12800 121. Martin LJ, Wong M, Hanaford A (2019) Neonatal brain Injury and genetic causes of adult-onset neurodegenerative disease in mice interact with Effects on Acute and late outcomes. Front Neurol 10:635. https://doi.org/10.3389/ fneur.2019.00635 122. References Pace MC, Xu G, Fromholt S, Howard J, Crosby K, Giasson BI, Lewis J, Borchelt DR (2018) Changes in proteome solubility indicate widespread proteostatic disruption in mouse models of neurodegenerative disease. Acta Neuropathol (Berl) 136:919–938. https://doi.org/10.1007/s00401-018-1895-y 152. Papuć E, Kurys-Denis E, Krupski W, Tatara M, Rejdak K (2015) Can antibod ies against glial derived antigens be early biomarkers of hippocampal demyelination and memory loss in Alzheimer’s Disease? J Alzheimers Dis JAD 48:115–121. https://doi.org/10.3233/JAD-150309 173. Sheng C, Lin L, Lin H, Wang X, Han Y, Liu S-L (2021) Altered gut microbiota in adults with subjective cognitive decline: the SILCODE Study. J Alzheimers Dis JAD 82:513–526. https://doi.org/10.3233/JAD-210259 p g 153. Papuć E, Rejdak K (2020) The role of myelin damage in Alzheimer’s disease pathology. Arch Med Sci AMS 16:345–351. https://doi.org/10.5114/ aoms.2018.76863 174. Shukla PK, Delotterie DF, Xiao J, Pierre JF, Rao R, McDonald MP, Khan MM (2021) Alterations in the gut-microbial-inflammasome-brain Axis in a mouse model of Alzheimer’s Disease. Cells 10:779. https://doi.org/10.3390/ cells10040779 154. Pedrero-Prieto CM, Frontiñán-Rubio J, Alcaín FJ, Durán-Prado M, Peinado JR, Rabanal-Ruiz Y (2021) Biological significance of the protein changes occur ring in the Cerebrospinal Fluid of Alzheimer’s Disease Patients: getting clues from Proteomic Studies. Diagn Basel Switz 11:1655. https://doi.org/10.3390/ diagnostics11091655 175. Simons M, Trajkovic K (2006) Neuron-glia communication in the control of oligodendrocyte function and myelin biogenesis. J Cell Sci 119:4381–4389. https://doi.org/10.1242/jcs.03242 176. Sinclair P, Baranova A, Kabbani N (2021) Mitochondrial disruption by amyloid Beta 42 identified by Proteomics and Pathway Mapping. Cells 10:2380. https://doi.org/10.3390/cells10092380 155. Piersson AD, Ibrahim B, Suppiah S, Mohamad M, Hassan HA, Omar NF, Ibra him MI, Yusoff AN, Ibrahim N, Saripan MI, Razali RM (2021) Multiparametric MRI for the improved diagnostic accuracy of Alzheimer’s disease and mild cognitive impairment: Research protocol of a case-control study design. PLoS ONE 16:e0252883. https://doi.org/10.1371/journal.pone.0252883 177. Singh DK, Ling E-A, Kaur C (2018) Hypoxia and myelination deficits in the developing brain. Int J Dev Neurosci Off J Int Soc Dev Neurosci 70:3–11. https://doi.org/10.1016/j.ijdevneu.2018.06.012 156. Poggi G, Boretius S, Möbius W, Moschny N, Baudewig J, Ruhwedel T, Has souna I, Wieser GL, Werner HB, Goebbels S, Nave K-A, Ehrenreich H (2016) Cortical network dysfunction caused by a subtle defect of myelination. Glia 64:2025–2040. https://doi.org/10.1002/glia.23039 178. References Operto G, Molinuevo JL, Cacciaglia R, Falcon C, Brugulat-Serrat A, Suárez- Calvet M, Grau-Rivera O, Bargalló N, Morán S, Esteller M, Study ALFA, Gispert JD (2019) Interactive effect of age and APOE-ε4 allele load on white matter myelin content in cognitively normal middle-aged subjects. NeuroImage Clin 24:101983. https://doi.org/10.1016/j.nicl.2019.101983 128. Mortamais M, Reynes C, Brickman AM, Provenzano FA, Muraskin J, Portet F, Berr C, Touchon J, Bonafé A, le Bars E, Maller JJ, Meslin C, Sabatier R, Ritchie K, Artero S (2013) Spatial distribution of cerebral white matter lesions predicts progression to mild cognitive impairment and dementia. PLoS ONE 8:e56972. https://doi.org/10.1371/journal.pone.0056972 g j 147. Ota K, Oishi N, Ito K, Fukuyama H, Study Group SEAD-J, Alzheimer’s Disease Neuroimaging Initiative (2016) Prediction of Alzheimer’s disease in amnestic mild cognitive impairment subtypes: Stratification based on imaging biomarkers. J Alzheimers Dis JAD 52:1385–1401. https://doi.org/10.3233/JAD-160145 129. Mossad O, Blank T (2021) Getting on in Old Age: how the gut microbiota interferes with brain innate immunity. Front Cell Neurosci 15:698126. https:// doi.org/10.3389/fncel.2021.698126 148. Ota M, Sato N, Kimura Y, Shigemoto Y, Kunugi H, Matsuda H (2019) Changes of myelin Organization in patients with Alzheimer’s Disease shown by 130. Mot AI, Depp C, Nave K-A (2018) An emerging role of dysfunctional axon- oligodendrocyte coupling in neurodegenerative diseases. Dialogues Clin Neurosci 20:283–292 (2023) 11:56 Maitre et al. Acta Neuropathologica Communications Page 17 of 18 q-Space myelin map imaging. Dement Geriatr Cogn Disord Extra 9:24–33. https://doi.org/10.1159/000493937 170. Sathe G, Albert M, Darrow J, Saito A, Troncoso J, Pandey A, Moghekar A (2021) Quantitative proteomic analysis of the frontal cortex in Alzheimer’s disease. J Neurochem 156:988–1002. https://doi.org/10.1111/jnc.15116 149. Othman T, Yan H, Rivkees SA (2003) Oligodendrocytes express functional A1 adenosine receptors that stimulate cellular migration. Glia 44:166–172. https://doi.org/10.1002/glia.10281 171. Sciaccaluga M, Megaro A, Bellomo G, Ruffolo G, Romoli M, Palma E, Costa C (2021) An unbalanced synaptic transmission: cause or consequence of the amyloid oligomers neurotoxicity? Int J Mol Sci 22:5991. https://doi. org/10.3390/ijms22115991 150. Otto G (2021) Myelin loss in AD. Nat Rev Neurosci 22:456–457. https://doi. org/10.1038/s41583-021-00492-2 172. Serrano-Regal MP, Luengas-Escuza I, Bayón-Cordero L, Ibarra-Aizpurua N, Alberdi E, Pérez-Samartín A, Matute C, Sánchez-Gómez MV (2020) Oligodendrocyte differentiation and myelination is potentiated via GABAB receptor activation. Neuroscience 439:163–180. https://doi.org/10.1016/j. neuroscience.2019.07.014 151. References Smith DH, Nakamura M, McIntosh TK, Wang J, Rodríguez A, Chen XH, Raghupathi R, Saatman KE, Clemens J, Schmidt ML, Lee VM, Trojanowski JQ (1998) Brain trauma induces massive hippocampal neuron death linked to a surge in beta-amyloid levels in mice overexpressing mutant amyloid precursor protein. Am J Pathol 153:1005–1010. https://doi.org/10.1016/ s0002-9440(10)65643-x 157. Polychronidou E, Avramouli A, Vlamos P (2020) Alzheimer’s Disease: the role of mutations in protein folding. Adv Exp Med Biol 1195:227–236. https://doi. org/10.1007/978-3-030-32633-3_31 179. Sock E, Wegner M (2019) Transcriptional control of myelination and remyelin ation. Glia 67:2153–2165. https://doi.org/10.1002/glia.23636 158. Pruvost M, Moyon S (2021) Oligodendroglial epigenetics, from lineage speci fication to activity-dependent myelination. Life Basel Switz 11:62. https://doi. org/10.3390/life11010062 180. Soto C, Pritzkow S (2018) Protein misfolding, aggregation, and conforma tional strains in neurodegenerative diseases. Nat Neurosci 21:1332–1340. https://doi.org/10.1038/s41593-018-0235-9 g 159. Querfurth HW, LaFerla FM (2010) Alzheimer’s disease. N Engl J Med 362:329– 344. https://doi.org/10.1056/NEJMra0909142 g 181. Stadelmann C, Timmler S, Barrantes-Freer A, Simons M (2019) Myelin in the Central Nervous System: structure, function, and Pathology. Physiol Rev 99:1381–1431. https://doi.org/10.1152/physrev.00031.2018 160. Resende R, Ferreiro E, Pereira C, Resende de Oliveira C (2008) Neurotoxic effect of oligomeric and fibrillar species of amyloid-beta peptide 1–42: involvement of endoplasmic reticulum calcium release in oligomer- induced cell death. Neuroscience 155:725–737. https://doi.org/10.1016/j. neuroscience.2008.06.036 y 182. Stephenson J, Nutma E, van der Valk P, Amor S (2018) Inflammation in CNS neurodegenerative diseases. Immunology 154:204–219. https://doi. org/10.1111/imm.12922 161. Rutsch A, Kantsjö JB, Ronchi F (2020) The gut-brain Axis: how microbiota and host Inflammasome Influence Brain Physiology and Pathology. Front Immunol 11:604179. https://doi.org/10.3389/fimmu.2020.604179 183. Stevens B, Porta S, Haak LL, Gallo V, Fields RD (2002) Adenosine: a neuron-glial transmitter promoting myelination in the CNS in response to action poten tials. Neuron 36:855–868. https://doi.org/10.1016/s0896-6273(02)01067-x i 162. Saez-Atienzar S, Masliah E (2020) Cellular senescence and Alzheimer disease: the egg and the chicken scenario. Nat Rev Neurosci 21:433–444. https://doi. org/10.1038/s41583-020-0325-z 184. Stojić-Vukanić Z, Hadžibegović S, Nicole O, Nacka-Aleksić M, Leštarević S, Leposavić G (2020) CD8 + T cell-mediated mechanisms contribute to the progression of neurocognitive impairment in both multiple sclerosis and Alzheimer’s Disease? Front Immunol 11:566225. https://doi.org/10.3389/ fimmu.2020.566225 163. Safaiyan S, Besson-Girard S, Kaya T, Cantuti-Castelvetri L, Liu L, Ji H, Schifferer M, Gouna G, Usifo F, Kannaiyan N, Fitzner D, Xiang X, Rossner MJ, Brendel M, Gokce O, Simons M (2021) White matter aging drives microglial diversity. Neuron 109:1100–1117e10. https://doi.org/10.1016/j.neuron.2021.01.027 i 185. References Xu J, Chen S, Ahmed SH, Chen H, Ku G, Goldberg MP, Hsu CY (2001) Amyloid- beta peptides are cytotoxic to oligodendrocytes. J Neurosci Off J Soc Neuro sci 21:RC118. https://doi.org/10.1523/JNEUROSCI.21-01-j0001.2001 p 199. Wang Z, Colognato H, Ffrench-Constant C (2007) Contrasting effects of mito genic growth factors on myelination in neuron-oligodendrocyte co-cultures. Glia 55:537–545. https://doi.org/10.1002/glia.20480 220. Yokota M, Saido TC, Tani E, Yamaura I, Minami N (1996) Cytotoxic fragment of amyloid precursor protein accumulates in hippocampus after global forebrain ischemia. J Cereb Blood Flow Metab Off J Int Soc Cereb Blood Flow Metab 16:1219–1223. https://doi.org/10.1097/00004647-199611000-00016 200. Wang J, Gu BJ, Masters CL, Wang Y-J (2017) A systemic view of Alzheimer dis ease - insights from amyloid-β metabolism beyond the brain. Nat Rev Neurol 13:612–623. https://doi.org/10.1038/nrneurol.2017.111 221. Yoo S-W, Agarwal A, Smith MD, Khuder SS, Baxi EG, Thomas AG, Rojas C, Moniruzzaman M, Slusher BS, Bergles DE, Calabresi PA, Haughey NJ (2020) Inhibition of neutral sphingomyelinase 2 promotes remyelination. Sci Adv 6:eaba5210. https://doi.org/10.1126/sciadv.aba5210 201. Wang Y-R, Wang Q-H, Zhang T, Liu Y-H, Yao X-Q, Zeng F, Li J, Zhou F-Y, Wang L, Yan J-C, Zhou H-D, Wang Y-J (2017) Associations between hepatic functions and plasma amyloid-Beta levels-implications for the capacity of liver in Peripheral amyloid-Beta clearance. Mol Neurobiol 54. https://doi. org/10.1007/s12035-016-9826-1 222. Yoon S-S, Jo SA (2012) Mechanisms of Amyloid-β peptide clearance: potential therapeutic targets for Alzheimer’s Disease. Biomol Ther 20:245–255. https:// doi.org/10.4062/biomolther.2012.20.3.245 g 202. Wang S-S, Zhang Z, Zhu T-B, Chu S-F, He W-B, Chen N-H (2018) Myelin injury in the central nervous system and Alzheimer’s disease. Brain Res Bull 140:162–168. https://doi.org/10.1016/j.brainresbull.2018.05.003 223. You Y, Joseph C, Wang C, Gupta V, Liu S, Yiannikas C, Chua BE, Chitranshi N, Shen T, Dheer Y, Invernizzi A, Borotkanics R, Barnett M, Graham SL, Klistorner A (2019) Demyelination precedes axonal loss in the transneuronal spread of human neurodegenerative disease. Brain J Neurol 142:426–442. https://doi. org/10.1093/brain/awy338 203. Wang F, Ren S-Y, Chen J-F, Liu K, Li R-X, Li Z-F, Hu B, Niu J-Q, Xiao L, Chan JR, Mei F (2020) Myelin degeneration and diminished myelin renewal contribute to age-related deficits in memory. Nat Neurosci 23:481–486. https://doi. org/10.1038/s41593-020-0588-8 224. Yu SP, Farhangrazi ZS, Ying HS, Yeh CH, Choi DW (1998) Enhancement of out ward potassium current may participate in beta-amyloid peptide-induced cortical neuronal death. Neurobiol Dis 5:81–88. https://doi.org/10.1006/ nbdi.1998.0186 204. References Ticinesi A, Tana C, Nouvenne A (2019) The intestinal microbiome and its relevance for functionality in older persons. Curr Opin Clin Nutr Metab Care 22:4–12. https://doi.org/10.1097/MCO.0000000000000521 213. Wu S, Liu X, Jiang R, Yan X, Ling Z (2021) Roles and mechanisms of gut micro biota in patients with Alzheimer’s Disease. Front Aging Neurosci 13:650047. https://doi.org/10.3389/fnagi.2021.650047 192. Timmler S, Simons M (2019) Grey matter myelination. Glia 67:2063–2070. https://doi.org/10.1002/glia.23614 214. Wu HM, Goate AM, O’Reilly PF (2021) Heterogeneous effects of genetic risk for Alzheimer’s disease on the phenome. Transl Psychiatry 11:406. https://doi. org/10.1038/s41398-021-01518-0 193. Tini G, Scagliola R, Monacelli F, La Malfa G, Porto I, Brunelli C, Rosa GM (2020) Alzheimer’s Disease and Cardiovascular Disease: a Particular Association. Cardiol Res Pract 2020:2617970. https://doi.org/10.1155/2020/2617970 215. Xia M, Cheng X, Yi R, Gao D, Xiong J (2016) The binding receptors of Aβ: an alternative therapeutic target for Alzheimer’s Disease. Mol Neurobiol 53:455–471. https://doi.org/10.1007/s12035-014-8994-0 194. van Duijn S, Bulk M, van Duinen SG, Nabuurs RJA, van Buchem MA, van der Weerd L, Natté R (2017) Cortical Iron reflects severity of Alzheimer’s Disease. J Alzheimers Dis JAD 60:1533–1545. https://doi.org/10.3233/JAD-161143 216. Xia W, Fancy SPJ (2021) Mechanisms of oligodendrocyte progenitor devel opmental migration. Dev Neurobiol 81:985–996. https://doi.org/10.1002/ dneu.22856 195. Vassar R, Kuhn P-H, Haass C, Kennedy ME, Rajendran L, Wong PC, Lichten thaler SF (2014) Function, therapeutic potential and cell biology of BACE pro teases: current status and future prospects. J Neurochem 130:4–28. https:// doi.org/10.1111/jnc.12715 217. Xin W, Chan JR (2020) Myelin plasticity: sculpting circuits in learning and memory. Nat Rev Neurosci 21:682–694. https://doi.org/10.1038/ s41583-020-00379-8 g j 196. Vaz M, Silvestre S (2020) Alzheimer’s disease: recent treatment strategies. Eur J Pharmacol 887:173554. https://doi.org/10.1016/j.ejphar.2020.173554 218. Xing YL, Röth PT, Stratton JAS, Chuang BHA, Danne J, Ellis SL, Ng SW, Kilpatrick TJ, Merson TD (2014) Adult neural precursor cells from the subventricular zone contribute significantly to oligodendrocyte regeneration and remyelin ation. J Neurosci Off J Soc Neurosci 34:14128–14146. https://doi.org/10.1523/ JNEUROSCI.3491-13.2014 197. Vegas-Suárez S, Simón J, Martínez-Chantar ML, Moratalla R (2022) Metabolic diffusion in neuropathologies: the relevance of Brain-Liver Axis. Front Physiol 13:864263. https://doi.org/10.3389/fphys.2022.864263 p g p y 198. Vos JP, Gard AL, Pfeiffer SE (1996) Regulation of oligodendrocyte cell survival and differentiation by ciliary neurotrophic factor, leukemia inhibitory factor, oncostatin M, and interleukin-6. Perspect Dev Neurobiol 4:39–52 219. References Stopińska K, Radziwoń-Zaleska M, Domitrz I (2021) The Microbiota-Gut- Brain Axis as a Key to Neuropsychiatric Disorders: a Mini Review. J Clin Med 10:4640. https://doi.org/10.3390/jcm10204640 164. Sams EC (2021) Oligodendrocytes in the aging brain. Neuronal Signal 5:NS20210008. https://doi.org/10.1042/NS20210008 186. Sukhorukov VS, Mudzhiri NM, Voronkova AS, Baranich TI, Glinkina VV, Illari oshkin SN (2021) Mitochondrial Disorders in Alzheimer’s Disease. Biochem Biokhimiia 86:667–679. https://doi.org/10.1134/S0006297921060055 165. Samudyata null, Castelo-Branco G, Liu J (2020) Epigenetic regulation of oli godendrocyte differentiation: from development to demyelinating disorders. Glia 68:1619–1630. https://doi.org/10.1002/glia.23820 187. Sulehria T, Corbett AM, Sharma N, Nagarajan D, Abushamma A, Gagle S, Johnson A (2020) Increasing Progenitor Cell Proliferation in the Sub-Ventricular Zone: a therapeutic treatment for progressive multiple sclerosis? Recent Pat Drug Deliv Formul 14:233–241. https://doi.org/10.2174/1872211314999201117130123 166. Sancho L, Contreras M, Allen NJ (2021) Glia as sculptors of synaptic plasticity. Neurosci Res 167:17–29. https://doi.org/10.1016/j.neures.2020.11.005 g j 167. Savonije K, Weaver DF (2023) The role of Tryptophan Metabolism in Alzheim er’s Disease. Brain Sci 13:292. https://doi.org/10.3390/brainsci13020292 188. Tamagno E, Bardini P, Guglielmotto M, Danni O, Tabaton M (2006) The various aggregation states of beta-amyloid 1–42 mediate different effects on oxida tive stress, neurodegeneration, and BACE-1 expression. Free Radic Biol Med 41:202–212. https://doi.org/10.1016/j.freeradbiomed.2006.01.021 168. Sanguinetti E, Collado MC, Marrachelli VG, Monleon D, Selma-Royo M, Pardo- Tendero MM, Burchielli S, Iozzo P (2018) Microbiome-metabolome signatures in mice genetically prone to develop dementia, fed a normal or fatty diet. Sci Rep 8:4907. https://doi.org/10.1038/s41598-018-23261-1 189. Tang W, Meng Z, Li N, Liu Y, Li L, Chen D, Yang Y (2020) Roles of gut microbiota in the regulation of hippocampal plasticity, inflammation, and Hippocampus-Dependent Behaviors. Front Cell Infect Microbiol 10:611014. https://doi.org/10.3389/fcimb.2020.611014 169. Santos LRD, Almeida JFF, Pimassoni LHS, Morelato RL, de Paula F (2020) The combined risk effect among BIN1, CLU, and APOE genes in Alzheimer’s disease. Genet Mol Biol 43:e20180320. https://doi. org/10.1590/1678-4685-GMB-2018-0320 (2023) 11:56 (2023) 11:56 Page 18 of 18 Maitre et al. Acta Neuropathologica Communications 190. Thorburne SK, Juurlink BH (1996) Low glutathione and high iron govern the susceptibility of oligodendroglial precursors to oxidative stress. J Neurochem 67:1014–1022. https://doi.org/10.1046/j.1471-4159.1996.67031014.x 212. Wu D, Tang X, Gu L-H, Li X-L, Qi X-Y, Bai F, Chen X-C, Wang J-Z, Ren Q-G, Zhang Z-J (2018) LINGO-1 antibody ameliorates myelin impairment and spatial memory deficits in the early stage of 5XFAD mice. CNS Neurosci Ther 24:381–393. https://doi.org/10.1111/cns.12809 191. References Wang X-L, Li L (2021) Cell type-specific potential pathogenic genes and functional pathways in Alzheimer’s Disease. BMC Neurol 21:381. https://doi. org/10.1186/s12883-021-02407-1 205. Wesenhagen KEJ, Teunissen CE, Visser PJ, Tijms BM (2020) Cerebrospinal fluid proteomics and biological heterogeneity in Alzheimer’s disease: a literature review. Crit Rev Clin Lab Sci 57:86–98. https://doi.org/10.1080/10408363.2019 .1670613 225. Yu W, Gao D, Jin W, Wang Z, Li Y, Peng X, Cong Y, Li C, Zhao A, Liu S, Qi S (2020) Intestinal Flora Dysbiosis aggravates cognitive dysfunction Associated with Neuroinflammation in Heart failure. J Card Fail 26:885–894. https://doi. org/10.1016/j.cardfail.2020.02.002 206. Westi EW, Jakobsen E, Voss CM, Bak LK, Pinborg LH, Aldana BI, Andersen JV (2022) Divergent Cellular Energetics, Glutamate Metabolism, and mitochon drial function between human and mouse cerebral cortex. Mol Neurobiol 59:7495–7512. https://doi.org/10.1007/s12035-022-03053-5 g j 226. Zendjabil M (2018) Circulating microRNAs as novel biomarkers of Alzheimer’s disease. Clin Chim Acta Int J Clin Chem 484:99–104. https://doi.org/10.1016/j. cca.2018.05.039 227. Zhan X, Jickling GC, Ander BP, Stamova B, Liu D, Kao PF, Zelin MA, Jin L-W, DeCarli C, Sharp FR (2015) Myelin Basic Protein Associates with AβPP, Aβ1–42, and amyloid plaques in cortex of Alzheimer’s Disease Brain. J Alzheimers Dis JAD 44:1213–1229. https://doi.org/10.3233/JAD-142013 207. Westwell-Roper C, Verchere CB (2019) Modulation of Innate immunity by amyloidogenic peptides. Trends Immunol 40:762–780. https://doi. org/10.1016/j.it.2019.06.005 208. Wiatrak B, Balon K, Jawień P, Bednarz D, Jęśkowiak I, Szeląg A (2022) The role of the Microbiota-Gut-Brain Axis in the development of Alzheimer’s Disease. Int J Mol Sci 23:4862. https://doi.org/10.3390/ijms23094862 228. Zhu X, Li B, Lou P, Dai T, Chen Y, Zhuge A, Yuan Y, Li L (2021) The Relationship between the gut microbiome and neurodegenerative Diseases. Neurosci Bull 37:1510–1522. https://doi.org/10.1007/s12264-021-00730-8 209. Wirths O, Weis J, Kayed R, Saido TC, Bayer TA (2007) Age-dependent axonal degeneration in an Alzheimer mouse model. Neurobiol Aging 28:1689–1699. https://doi.org/10.1016/j.neurobiolaging.2006.07.021 229. Zhang X, Sun Y, Li W, Liu B, Wu W, Zhao H, Liu R, Zhang Y, Yin Z, Yu T, Qing Z, Zhu B, Xu Y, Nedelska Z, Hort J, Zhang B, Alzheimer’s Disease Neuroimaging Initiative (2019) Characterization of white matter changes along fibers by automated fiber quantification in the early stages of Alzheimer’s disease. NeuroImage Clin 22:101723. https://doi.org/10.1016/j.nicl.2019.101723 210. Wissler Gerdes EO, Zhu Y, Weigand BM, Tripathi U, Burns TC, Tchkonia T, Kirkland JL (2020) Cellular senescence in aging and age-related diseases: implications for neurodegenerative diseases. Int Rev Neurobiol 155:203–234. https://doi.org/10.1016/bs.irn.2020.03.019 211. References Wu Y, Ma Y, Liu Z, Geng Q, Chen Z, Zhang Y (2017) Alterations of myelin morphology and oligodendrocyte development in early stage of Alzheimer’s disease mouse model. Neurosci Lett 642:102–106. https://doi.org/10.1016/j. neulet.2017.02.007 Publisher’s Note S i N i Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W4366089414	https://link.springer.com/content/pdf/10.1007/s00410-023-02012-0.pdf	English	null	Origin of the Paleoproterozoic basaltic dikes from the central and eastern Dharwar Craton and sills and volcanics from the adjoining Cuddapah Basin, southern India	Contributions to mineralogy and petrology	2,023	cc-by	21,329	Contributions to Mineralogy and Petrology (2023) 178:28 https://doi.org/10.1007/s00410-023-02012-0 Contributions to Mineralogy and Petrology (2023) 178:28 https://doi.org/10.1007/s00410-023-02012-0 ORIGINAL PAPER Abstract Reverse fractionation modeling considering energy-constrained assimilation-fractional crystallization is performed to esti- mate primary magma compositions, degree of crustal contamination, pressure–temperature of equilibrium with mantle, and potential temperatures for the origin of the Paleoproterozoic (~ 2.37–1.88 Ga) basaltic dikes in central and eastern Dharwar Craton and sills and volcanics in the adjoining Cuddapah Basin, southern India. Mineral thermobarometry indicates that the dikes crystallized at upper crustal conditions (~ 1–6 kbar/ ~ 1120–1210 °C). Hence, the reverse fractionation calculations are performed at low pressures by adding olivine + plagioclase + clinopyroxene, olivine + plagioclase and only olivine in equilibrium with melt, and simultaneously subtracting an upper crustal partial melt in small steps until the melt is multiply saturated with lherzolite at a high pressure. The results indicate that the basalts are 5–30% contaminated, and their enriched light rare earth element (REE) patterns can be attributed to upper crustal assimilation. The upper crust was pre-heated to 665–808 °C during dike emplacement. The primary magmas of all basalts were last equilibrated with spinel lherzolite at 10–16.5 kbar/1291–1366 °C, and they resemble pooled polybaric incremental melts generated along a ~ 1450 °C adiabat. The estimated mantle potential temperatures (1293–1515 °C) are similar to Paleoproterozoic ambient mantle temperatures. All basalts and their primary magmas show lower chondrite-normalized DyN/YbN ratios than the plume-derived mid-Proterozoic Mackenzie dikes of Canadian Shield, and the primary magmas show flat REE patterns indicating spinel lherzolite melting. The low estimated potential temperatures, low DyN/YbN ratios, and a spinel-bearing mantle source are at odds with an origin of the basalts from mantle plumes. Keywords Dharwar Craton · Paleoproterozoic dike swarm · Primary magma · Basalt · Lherzolite · C Origin of the Paleoproterozoic basaltic dikes from the central and eastern Dharwar Craton and sills and volcanics from the adjoining Cuddapah Basin, southern India Nilanjan Chatterjee1 Received: 31 May 2022 / Accepted: 6 April 2023 / Published online: 17 April 2023 © The Author(s) 2023 1 Department of Earth, Atmospheric and Planetary Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Introduction associated with a mantle plume relative to the ambient mantle, ∆ TP, is ~ 100–250 °C (McKenzie and Bickle 1988; Watson and McKenzie 1991; Kinzler and Grove 1992a,b; Presnall et al. 2002; Herzberg et al. 2007; Putirka et al. 2007; Krein et al. 2021). Herzberg et al. (2010) concluded from petrological modeling that the TP of ambient mantle was ~ 1500–1600 °C at 2.5–3.0 Ga that decreased to the present-day value of ~ 1350 °C in accordance to the Earth’s thermal history model of Korenaga (2008). High potential temperatures of ~ 1700 °C estimated for the Archean and Paleoproterozoic komatiites (Herzberg 2022, and references therein) have been used as evidence to support their origin from mantle plumes.i Flood basalt volcanism is thought to result from high degrees of melting in the upper mantle caused by upwelling of a hot mantle plume from depth (Morgan 1971; Sleep 1990; Davies 1999). The hallmark of a mantle plume is a high potential temperature, TP, defined as the temperature of the mantle if it were to adiabatically decompress and reach the Earth’s surface without melting. The excess potential temperature Communicated by Othmar Müntener. * Nilanjan Chatterjee nchat@mit.edu 1 Department of Earth, Atmospheric and Planetary Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Communicated by Othmar Müntener. Communicated by Othmar Müntener. * Nilanjan Chatterjee nchat@mit.edu * Nilanjan Chatterjee nchat@mit.edu The origin of radiating mafic dike swarms such as the mid-Proterozoic Mackenzie swarm of the Canadian Shield has been linked to mantle plumes (Ernst and Baragar 1992; Baragar et al. 1996). Several researchers contend that the (0121 Contributions to Mineralogy and Petrology (2023) 178:28 Page 2 of 25 28 originated from mantle plumes (Halls et al. 2007; French et al. 2008; Ernst and Srivastava 2008; French and Hea- man 2010; Kumar et al. 2012a,b; Belica et al. 2014; Mishra 2015; Stark et al. 2019). Their conclusions are based on different Paleoproterozoic (~ 2.37–1.89 Ga) basaltic dike swarms intruding the central and eastern Dharwar Cra- ton (CDC and EDC, Fig. Introduction 2013; Chatterjee and Sheth 2015; Till 2017; Chatterjee 2021; Krein et al. 2021). There is trace element evidence of upper crustal contamination in the basalts. Hence, the energy-constrained assimilation-frac- tional crystallization (EC-AFC) formulation of Spera and Bohrson (2001) and Bohrson and Spera (2001) is incorpo- rated in the modeling that provides estimates of the degree of crustal contamination as well as the temperature of the upper crust during magmatism. Abundances of the trace ele- ments including Ni, Rb and the rare earth elements (REE) in the primary magmas are also modeled, providing further insight into the origin the CDC/EDC and Cuddapah Basin basalts. The EDC is separated from the Eastern Ghats Belt to the east by the crescent-shaped Cuddapah Basin that contains a sequence of gently east-dipping, Proterozoic sedimentary rocks (Nagaraja Rao et al. 1987) (Fig. 1). Near the base of the sequence in the eastern part of the basin, the sediments are intercalated with basaltic pillow lavas in the Vempalle Formation, and basaltic sills and tuffs in the overlying Tadpatri Formation (Sheppard et al. 2017, and references therein). The basaltic exposures are parallel to the arcuate southwestern margin of the basin and coincide with an ellip- tical region of gravity high (− 55 mGal) surrounded by grav- ity lows (− 100 mGal) that indicate the presence of a dense (~ 3.0 g cm−3) lopolithic intrusion in the upper crust (NGRI 1978; Bhattacharji and Singh 1984; Bhattacharji 1987; Singh et al. 2004). A sill (1899 ± 20 Ma, Anand et al. 2003; 1885.4 ± 3.1 Ma, French et al. 2008) in the lower part and a felsic tuff (1862 ± 9 Ma, Sheppard et al. 2017) in the upper part of the Tadpatri Formation provide evidence for a pro- tracted ~ 30 Myr period of volcanism during sediment depo- sition. The basin may have started forming before ~ 1.9 Ga by rifting between the EDC and the Napier complex of East Antarctica (Mohanty 2011), and it evolved into a foreland basin after collision with the Eastern Ghats Belt in the late Paleoproterozoic (Collins et al. 2015).if Paleoproterozoic mafic dike swarms of different trends and ages are widespread in the CDC and the EDC (Halls 1982; Murty et al. 1987; Radhakrishna and Joseph 1996; Poornachandra Rao 2005; Halls et al. 2007; French and Heaman 2010; Söderlund et al. 2019; Samal et al. 2021) (Fig. 1). Introduction (2019) showed that the pre-2.08 Ga swarms were originally linear, and the plume center reconstructions based on the current orientations of the dikes are incorrect. Furthermore, Anand et al. (2003) estimated a TP of ~ 1500 °C for the ~ 1.89 Ga old sills and volcanics within the adjoining Cuddapah Basin that are genetically related to the ~ 1.89–1.88 Ga old EDC dikes, and explained their result by secular cooling of the Earth without invoking a plume. Sheppard et al. (2017) also presented geological arguments to preclude the involvement of a plume in the origin of the Cuddapah Basin sills and volcanics. In addition, Shellnutt et al. (2018) concluded from isotopic and trace element data that the ~ 1.88 Ga old dikes from the neighboring Bastar Craton, genetically related to the ~ 1.89–1.88 Ga old EDC dikes and Cuddapah Basin sills and volcanics, originated from a subcontinental lithospheric mantle source, not from an asthenospheric source. Srivas- tava et al. (2015) demonstrated the futility of trace element discrimination diagrams that often indicate incorrect or ambiguous tectonic settings. This study attempts to estimate mantle potential temperatures for the origin of the CDC/ EDC dikes and Cuddapah Basin sills and volcanics using their major element compositions. The primary magmas of the basalts and their pressure–temperature (P–T) conditions of equilibrium with mantle are modeled with the reverse fractionation technique that has been previously used for mid-ocean ridge, ocean island, arc, and flood basalts (Till et al. 2012, 2013; Grove et al. 2013; Chatterjee and Sheth 2015; Till 2017; Chatterjee 2021; Krein et al. 2021). There is trace element evidence of upper crustal contamination in the basalts. Hence, the energy-constrained assimilation-frac- tional crystallization (EC-AFC) formulation of Spera and Bohrson (2001) and Bohrson and Spera (2001) is incorpo- rated in the modeling that provides estimates of the degree of crustal contamination as well as the temperature of the upper crust during magmatism. Abundances of the trace ele- ments including Ni, Rb and the rare earth elements (REE) in the primary magmas are also modeled, providing further insight into the origin the CDC/EDC and Cuddapah Basin b l 1991; Chardon et al. 2011; Manikyamba and Kerrick 2012; Jayananda et al. 2013a,b). The oldest rocks representing the cratonic nucleus occur in the western Dharwar Craton (WDC). Introduction The individual dikes can be traced in length from a few meters to hundreds of kilometers, and their widths vary between 1 m and ~ 400 m. At least nine different dike swarms have been identified (Söderlund et al. 2019; Samal et al. 2021). The dominantly ENE- to NE-trending (some ESE-trending at the SGT contact) ~ 2.37 Ga old dikes of the Bangalore-Karimnagar swarm orthogonally cut across the NNW-SSE structural grain of the craton. Introduction A steep mylonitic shear zone along the eastern margin of the Chitradurga schist belt has been traditionally considered the eastern boundary of WDC (Swami Nath et al. 1976; Gupta et al. 2003). Based on recent petrologic, geo- chronologic and isotopic data, the region to the east of the shear zone has been divided into the central and the eastern Dharwar cratonic blocks (CDC and EDC) along the Kolar- Kadiri-Hungund belt (Peucat et al. 2013; Jayananda et al. 2013a). Charnockites near the southern margin of CDC/ EDC originated by metamorphism of magmatic protoliths at ~ 2.48 Ga as a result of a collision between the CDC/EDC block and the Southern Granulite Terrane (SGT, Ghosh et al. 2004; Clark et al. 2009). geometric reconstructions of the piercing points of radial dike swarms across continents using geochronological and paleomagnetic data. However, considering oroclinal bending due to later tectonic deformation, Söderlund et al. (2019) showed that the pre-2.08 Ga swarms were originally linear, and the plume center reconstructions based on the current orientations of the dikes are incorrect. Furthermore, Anand et al. (2003) estimated a TP of ~ 1500 °C for the ~ 1.89 Ga old sills and volcanics within the adjoining Cuddapah Basin that are genetically related to the ~ 1.89–1.88 Ga old EDC dikes, and explained their result by secular cooling of the Earth without invoking a plume. Sheppard et al. (2017) also presented geological arguments to preclude the involvement of a plume in the origin of the Cuddapah Basin sills and volcanics. In addition, Shellnutt et al. (2018) concluded from isotopic and trace element data that the ~ 1.88 Ga old dikes from the neighboring Bastar Craton, genetically related to the ~ 1.89–1.88 Ga old EDC dikes and Cuddapah Basin sills and volcanics, originated from a subcontinental lithospheric mantle source, not from an asthenospheric source. Srivas- tava et al. (2015) demonstrated the futility of trace element discrimination diagrams that often indicate incorrect or ambiguous tectonic settings. This study attempts to estimate mantle potential temperatures for the origin of the CDC/ EDC dikes and Cuddapah Basin sills and volcanics using their major element compositions. The primary magmas of the basalts and their pressure–temperature (P–T) conditions of equilibrium with mantle are modeled with the reverse fractionation technique that has been previously used for mid-ocean ridge, ocean island, arc, and flood basalts (Till et al. 2012, 2013; Grove et al. Introduction 1) of the Indian Shield, believed to be remnants of ancient flood basalt provinces, also 32 36 36 36 40 44 48 36 12o 16o 14o 80o 78o 77o Chitradurga Schist Belt Closepet Granite Cuddapah Basin Eastern Ghats Belt Southern Granulite Terrane Bay of Bengal Chennai Bengaluru Hyderabad Dhone Tadpatri Tirupati Anantpur 8-3(e) 10-6(e) 24-1(e) 23-2(e) EDC9/28(h) EDC9/25(h) EDC9/13(h) EDC9/16(h) EDC 9/12(h) EDC9/6(h) D9A(a) D88(b) D86(b) 2/14 1A D11 D28(a) D47(b) D84(a) EDC9/23(h) EDC9/9(h) EDC8/9(h) D75(a) GD24(g) HD12 (g) HD14 (g) DC08/4(i) JEF00-55(f) D77(a) EDD09/21(j) EDD09/14 (j) DC12/09(j) DC12/05(j) DC12/02(j) D89(a) D30(a) K88(a) 6/33 D8A(a) B37(c) T98MA94(d) T98MA74(d) V99MA81(d) V99MA80(d) V99MA04(d) 2.37 Ga 2.25 Ga 2.22 Ga 2.21 Ga 2.08 Ga 1.88 Ga Dikes Gneiss EDC CDC WDC Sanukitoids and anatectic granite Greenstone and schist belts Basalt sills and volcanics Cuddapah sediments 25o 20o 15o 10o 70o 75o 80o 85o Bay of Bengal Arabian Sea INDIA BuC AC SC BC DC Fig. 1 Geological map of a part of the Dharwar Craton and Cud- dapah Basin (box in inset) after Geological Survey of India (GSI 1998) with boundaries between western (WDC), central (CDC) and eastern (EDC) parts of the craton (thick dashed lines) and for sanukitoids and anatectic granites from Chadwick et al. (2000) and Jayananda et al. (2018), dike swarms (grey lines) from Halls et al. (2007), and Moho depth contours (km, red dashed lines) from Das et al. (2015). Location of the studied samples of dikes, sills and volcanics are shown (letter in parentheses after the sample names refer to sources reporting bulk compositions: a—Murty et al. 1987, b—Rao et al. 1995, c—Chatterjee and Bhattacharji 1998, d—Anand et al. 2003, e—Halls et al. 2007, f—French and Heaman 2010, g—Kumar et al. 2012b, h, i and j—Sriv- astava et al. 2014a,b, 2015). Abbreviations for the cratons are: AC Aravalli, BC Bastar, BuC Bundelkhand, EDC/CDC/ WDC eastern/central/western Dharwar, and SC—Singhbhum 25o 20o 15o 10o 70o 75o 80o 85o Bay of Bengal Arabian Sea INDIA BuC AC SC BC DC Bay of Bengal Cuddapah Basin 1 3 3 Contributions to Mineralogy and Petrology (2023) 178:28 Page 3 of 25 28 geometric reconstructions of the piercing points of radial dike swarms across continents using geochronological and paleomagnetic data. However, considering oroclinal bending due to later tectonic deformation, Söderlund et al. Previous petrological work The Cuddapah Basin sills and volcanics mostly comprise subalkaline tholeiitic basalts (Chatterjee and Bhattacharji 1998; Anand et al. 2003). A few of the Vempalle samples are alkalic. The major sill complex in the Tadpatri Formation consists of basaltic rocks with mafic xenoliths composed of olivine + orthopyroxene + clinopyroxene + plagioclase (Ol + Opx + Cpx + Pl) near the base, grading into leuco- cratic gabbro near the top. The minor sills consist of non- cumulate, differentiated basalts. In general, the Cuddapah Basin basalts can be related by fractional crystallization of olivine, clinopyroxene and plagioclase. Thermobarometry shows that the basalts crystallized at a pressure of ~ 5 kbar (~ 18 km depth) and temperatures of 1019–1154 °C (Chat- terjee and Bhattacharji 1998). These P–T conditions are con- sistent with differentiation within a crustal magma chamber under Cuddapah Basin postulated from gravity data (NGRI 1978). The basalts have low loss-on-ignition values indi- cating minor hydrothermal alteration (Anand et al. 2003). They show negative Nb–Ta anomalies in their incompat- ible element patterns and high La/Nb (1.3–3.8, cf. primitive mantle: 0.99) and Th/Nb (0.23–1.45, cf. primitive mantle: 0.12) ratios (Anand et al. 2003; McDonough and Sun 1995). Mixing models based on La/Nb and Ce/Y ratios indicate that the non-cumulate basalts comprising most of the Cuddapah Basin lavas and sills are ~ 10–15% contaminated and some minor sills are 20–35% contaminated by the local granitic crust (Anand et al. 2003). The isotopic compositions of the basalts (εNd(t) = − 10 to + 1, 87Sr/86Sri = 0.7056 to 0.7082) are also consistent with upper crustal contamination (Anand et al. 2003). In addition, based on Fe-Nd and REE modeling, Anand et al. (2003) suggested ~ 10–15% partial melting of spinel lherzolite for the generation of the primary magmas of the basalts. They estimated a mantle potential temperature of ~ 1500 °C, and an initially 120 km-thick lithosphere that was thinned to 70 km during magma generation. Moreover, Anand et al. (2003) concluded that the ~ 1500 °C potential Several researchers have correlated the CDC/EDC dike swarms with swarms on other continents using U–Pb geo- chronological and paleomagnetic data, and have recon- structed the location of plume centers from which the dike swarms supposedly originated (Halls et al. 2007; French et al. 2008; French and Heaman 2010; Kumar et al. 2012a,b; Stark et al. 2019). Geological setting The Dharwar Craton (~ 600,000 km2) in southern India is one of the several Archean cratonic blocks that comprise the Indian shield (Naqvi and Rogers 1987) (Fig. 1). It is composed of ~ 3.4–3.0 Ga old tonalite-trondhjemite-gran- odiorite (TTG) gneisses and Neoarchean greenstone belts with basaltic volcanics that are intruded by late Neoarchean calc-alkaline and potassic granitoids (Friend and Nutman 1 3 3 Contributions to Mineralogy and Petrology (2023) 178:28 Page 4 of 25 28 These dikes were emplaced within a short interval of < 5 Myr (2369–2365 Ma, Halls et al. 2007; French and Hea- man 2010; Kumar et al. 2012a; Liao et al. 2019; Söderlund et al. 2019). The N- to NNE-trending ~ 2.25 Ga old dikes of the Ippaguda-Dhiburahalli swarm to the north and south of Cuddapah Basin were also emplaced within a short, ~ 6 Myr time interval (2257–2251 Ma, Nagaraju et al. 2018a; Söder- lund et al. 2019). The N- to NNW-trending ~ 2.22 Ga old dikes of the Kandlamadugu swarm includes the well-known Kandlamadugu dike to the southwest of Cuddapah Basin and the ~ 400 km long, ~ 2.21–2.22 Ga old, arcuate Nelahalu dike parallel to the western margin of the CDC (French and Heaman 2010; Kumar et al. 2012b; Söderlund et al. 2019). The subparallel NW- to WNW-trending ~ 2.21 Ga old dikes of the Anantapur-Kunigal swarm including the Somala dike, and the NW- to WNW-trending ~ 2.18 Ga old dikes of the Mahabubnagar-Dandeli swarm including the Dandeli and Bandepalem dikes were emplaced within ~ 30 Myr each other, and the younger dikes probably intruded through some of the older magma pathways (French and Heaman 2010; Nagaraju et al. 2018a,b; Söderlund et al. 2019). Several NE-, NW- and N-trending ~ 2.08 Ga old dikes of the Devar- abanda swarm around the Cuddapah Basin form a radial swarm with the center inside the basin (Kumar et al. 2015; Söderlund et al. 2019). Two ENE-trending ~ 1.89–1.88 Ga old dikes of the Hampi swarm to the west of the basin (Chatterjee and Bhattacharji 2001; Halls et al. 2007) are coeval with the Tadpatri sill within Cuddapah Basin (Anand et al. 2003; French et al. 2008). Geological setting Some of the ~ 2.08 Ga old dikes and the ~ 1.89–1.88 Ga old dikes are parallel to the older ~ 2.37 Ga and ~ 2.21 Ga old dikes, indicating that the younger dikes were perhaps emplaced by reactivation of older magma pathways and preexisting fractures (cf. Bhat- tacharji 1987). In addition, several NW-trending ~ 1.84 Ga old dikes of the Dharmapuri swarm (Belica et al. 2014) and NW- to WNW-trending ~ 1.79 Ga old dikes of the Pebbair swarm (Söderlund et al. 2019) are subparallel to the ~ 2.21 Ga old dikes, and may have also intruded through preexisting magma pathways. with the ~ 2.41 Ga old dikes of the Yilgarn Craton. How- ever, Belica et al. (2014) argued against a link between the Yilgarn Craton and the Dharwar Craton at 2.41–2.37 Ga cit- ing age disparity and a ~ 25° latitudinal separation between the two cratons. French and Heaman (2010) also proposed that the Dharwar and Yilgarn dikes are unrelated, and they were emplaced on different continental masses through discrete events. Thus, the locations of the purported plume centers are highly uncertain. Furthermore, Söderlund et al. (2019) argued against the involvement of plumes for the pre- 2.08 Ga old CDC/EDC dikes by showing that the swarms were initially linear, and their fan-shaped orientations origi- nated by later tectonic deformation. Hence, the plume center reconstructions are flawed. Previous petrological work These studies suggest that the ~ 2.37 Ga (Bangalore-Karimnagar), ~ 2.21–2.18 Ga (Anantapur-Kuni- gal and Mahabubnagar-Dandeli) and ~ 1.89 Ga (Hampi) old dike swarms originated from mantle plumes with centers located ~ 300 km west, ~ 1000 km NNW, and ~ 600 km east of the CDC/EDC, respectively. Halls et al. (2007) proposed the location of the ~ 2.37 Ga old plume center to the west of the Dharwar Craton by correlating the Dharwar dikes 1 3 1 3 Page 5 of 25 Contributions to Mineralogy and Petrology (2023) 178:28 28 temperature can be explained by secular cooling of the Earth without the involvement of a hot mantle plume. and D47 is a trachyandesite (Murty et al. 1987). In addi- tion, the tholeiitic basalt sample D8A is also from an ENE- trending dike west of the basin (Fig. 1), but it belongs to the younger ~ 1.89–1.88 Ga old Hampi swarm (1879 Ma, Chatterjee and Bhattacharji 2001). It has a bulk composi- tion (Murty et al. 1987) similar to the Dike-25 sample studied by Halls et al. (2007). The CDC and EDC dikes are mostly composed of basalts and basaltic andesites. A few have picritic compositions likely due to their cumulate nature. The dikes of all ages show LILE and LREE enrichment and negative Nb–Ta anomalies in normalized incompatible element plots, and high Th/Nb ratios (0.23–0.73) (Kumar et al. 2012a,b; Sriv- astava et al. 2014a,b, 2015; Liao et al. 2019). In a Th/Yb versus Nb/Yb diagram, the ENE- to NE-trending dikes of the ~ 2.37 Ga old Bangalore-Karimnagar swarm and the N- to NNW-trending dikes of the ~ 2.22 Ga old Kandlamadugu swarm plot above the MORB-OIB array and the data trend toward upper continental crust (Kumar et al. 2012a, b). These characteristics have been attributed to AFC-type frac- tionation of low-Th/Nb parental melts, which are particu- larly susceptible to Th enrichment and Nb depletion through crustal contamination (Pearce 2008; Kumar et al. 2012a). In addition, the εNd(t) values of the Karimnagar dikes (Ban- galore-Karimnagar swarm, − 0.7 to + 0.6, Liao et al. 2019) and the Nelahalu dike (Kandlamadugu swarm, − 2.9 to − 1.7, Kumar et al. 2012b) have been attributed to crustal contami- nation or an isotopically heterogeneous mantle source. y All of the other samples are also tholeiitic basalts. One sample (D75) from an NNE-trending dike near the south- western margin of Cuddapah Basin (Fig. Previous petrological work 1) belongs to the ~ 2.25 Ga old Ippaguda-Dhiburahalli swarm, though it has a bulk composition (Murty et al. 1987) similar to some ~ 2.22 Ga old N- to NNW-trending dikes of the Kandlamadugu swarm (Kumar et al. 2012b). Three sam- ples (D89, D77 and D30) are from NW-trending dikes parallel to the southwestern margin of Cuddapah Basin (Fig. 1). These dikes cross-cut the Bangalore-Karimnagar and Ippaguda-Dhiburahalli swarms, and are members of the ~ 2.21 Ga old Anantapur-Kunigal swarm (French and Heaman 2010). Sample D30 has a variable bulk composi- tion with MgO contents between 8.3 wt% (Murty et al. 1987) and 11.2 wt% (Chatterjee and Bhattacharji 2001). In addition, two samples (6/33 and K88) are also from NW-trending dikes, but they are located near the north- western margin of Cuddapah Basin (Fig. 1) and they belong to the younger, ~ 2.08 Ga old Devarabanda radial dike swarm (2083–2080 Ma, Kumar et al. 2015; Söderlund et al. 2019). The origin of the ENE- to NE-trending dikes of the ~ 2.37 Ga old Bangalore-Karimnagar swarm, N- to NNW-trending dikes of the ~ 2.22 Ga old Kandlamadugu swarm, and NW- to WNW-trending dikes of the ~ 2.21 Ga old Anantapur-Kunigal swarm were modeled by ~ 15–25% batch melting of primitive mantle sources (Srivastava et al. 2014a, b, 2015). Furthermore, Srivastava et al. (2015) used chondrite-normalized DyN/YbN ratios and available petro- genetic models to suggest that the ~ 2.37 Ga old Bangalore- Karimnagar swarm originated by melting of spinel lherzo- lite, whereas the ~ 2.21 Ga old Anantapur-Kunigal, ~ 2.18 Ga old Mahabubnagar-Dandeli, and ~ 1.89–1.88 Ga old Hampi swarms originated by melting of transitional spinel-garnet lherzolite. Analytical methods Textural studies and mineral analyses were performed on a JEOL JXA-733 Superprobe electron probe microana- lyzer (EPMA) at Massachusetts Institute of Technology, Cambridge, MA, USA operating with a 15 kV accelerat- ing voltage, a 10 nA beam current, and 1–10 μm beam diameter. Typical counting times were 20–40 s per element that yielded accumulated counts with 1σ standard devia- tions of 0.3–1.0% for major elements and 1–5% for minor elements from counting statistics. The raw data were cor- rected for matrix effects with the CITZAF package (Arm- strong 1995). Sample locations, age and bulk compositions Sixteen dike samples were studied in detail for the purpose of mineral thermobarometry. Nine of these samples (D84, D86, D88, 1A, 2/14, D9A, D11, D28, and D47) are from ENE-trending dikes near the western, southwestern and southern margins of Cuddapah Basin (Fig. 1). One of the dikes (D11) has been previously dated at 2369 Ma (dike JEF-99–7, French and Heaman 2010). All of the dikes in this group are inferred to be of the same age and members of the ~ 2.37 Ga old Bangalore-Karimnagar swarm. Sam- ples D86, D88 and D9A are tholeiitic basalts (Rao et al. 1995), and D88 has a similar bulk composition to sev- eral samples (EDC9/6,9,13) analyzed by Srivastava et al. (2014a). D84 is a basaltic andesite, D28 is a picrobasalt, Petrography and mineral chemistry The analyzed samples primarily consist of augite and pla- gioclase with ilmenite and magnetite as common acces- sory minerals (Fig. 2, Table 1). Minor orthopyroxene and pigeonite are present in some samples. Olivine and Mg-rich amphibole are rare. Most of the samples have equigranular, sub-ophitic and hypidiomorphic textures (Fig. 2a, d). 1 3 1 3 Contributions to Mineralogy and Petrology (2023) 178:28 28 Page 6 of 25 28 ENE‑ to NE‑trending dikes of the ~ 2.37 Ga old Bangalore‑Karimnagar swarm These dikes are dominantly composed of normally zoned augite and plagioclase with accessory ilmenite, magnet- ite, titanite, apatite and pyrite. The augite cores in samples D86, D88, 1A, 2/14 and D11 have a restricted composition range (En41-49Fs12-16Wo35-46), whereas the augite cores are relatively Fe-rich in D84 (En46Fs21Wo33) and Mg-rich in D9A and D28 (En49-50Fs10-14Wo37-41) (Fig. 3a, Table S1). Samples D9A and D28 also contain Mg-rich orthopy- roxene (En64-71Fs26-31Wo4-5) and plagioclase with cores h l i (A Ab ) h i h h l D9A (Murty et al. 1987) are related to excess accumula- tion of Mg-rich orthopyroxene. Orthopyroxene (Fe-rich) also occurs in D86 (En28Fs67Wo4), and pigeonite (core: En60-64Fs26-39Wo7-10) occurs in D84, D86, D88 and D11 (Fig. 3a). The jadeite and aegirine contents of the pyrox- enes are < 2.5%. Minor quartz is present in samples D84 and D86. Clinopyroxene is commonly rimmed by actinolite and ferrohornblende. Secondary chlorite, epidote and albite are also present. Clinopyroxene is absent in sample D47 that contains ferroedenite, Fe-rich orthoamphibole and Ca-poor plagioclase (An30Ab70). Fig. 2 Back-scattered electron images of basaltic dike samples (names at upper right corners) from the CDC and EDC show- ing a, d ophitic to sub-ophitic texture dominated by plagio- clase laths and clinopyroxene crystals, b orthopyroxene between olivine and clinopyrox- ene, c cumulus clinopyroxene with interstitial plagioclase, e olivine overgrowth on orthopy- roxene, and f plagioclase and amphibole inclusions in olivine Fig. 2 Back-scattered electron images of basaltic dike samples (names at upper right corners) from the CDC and EDC show- ing a, d ophitic to sub-ophitic texture dominated by plagio- clase laths and clinopyroxene crystals, b orthopyroxene between olivine and clinopyrox- ene, c cumulus clinopyroxene with interstitial plagioclase, e olivine overgrowth on orthopy- roxene, and f plagioclase and amphibole inclusions in olivine ENE‑ to NE‑trending dikes of the ~ 2.37 Ga old Bangalore‑Karimnagar swarm D9A (Murty et al. 1987) are related to excess accumula- tion of Mg-rich orthopyroxene. Petrography and mineral chemistry Orthopyroxene (Fe-rich) also occurs in D86 (En28Fs67Wo4), and pigeonite (core: En60-64Fs26-39Wo7-10) occurs in D84, D86, D88 and D11 (Fig. 3a). The jadeite and aegirine contents of the pyrox- enes are < 2.5%. Minor quartz is present in samples D84 and D86. Clinopyroxene is commonly rimmed by actinolite and ferrohornblende. Secondary chlorite, epidote and albite are also present. Clinopyroxene is absent in sample D47 that contains ferroedenite, Fe-rich orthoamphibole and Ca-poor plagioclase (An30Ab70). Radial dikes of the ~ 2.08 Ga old Devarabanda swarm The NW-trending dike K88 is medium-grained and has an ophitic texture. It dominantly consists of augite (En47Fs15Wo39) and plagioclase (core: An71Ab29) with minor olivine (Fo67), orthopyroxene (En68Fs29Wo4) and amphibole (magnesiohastingsite, Mg/(Mg + Fe) = 0.65), and acces- sory chlorite, ilmenite and magnetite (Fig. 3a, b, Table S1). Orthopyroxene occurs as discrete crystals, exsolution lamellae in augite, and at the rims of olivine and clinopy- roxene. Olivine-hosted inclusions of plagioclase, pyroxene and amphibole (Fig. 2e, f) belong to an older generation of basalt, and the olivine overgrowth probably formed through influx of a younger batch of primitive magma. This suggests that dike K88 was probably emplaced by reactivation of an old magma pathway. Another NW-trending dike 6/33 con- tains augite with lower Mg (En38Fs25Wo38) and less calcic plagioclase (core An63Ab37) compared to K88 (Fig. 3a, b). It also contains Mg-rich pigeonite (En71Fs19Wo10), but it lacks olivine, orthopyroxene and hornblende. The NW-trending dike K88 is medium-grained and has an ophitic texture. It dominantly consists of augite (En47Fs15Wo39) and plagioclase (core: An71Ab29) with minor olivine (Fo67), orthopyroxene (En68Fs29Wo4) and amphibole (magnesiohastingsite, Mg/(Mg + Fe) = 0.65), and acces- sory chlorite, ilmenite and magnetite (Fig. 3a, b, Table S1). ENE‑ to NE‑trending dikes of the ~ 2.37 Ga old Bangalore‑Karimnagar swarm These dikes are dominantly composed of normally zoned augite and plagioclase with accessory ilmenite, magnet- ite, titanite, apatite and pyrite. The augite cores in samples D86, D88, 1A, 2/14 and D11 have a restricted composition range (En41-49Fs12-16Wo35-46), whereas the augite cores are relatively Fe-rich in D84 (En46Fs21Wo33) and Mg-rich in D9A and D28 (En49-50Fs10-14Wo37-41) (Fig. 3a, Table S1). Samples D9A and D28 also contain Mg-rich orthopy- roxene (En64-71Fs26-31Wo4-5) and plagioclase with cores that are more calcic (An79Ab21) than in the other samples (An52-68Ab32-49) (Fig. 3a, b), and D28 contains olivine with Fo70 composition (Fig. 2a, b, Table S1). The high bulk MgO (13.8 wt%) and low Al2O3 and CaO contents of sample 1 3 3 Contributions to Mineralogy and Petrology (2023) 178:28 Page 7 of 25 28 5 28 Ol olivine, Cpx clinopyroxene, Pgt pigeonite, Opx orthopyroxene, Pl plagioclase, Amp amphibole, Mag magnetite, Ilm ilmenite Latitude/Longitude Ol Cpx Pgt Opx Pl Amp Mag Ilm ENE-trending dikes (~ 2.37 Ga old Bangalore-Karimnagar swarm) D84 13o37′20"N, 78o57′58"E X X X X X D86 13o35′06"N, 79o01′16"E X X X X X X D88 13o37′40"N, 79o04′53"E X X X X X 1A 14o09′47"N, 78o12′46"E X X X X 2/14 13o59′08"N, 77o34′00"E X X X X D9A 14o27′59"N, 77o19′32"E X X X X D11 14o14′17"N, 77o31′41"E X X X X X D28 14o33′07"N, 77o39′42"E X X X X X X D47 14o24′35"N, 77o54′14"E X X NNE-trending dike (~ 2.25 Ga old Ippaguda-Dhiburahalli swarm) D75 14o10′33"N, 78o30′00"E X X X X NW-trending dikes (~ 2.21 Ga old Anantapur-Kunigal swarm) D77 14o07′51"N, 78o27′02"E X X X D89 13o39′16"N, 79o07′21"E X X X D30 14o37′46"N, 77o41′19"E X X X X X X NW-trending dikes (~ 2.08 Ga old Devarabanda swarm) 6/33 15o22′46"N, 77o37′18"E X X X X X K88 15o25′17"N, 77o43′14"E X X X X X X X ENE-trending dike (~ 1.89–1.88 Ga old Hampi swarm) D8A 14o31′58"N, 77o25′08"E X X X X X X Table 1 Mineral assemblages in the CDC and EDC basaltic dikes Table 1 Mineral assemblages in the CDC and EDC basaltic dikes Ol olivine, Cpx clinopyroxene, Pgt pigeonite, Opx orthopyroxene, Pl plagioclase, Amp amphibole, Mag magnetite, Ilm ilmenite N‑ to NNE‑trending dikes of the ~ 2.25 Ga old Ippaguda‑Dhiburahalli swarm An68Ab32, rim: An25Ab75) (Figs. 2d, 3a-d). It also contains secondary orthoamphibole, chlorite and mica, and accessory ilmenite and magnetite. The NNE-trending dike D75 consists of augite with thick rims of actinolite and ferrohornblende, plagioclase, and accessory epidote, ilmenite and pyrite. The augite and plagioclase cores have compositions of En42Fs23Wo34 and An72Ab28, respectively (Fig. 3a, b, Table S1). The sample also contains pigeonite (En61Fs28Wo11) and minor quartz. NW‑ to WNW‑trending dikes of the ~ 2.21 Ga old Anantapur‑Kunigal swarm Dikes D89 and D77 locally cross-cut the ~ 2.25 Ga old dike D75. They are mineralogically similar to D75, and are also composed of augite with thick rims of actinolite and ferrohornblende, plagioclase, and accessory epidote, ilmenite and pyrite. However, compared to D75, the augite (En46-47Fs16Wo37-38) and plagioclase (An60-64Ab36-40) cores in D89 and D77 are Mg-rich and Ca-poor, respectively (Fig. 3a, b, Table S1). Sample D30 has an equigranular sub-ophitic texture, and it consists of reverse-zoned augite (En52-55Fs9-15Wo34-36) and pigeonite (En59-67Fs22-31Wo10-11), oscillatory zoned orthopyroxene (core: En73-80Fs15-22Wo5, rim: En60Fs36Wo4), and normally zoned plagioclase (core: 1 3 3 Contributions to Mineralogy and Petrology (2023) 178:28 28 Page 8 of 25 28 Page 8 of 25 ENE ~2.37 Ga NNE ~2.25 Ga NW ~2.21 Ga NW ~2.08 Ga ENE ~1.89 Ga Hd Di Wo En Fs Aug Pgt a) ( Or Ab An b) ( 0 20 40 60 80 100 0 300 600 900 1200 Core-rim (microns) Orthopyroxene (D30) En Fs Wo c) ( 0 20 40 60 80 0 50 100 150 200 Core-rim (microns) Plagioclase (D30) An Ab Or d) ( Fig. 3 Composition of minerals in the CDC and EDC basaltic dikes: a, c pyroxenes, and b, d plagioclase. c, d Compositional variation from core to rim of orthopyroxene and plagioclase in sample D30 ENE ~2.37 Ga NNE ~2.25 Ga NW ~2.21 Ga NW ~2.08 Ga ENE ~1.89 Ga Hd Di Wo En Fs Aug Pgt a) ( 0 20 40 60 80 100 0 300 600 900 1200 Core-rim (microns) Orthopyroxene (D30) En Fs Wo c) ( 0 20 40 60 80 0 50 100 150 200 Core-rim (microns) Plagioclase (D30) An Ab Or d) ( 0 20 40 60 80 100 0 300 600 900 1200 Core-rim (microns) Orthopyroxene (D30) En Fs Wo c) ( d) ( c) ( a) ( Orthopyroxene (D30) d) ( 0 20 40 60 80 0 50 100 150 200 Core-rim (microns) Plagioclase (D30) An Ab Or ) ( En En Fs Or Ab An b) ( b) ( Ab Fig. 3 Composition of minerals in the CDC and EDC basaltic dikes: a, c pyroxenes, and b, d plagioclase. c, d Compositional variation from core to rim of orthopyroxene and plagioclase in sample D30 ENE‑ to NE‑trending dikes of the ~ 1.89–1.88 Ga old Hampi swarm from the Cpx-bulk Fe2+-Mg distribution coefficient, KD(Fe2+-Mg), the equilibrium value of which is 0.28 ± 0.08 (Putirka 2008). So, a knowledge of the bulk Fe2+ content (or the bulk Fe3+/Fe ratio) is necessary. For samples that contain equilibrium olivine and the equilibrium temperature is independently known, the bulk Fe3+/Fe ratio can be calcu- lated with Eq. 8 of Blundy et al. (2020). Olivine-melt equi- librium is assessed by comparing the observed value of the olivine-bulk Mn-Mg distribution coefficient, KD(Mn-Mg), which is relatively constant over a wide range of P–T-fO2 conditions, with the equilibrium value predicted by the lattice strain model (Blundy et al. 2020). If the observed KD(Mn-Mg) shows disequilibrium, the bulk composition is adjusted by adding or subtracting olivine until olivine is in equilibrium with the bulk (Blundy et al. 2020). This method was attempted on the olivine-bearing samples K88 and D28, the latter with a temperature of 1172 °C determined from Cpx composition only (see below). However, the olivines (Fo67-70) in these samples are not in equilibrium with the bulk, as indicated by the higher observed KD(Mn-Mg) val- ues (0.8–1.0) than predicted (0.26–0.27) at 1172 °C, and Eq. 8 of Blundy et al. (2020) yields negative Fe3+/∑Fe ratios (− 1.35 and − 1.65). Very large corrections (40–45% olivine The ENE-trending dike D8A contains abundant cumulus augite (core: En50Fs14Wo36) and compositionally zoned plagioclase (core: An49Ab51). Augite accounts for > 50% of the rock volume (Fig. 2c). The sample also contains minor pigeonite (En51Fs41Wo8) and hornblende (Mg/ (Mg + Fe) = 0.64), secondary albite and mica, and accessory ilmenite and magnetite. Methods The P–T conditions of crystallization were determined with the clinopyroxene-anhydrous liquid thermobarometer of Putirka et al. (1996) using mineral compositions in Table S1 and bulk compositions in Murty et al. (1987) and Rao et al. (1995). The quoted uncertainties in the P–T calculated with this thermobarometer are ± 1.4 kbar and ± 27 °C. Appli- cation of this thermobarometer requires that the Cpx is in equilibrium with the bulk (liquid). Equilibrium is assessed 3 Contributions to Mineralogy and Petrology (2023) 178:28 Page 9 of 25 28 subtraction) are required to the bulk to bring it in Mn-Mg equilibrium with olivine, and the Fe3+/∑Fe ratios after correction are unreasonably high (0.36–0.58). So, Blundy et al.’s method was not applied to determine bulk Fe3+/∑Fe ratios. Instead, the bulk Fe3+/∑Fe ratios were determined using the spinel-ilmenite equilibrium. First, oxygen fugacity values were calculated from coexisting spinel and ilmenite compositions (program QUILF4, Andersen and Lindsley 1988). Then, Eq. 6b of Putirka (2016a) was used to calcu- late ln(XFe2O3/XFeO) of the melt. Fe3+/Fe2+ equals 2XFe2O3/ XFeO, and Fe3+/∑Fe is 1/(1 + 1/(Fe3+/Fe2+)). For samples in which coexisting spinel and ilmenite are absent, the average bulk Fe3+/∑Fe ratio of the other samples was used in the calculations. and 1166–1192 °C (± 58 °C) (Table 3). The Higgins et al. (2022) and Jorgenson et al. (2022) methods also yielded low pressures (1 bar-2 kbar), but with lower uncertainties (± 1.1–1.5 kbar, and ± 0.3–2.0 kbar). The temperature esti- mates are lower with both the methods of Higgins et al. (2022) (1016–1150 °C, ± 20–89 °C) and Jorgenson et al. (2022) (1115–1160 °C, ± 27–88 °C) compared to Putirka (2008), but all three methods have overlapping uncertain- ties (Table 3). Spinel and ilmenite equilibrated at subsoli- dus temperatures (457–661 °C) and oxygen fugacity values below the fayalite-magnetite-quartz buffer (∆FMQ between -0.4 and -3.2), and the calculated range of Fe3+/∑Fe ratios is 0.06–0.12. In the NNE-trending dike D75 from the ~ 2.25 Ga old Ippaguda-Dhiburahalli swarm, the estimated P–T of clino- pyroxene crystallization are 0.9 kbar and 1119 °C with the Cpx-only formulations of Putirka (2008). The methods of Higgins et al. (2022) (2.0 ± 1.1 kbar, 1032 ± 82 °C) and Jor- genson et al. (2022) (1 bar, 1112 ± 21 °C) yielded similar results. The P–T were also calculated with the Cpx-composition thermobarometer of Putirka (2008). The T-dependent baro- metric expression (Eq. Methods 32a) and P-dependent thermometric expression (Eq. 32d) of Putirka (2008) were solved simulta- neously to obtain P–T. These equations are based on multi- ple regression of clinopyroxene compositions obtained from partial melting experiments on basalts in the P–T range of 1 bar-75 kbar/800–2200 °C. The equations use the enstatite- ferrosilite and diopside-hedenbergite components and cation proportions of clinopyroxene calculated on the basis of 6 oxygen atoms. The quoted uncertainties are ± 3.1 kbar and ± 58 °C for clinopyroxene crystallizing from anhydrous melts. In addition, the P–T were also calculated with the random forest machine learning-based algorithms of Hig- gins et al. (2022) and Jorgenson et al. (2022) that provided independent estimates of the uncertainties. Jorgensen et al. (2022) use the same methodology as Higgins et al. (2022), but they use an expanded dataset that includes Cpx in equi- librium with alkalic liquids. These thermobarometers are also based on clinopyroxene compositions obtained from partial melting experiments on basalts that cover a P–T range of 0.002–30 kbar/750–1250 °C. In the NW-trending ~ 2.21 Ga old Anantapur-Kuni- gal dikes, clinopyroxene crystallized at P–T conditions of 4.0–5.7 kbar and 1169–1204 °C, as estimated with the Cpx-anhydrous liquid formulations of Putirka et al. (1996) (Table 2). The Cpx-only formulations of Putirka (2008) yielded P–T of 2.6–5.4 kbar (± 3.1 kbar) and 1169–1211 °C (± 58 °C) (Table 3). The Higgins et al. (2022) method yielded similar results (2–7 kbar, ± 0.3–4.0 kbar, 1016–1200 °C, ± 29–76 °C). The Jorgenson et al. (2022) method also yielded similar results (1 bar-2 kbar, ± 0.5–10.0 kbar, 1135–1210 °C, ± 29–79 °C), but very high uncertain- ties in pressure for sample D30 (Table 3) that may be related to the high Cr2O3 (> 1 wt%) content of the clinopyroxene (Table S1). In sample D30, the Cpx-Opx thermobarometer of Putirka (2008) yielded P–T of 5.5–5.7 kbar (± 3.7 kbar) and 1150–1178 °C (± 60 °C) (Table 2). Thus, the P–T results for sample D30 with the Cpx-anhydrous liquid, Cpx-only and Cpx-Opx formulations are consistent with each other. Sample D30 also registered a spinel-ilmenite equilibration temperature of 638 °C and ∆FMQ of -1.06 with a corre- sponding Fe3+/∑Fe ratio of 0.11. The two-pyroxene thermobarometer of Putirka (2008) (uncertainties: ± 3.7 kbar and ± 60 °C) was applied to calculate P–T in sample D30 that contains Cpx and Opx showing equilibrium Fe–Mg distribution (KD(Fe2+-Mg) = 1.09 ± 0.14). Methods In addition, samples K88 and D8A contain Mg-rich amphibole, whose compositions were used to calculate temperatures with the thermometer of Putirka (2016b) (uncertainty: ± 30 °C). In the NW-trending dike K88 from the ~ 2.08 Ga old Devarabanda swarm, amphibole crystallized at a tempera- ture of 986 °C, and in dike 6/33 from the same swarm, spinel and ilmenite equilibrated at a temperature of 791 °C and a ∆FMQ value of − 0.49 (Table 2). Results (1987) Compositions used KD(Fe2+-Mg) P (kbar) T (oC)a T (oC)b ∆FMQb Fe3+/∑Fec ENE-trending dikes (~ 2.37 Ga old Bangalore-Karimnagar swarm) D88 Cpx core, bulkd 0.30 1.5 1158 Spl, Ilm 554 − 2.32 0.06 D86 Cpx outer core, bulkd 0.31 0.8 1119 Spl, Ilm 661 − 0.37 0.12 D84 Spl, Ilm 623 − 1.97 0.06 1A Spl, Ilm 645 − 2.02 2/14 Spl, Ilm 642 − 1.93 D11 Spl, Ilm 457 − 3.17 NW-trending dikes (~ 2.21 Ga old Anantapur-Kunigal swarm) D77 Cpx core, bulkd,e 0.37 5.0 1182 D89 Cpx core, bulkd 0.34 5.7 1197 Cpx core, bulke 0.31 4.0 1169 D30 Cpx outer core, bulke 0.27 4.4 1204 Cpx rim, bulke 0.36 4.1 1202 Spl, Ilm 638 − 1.06 0.11 Cpx and Opx outer core 1.14 5.7 1178 Cpx rim, Opx core 1.16 5.6 1154 Cpx rim, Opx outer core 1.11 5.5 1150 NW-trending dikes (~ 2.08 Ga old Devarabanda swarm) K88 Amp 986 6/33 Spl, Ilm 791 -0.49 ENE-trending dike (~ 1.89–1.88 Ga old Hampi swarm) D8A Cpx core, bulkd,e 0.32 5.1 1202 Spl, Ilm 665 − 0.54 0.16 Amp 785 Table 2 Mineral-liquid and mineral thermobarometry of the CDC and EDC basalts Formulations: Cpx-bulk (anhydrous liquid): equilb. KD(Fe2+-Mg) = 0.28 ± 0.08, Putirka et al. (1996), Eq. T1 (± 27 °C) and P1 (± 1.4 kbar); Spl-Ilm: Andersen and Lindsley (1988); Cpx-Opx: equilb. KD(Fe2+-Mg) = 1.09 ± 0.14, Putirka (2008), Eq. 37 (± 60 °C) and 38 (± 3.7 kbar); Amp: Putirka (2016b), Eq. 5 (± 30 °C); awith Cpx-bulk, Cpx-Opx, or Amp; bwith Spl-Ilm; cwith Eq. 6b of Putirka (2016a); bulk compositions from dRao et al. (1995), eMurty et al. (1987) Formulations: Cpx-bulk (anhydrous liquid): equilb. KD(Fe2+-Mg) = 0.28 ± 0.08, Putirka et al. (1996), Eq. T1 (± 27 °C) and P1 (± 1.4 kbar); Spl-Ilm: Andersen and Lindsley (1988); Cpx-Opx: equilb. KD(Fe2+-Mg) = 1.09 ± 0.14, Putirka (2008), Eq. 37 (± 60 °C) and 38 (± 3.7 kbar); Amp: Putirka (2016b), Eq. 5 (± 30 °C); awith Cpx-bulk, Cpx-Opx, or Amp; bwith Spl-Ilm; cwith Eq. 6b of Putirka (2016a); bulk compositions from dRao et al. (1995), eMurty et al. (1987) Table 3 Cpx thermobarometry of the CDC and EDC basalts Cpx composition thermobarometers of aPutirka (2008), Eq. 32a (± 3.1 kbar) and 32d (± 58 °C); bHiggins et al. (2022); cJorgenson et al. Table 3 Cpx thermobarometry of the CDC and EDC basalts Results In the ENE-trending dike D8A from the ~ 1.89–1.88 Ga old Hampi swarm, clinopyroxene crystallized at P–T con- ditions of 5.1 kbar and 1202 °C, as estimated with the Cpx-anhydrous liquid formulations of Putirka et al. (1996) (Table 2). With the Cpx-only formulations of Putirka (2008), the P–T are 3.8 kbar (± 3.1 kbar) and 1192 °C (± 58 °C) (Table 3). The Higgins et al. (2022) (5.0 ± 2.4 In the ENE-trending ~ 2.37 Ga old Bangalore-Karimna- gar dikes, clinopyroxene crystallized at P–T conditions of 0.8–1.5 kbar and 1119–1158 °C, as estimated with the Cpx-anhydrous liquid formulations of Putirka et al. (1996) (Table 2). Using only Cpx compositions, Putirka’s (2008) formulations yielded P–T of 1.0–4.9 kbar (± 3.1 kbar) 1 3 Contributions to Mineralogy and Petrology (2023) 178:28 28 Page 10 of 25 Formulations: Cpx-bulk (anhydrous liquid): equilb. KD(Fe2+-Mg) = 0.28 ± 0.08, Putirka et al. (1996), Eq. T1 (± 27 °C) and P1 (± 1.4 kbar); Spl-Ilm: Andersen and Lindsley (1988); Cpx-Opx: equilb. KD(Fe2+-Mg) = 1.09 ± 0.14, Putirka (2008), Eq. 37 (± 60 °C) and 38 (± 3.7 kbar); Amp: Putirka (2016b), Eq. 5 (± 30 °C); awith Cpx-bulk, Cpx-Opx, or Amp; bwith Spl-Ilm; cwith Eq. 6b of Putirka (2016a); bulk compositions from dRao et al. (1995), eMurty et al. Results (2022) Composition P (kbar)a T (oC)a P (kbar)b T (oC)b P (kbar)c T (oC)c ENE-trending dikes (~ 2.37 Ga old Bangalore-Karimnagar swarm) D88 Cpx core 4.9 1192 2.0 ± 1.4 1110 ± 67 0.0 ± 0.3 1132 ± 30 D86 Cpx outer core 4.6 1166 2.0 ± 1.4 1016 ± 89 0.0 ± 1.0 1115 ± 38 D9A Cpx core 1.0 1183 2.0 ± 1.5 1038 ± 47 1.0 ± 2.0 1160 ± 88 D28 Cpx core 2.4 1172 2.0 ± 1.1 1150 ± 20 0.7 ± 2.0 1148 ± 27 NNE-trending dike (~ 2.25 Ga old Ippaguda-Dhiburahalli swarm) D75 Cpx average 0.9 1119 2.0 ± 1.1 1032 ± 82 0.0 ± 0.0 1112 ± 21 NW-trending dikes (~ 2.21 Ga old Anantapur-Kunigal swarm) D77 Cpx core 3.3 1169 2.0 ± 0.3 1016 ± 70 0.0 ± 0.5 1135 ± 29 D89 Cpx core 5.2 1190 2.0 ± 1.3 1083 ± 76 0.0 ± 0.8 1142 ± 31 D30 Cpx core 2.6 1207 7.0 ± 4.0 1200 ± 29 2.0 ± 10.0 1210 ± 79 Cpx outer core 4.1 1211 7.0 ± 2.9 1193 ± 30 0.5 ± 6.3 1184 ± 60 Cpx rim 5.4 1207 3.8 ± 2.7 1146 ± 40 0.0 ± 1.3 1140 ± 33 ENE-trending dike (~ 1.89–1.88 Ga old Hampi swarm) D8A Cpx core 3.8 1192 5.0 ± 2.4 1150 ± 48 0.3 ± 2.7 1148 ± 37 1 3 Contributions to Mineralogy and Petrology (2023) 178:28 Page 11 of 25 2 f 25 28 28 kbar, 1150 ± 48 °C) and Jorgenson et al. (2022) methods (0.3 ± 2.7 kbar, 1148 ± 37 °C) yielded similar results with lower uncertainties. Amphibole crystallized at a temperature of 785 °C, and spinel-ilmenite equilibrated at a tempera- ture of 665 °C and ∆FMQ of -0.54 with a corresponding Fe3+/∑Fe ratio of 0.16. Ol-Pl-Cpx cotectics. Thirty (including the six that plot on their cotectics) of the 88 samples required ≤ 4% normative olivine subtraction, and these were selected for primary magma modeling (Table S2, locations in Fig. 1). The other samples were not selected as their excess crystal contents were deemed too high for meaningful adjustment to the bulk composition. ∑ In summary, clinopyroxene crystallized at P–T condi- tions of 0.8–5.7 kbar and 1119–1211 °C in all samples, esti- mated using the formulations of Putirka et al. (1996) and Putirka (2008). Sample selection At low pressures such as the ≤ 6 kbar pressures of crystal- lization calculated above, primitive basalts evolve by crystal- lizing olivine, followed by Ol + Pl, and then by Ol + Pl + Cpx with decreasing temperature (Kinzler and Grove 1992a). The olivine control line, the Ol-Pl cotectic, and the Ol-Pl-Cpx cotectic together define the fractionation path of the basalt. Any basalt whose composition has been modified by excess crystal accumulation (i.e., a “cumulate” sample) would show displacement from its fractionation path when plotted in the Ol-Pl-Cpx (from Qz) and Ol-Cpx-Qz (from Pl) pseudoter- nary projections of the basalt tetrahedron according to the methods of Tormey et al. (1987) and Grove (1993). For the purpose of modeling, a total of 88 basalt samples from the literature were considered (Murty et al. 1987; Rao et al. 1995; Chatterjee and Bhattacharji 1998; Anand et al. 2003; Halls et al. 2007; French and Heaman 2010; Kumar et al. 2012a,b; Srivastava et al. 2014a,b, 2015). The bulk com- position of each sample and its plagioclase lherzolite mul- tiple saturation point (PL-MSP) at 1 bar-10 kbar pressures predicted by the parameterized expressions of Kinzler and Grove (1992a) were plotted in the pseudoternary projections. The position of the PL-MSP and the constraints of Yang et al. (1996) define the position of the Ol-Pl-Cpx cotectic at different pressures in the projections. Six of the samples plot on their corresponding Ol-Pl-Cpx cotectics, indicating that they represent unmodified basaltic liquids. The other samples are variably displaced from their cotectics. The bulk compositions of these samples were adjusted by subtracting normative olivine so that they plotted on their respective The selected samples are all subalkaline tholeiitic basalts (Mg# 37–55) according to the total alkali versus silica clas- sification (Fig. 4a). There is a broad negative correlation between MgO and FeOT (Fig. 4b). The samples show chemi- cal index of alteration (CIA) values of 37–41, and they are tightly clustered around average unaltered basalt and gab- bro in an A-CN-K plot (Nesbitt and Young 1982; Babechuk et al. 2014) (Fig. 4c). Hence, these samples have not been altered by kaolinitization of feldspar, and their bulk K con- tents have largely remained unchanged since formation. In a Th/Yb versus Nb/Yb diagram (Fig. 4d), the samples plot above the MORB-OIB array and toward upper continental crust, and the trend of the data coincides with a model AFC trend of Pearce (2008). Results The machine learning-based algorithms of Higgins et al. (2022) and Jorgenson et al. (2022) yielded similar pressures (1 bar-5 kbar) with lower uncertainties (except for sample D30), but a larger range of temperatures (1016–1210 °C). Spinel and ilmenite equilibrated at sub- solidus temperatures (~ 460–665 °C, excluding 6/33) and ∆FMQ values between -0.5 and -3. The average Fe3+/∑Fe ratio of the samples is 0.1. These results clearly indicate that the dikes crystallized within the upper crust. Six of the selected samples are from inside the Cud- dapah Basin: three from the Tadpatri sills (minor sills near Krishnagiri and Yeraguntla-Vempalle, and top part of the major sill near Pulivendla), and three from the Vempalle volcanics near Gattimanikonda (Chatterjee and Bhattacharji 1998; Anand et al. 2003). They plot on their respective Ol- Pl-Cpx cotectics at 1 bar-6.5 kbar pressures (Table 4, S3), consistent with the ~ 5 kbar pressure of crystallization esti- mated by Chatterjee and Bhattacharji (1998). y j j ( ) From the remainder of the selected samples, 14 are from the ENE- to NE-trending dikes of the ~ 2.37 Ga old Banga- lore-Karimnagar swarm (four from CDC, 10 from EDC, Rao et al. 1995; Halls et al. 2007; Srivastava et al. 2014a), five are from the N- to NNW-trending dikes of the ~ 2.22 Ga old Kandlamadugu swarm (three from CDC, two from EDC, French and Heaman 2010; Kumar et al. 2012b; Srivastava et al. 2014b), and five are from the NW- to WNW-trending dikes of the ~ 2.21 Ga old Anantapur-Kunigal swarm (three from CDC, two from EDC, Srivastava et al. 2015). Srivas- tava et al. (2014a) identified two groups of basalts among the Bangalore-Karimnagar dikes based on chondrite-normalized LaN/LuN ratios (~ 2 and > 2). The 14 selected samples of Bangalore-Karimnagar dikes include 11 from the low LaN/ LuN group and 3 from the high LaN/LuN group. The selected dike samples from all swarms plot on their 1 bar-3 kbar Ol-Pl-Cpx cotectics (Table 4, S3), indicating crystallization pressures similar to those obtained from thermobarometry. Sample selection A similar (but less obvious) trend toward upper continental crust is also observed in a Zr/Y versus Nb/Y diagram (Fig. 4e). In a Ce/Y versus La/Nb dia- gram (Fig. 4f), the data plot along a mixing line between melts of primitive mantle and an average CDC/EDC ana- tectic granite, as shown previously for the Cuddapah Basin 1 3 1 Contributions to Mineralogy and Petrology (2023) 178:28 28 Page 12 of 25 Table 4 P–T of crystallization and primary magma equilibration with lherzolite for the CDC, EDC and Cuddapah Basin basalts a primary magma (Mg# 73) equilibrated with Fo90 olivine; bcalculated with Eq. Sample selection 16 of Putirka (2008); cdegree of fractionation; dT0: initial temper- ature of assimilant (wall rock); eT1: magma temperature at which assimilation begins (assimilant reaches solidus assumed at 900 °C), emaximum ratio of Ma (mass of partial melt of assimilant mixing with magma) to Mc (mass crystallized from magma); fdegree of assimilation of crustal melt; gaverage TTG gneiss from EDC (a) and CDC (b) Crystallization Reverse FC Reverse EC-AFC Primary magmaa Primary magmaa P Tb Depth Fc P T Depth Fc AsmT0 d MagT1 e Ma/Mc e Asmf Crustg P T Depth kbar oC km % kbar oC km % oC oC % kbar oC km ENE- to NE-trending dikes (~ 2.37 Ga old Bangalore-Karimnagar swarm) D88 3 1150 11 64.1 8.5 1272 31 63.3 800 1179 0.391 12.2 a 11 1297 38 8–3 0.001 1107 0.004 68.8 13 1319 44 67.9 665 1138 0.307 17.5 a 16 1362 55 10–6 0.001 1102 0.004 73.0 11 1302 40 72.5 720 1154 0.506 27.0 b 16 1363 57 23–2 0.5 1146 2 63.3 9 1280 33 62.6 781 1162 0.235 7.2 b 11 1305 40 24–1 2 1151 7 65.2 9.5 1286 35 64.3 777 1174 0.324 11.2 b 12 1311 41 EDC9/6 0.001 1130 0.004 66.8 10 1292 36 65.9 750 1155 0.315 12.6 a 13 1322 44 EDC9/12 1.5 1131 5 68.5 11 1303 39 67.2 718 1159 0.300 14.1 a 14 1340 49 EDC9/13 2 1152 7 61.0 8 1268 29 59.2 808 1187 0.459 14.4 a 12 1311 41 EDC9/16 2.5 1141 9 67.1 9.5 1285 35 65.5 755 1173 0.364 15.3 a 14 1335 47 EDC9/25 1.5 1135 5 67.4 9 1279 33 66.0 758 1169 0.377 15.6 a 13 1328 46 EDC9/28 1 1156 4 58.0 9.5 1287 35 57.7 788 1169 0.219 6.3 a 11 1306 39 EDC8/9 1 1128 4 67.1 13 1320 44 66.5 692 1151 0.233 11.8 b 15 1351 52 EDC9/9 3 1127 11 65.6 8.5 1270 31 62.8 780 1194 0.643 29.5 a 15 1349 52 EDC9/23 2.5 1125 9 64.8 11 1299 39 63.5 725 1160 0.376 18.1 a 15 1348 52 N- to NNW-trending dikes (~ 2.22 Ga old Kandlamadugu swarm) JEF-00–55 2.5 1133 9 65.7 9.5 1284 35 64.1 760 1177 0.459 20.3 a 15 1345 50 HD12 1.5 1128 5 53.7 13 1314 45 52.8 723 1151 0.284 12.7 b 15 1338 51 HD14 0.001 1114 0.004 58.6 12 1310 43 57.0 710 1147 0.350 17.3 b 16 1352 54 GD24 2 1137 7 53.0 12 1309 43 50.9 735 1162 0.299 12.9 b 15 1344 52 DC08/4 1.5 1117 5 72.1 10 1290 36 71.4 717 1155 0.383 19.2 a 14 1333 47 NW- to WNW-trending dikes (~ 2.21 Ga old Anantapur-Kunigal swarm) EDD09/14 3 1123 11 74.0 12 1309 41 73.6 680 1153 0.296 16.2 a 15 1352 52 EDD09/21 2.5 1130 9 63.3 13 1323 46 61.8 716 1167 0.361 18.3 a 17 1372 58 DC12/02 2 1139 7 62.3 11 1303 40 61.4 720 1156 0.206 8.9 b 13 1327 46 DC12/05 1 1154 4 54.2 11 1304 40 52.3 762 1165 0.169 5.6 b 13 1327 46 DC12/09 0.001 1151 0.004 49.5 11 1297 38 48.1 778 1162 0.162 4.8 b 12 1315 43 Vempalle volcanics, Cuddapah Basin (> 1.89 Ga) V99MA04 5 1172 18 56.8 8 1266 29 55.7 801 1185 0.225 5.9 a 10 1291 36 V99MA80 0.001 1110 0.004 64.1 9 1276 33 62.3 760 1159 0.519 23.4 a 14 1330 47 V99MA81 2.5 1144 9 61.5 9 1277 33 59.0 775 1180 0.415 16.0 a 14 1332 47 Tadpatri sills, Cuddapah Basin (~ 1.89 Ga) B37 4 1152 15 71.6 8.5 1274 31 71.5 757 1168 0.215 7.8 a 11 1304 39 T 98MA74 5.5 1171 20 63.5 8 1267 29 61.9 800 1206 0.418 14.0 a 12 1317 43 T98MA94 6.5 1188 24 57.7 12 1315 43 56.8 800 1224 0.421 14.2 a 16 1359 53 a primary magma (Mg# 73) equilibrated with Fo90 olivine; bcalculated with Eq. Sample selection 16 of Putirka (2008); cdegree of fractionation; dT0: initial temper- ature of assimilant (wall rock); eT1: magma temperature at which assimilation begins (assimilant reaches solidus assumed at 900 °C), emaximum ratio of Ma (mass of partial melt of assimilant mixing with magma) to Mc (mass crystallized from magma); fdegree of assimilation of crustal melt; gaverage TTG gneiss from EDC (a) and CDC (b) In the Th/Yb versus Nb/Yb and Ce/Y versus La/Nb dia- grams (Fig. 4d, f), the data plot beyond the range of mixing between primitive melts and lower continental crust, indi- cating that the lower crust was not the contaminant. A good match with the model AFC trend of Pearce (2008) (Fig. 4d) shows that the primitive melts were probably contaminated basalts by Anand et al. (2003). The CDC/EDC samples show similar Th/Nb (0.20–0.60) and La/Nb (1.4–3.7) ratios to the Cuddapah Basin samples (0.23–0.69 and 1.9–2.6) indicating that upper crustal contamination may be similar in the two groups (Fig. 4d, f, Table S2). 1 3 Contributions to Mineralogy and Petrology (2023) 178:28 Page 13 of 25 28 Page 13 of 25 28 28 Contributions to Mineralogy and Petrology (2023) 178:28 Page 13 of 25 2 with the upper crust through an AFC-type process. However, contamination of the primitive melts during passage through lith h t i i f ibl t l t i l i d b the contamination occurred before the primary magm crystallized. 0.01 0.1 1 10 0.1 1 10 100 Th/Yb Nb/Yb (d) PM UCC LCC 2 2.5 3 3.5 4 4.5 5 5.5 44 46 48 50 52 54 Na2O + K2O (wt%) SiO2 (wt%) ENE to NE 2.37 Ga N to NNW 2.22 Ga NW to WNW 2.21 Ga Vempalle >1.9 Ga Tadpatri 1.9 Ga basalt basalc andesite (a) 10 11 12 13 14 15 16 4 5 6 7 8 FeOT (wt%) MgO (wt%) (b) 0.01 0.1 1 10 0 1 1 Nb/Y Zr/Y (e) PM UCC LCC 0 1 2 3 4 5 6 0 2 4 6 8 La/Nb Ce/Y (f) PM EDC granite CDC granite LCC UCC Al2O3 CaO* + Na2O K2O Basalt Gabbro (c) Fig. 4 Bulk composition of the selected CDC/EDC and Cuddapah Basin basalts (data from Rao et al. 1995, Halls et al. 2007, and Srivastava et al. 2014a for ENE- to NE-trending dikes, French and Heaman 2010, Kumar et al. 2012b, and Srivastava et al. Sample selection 2014b for N- to NNW-trending dikes, Srivastava et al. 2015 for NW- to WNW- trending dikes, and Chatterjee and Bhattacharji 1998, and Anand et al. 2003 for Vempalle volcanics and Tadpatri sills within Cud- dapah Basin), a Total alkali versus silica after Le Bas et al. (1986), subalkalic-alkalic boundary from Macdonald and Katsura (1964), b MgO versus total FeO, c A-CN-K plot, average gabbro and basa (red dots) from Nesbitt and Young (1982) and Babechuk et al. (2014 d MORB-OIB array and dashed line with arrow representing AF trend from Pearce (2008), e Plume array after Condie (2005), f Ave age CDC and EDC anatectic granites (green dots) from Jayanand et al. (2018), dashed line with arrow shows mixing trend of prim tive melts with average CDC/EDC granite. d-f PM – primitive mant (McDonough and Sun 1995), and UCC and LCC – upper and low continental crust (Taylor and McLennan 1995) 2 2.5 3 3.5 4 4.5 5 5.5 44 46 48 50 52 54 Na2O + K2O (wt%) SiO2 (wt%) ENE to NE 2.37 Ga N to NNW 2.22 Ga NW to WNW 2.21 Ga Vempalle >1.9 Ga Tadpatri 1.9 Ga basalt basalc andesite (a) 10 11 12 13 14 15 16 4 5 6 7 8 FeOT (wt%) MgO (wt%) (b) (b) 2 ( ) Al2O3 CaO* + Na2O K2O Basalt Gabbro (c) 0.01 0.1 1 10 0.1 1 10 100 Th/Yb Nb/Yb (d) PM UCC LCC (d) (c) 0.01 0.1 1 10 0 1 1 Nb/Y Zr/Y (e) PM UCC LCC 0 1 2 3 4 5 6 0 2 4 6 8 La/Nb Ce/Y (f) PM EDC granite CDC granite LCC UCC b MgO versus total FeO, c A-CN-K plot, average gabbro and basalt (red dots) from Nesbitt and Young (1982) and Babechuk et al. (2014), d MORB-OIB array and dashed line with arrow representing AFC trend from Pearce (2008), e Plume array after Condie (2005), f Aver- age CDC and EDC anatectic granites (green dots) from Jayananda et al. (2018), dashed line with arrow shows mixing trend of primi- tive melts with average CDC/EDC granite. d-f PM – primitive mantle (McDonough and Sun 1995), and UCC and LCC – upper and lower continental crust (Taylor and McLennan 1995) Fig. 4 Bulk composition of the selected CDC/EDC and Cuddapah Basin basalts (data from Rao et al. 1995, Halls et al. 2007, and Srivastava et al. Calculation method: reverse FC model magma assimilates tonalitic crust much more efficiently if the crust is initially at 800 °C than at 200 °C. The AFC process is complicated by localized convective heat transfer and dynamic processes including melt segregation by com- paction, deformation, and buoyancy instabilities, but these local processes do not have large effects on the overall heat sharing and melting relationships (Annen and Sparks 2002). Assuming that the CDC/EDC and Cuddapah Basin primary magmas were already contaminated before crystallization during passage through lithosphere, their composition and P–T of equilibration with the metasomatized mantle were determined through simple low-pressure reverse fractional crystallization (Reverse FC) calculations (Till et al. 2012; Chatterjee and Sheth 2015; Till 2017; Krein et al. 2021) (Fig. S1). Starting from the composition of the sample, its low- pressure fractionation path was modeled backward by adding Ol + Pl + Cpx (stage 1), Ol + Pl (stage 2) and Ol-only (stage 3) in small steps (step size < 0.5%) until the melt reached its lherzolite MSP at a high pressure. Equilibrium Fe2+-Mg dis- tribution between olivine-liquid (KD(Fe2+-Mg) = 0.3, Roeder and Emslie 1970) and Cpx-liquid (KD(Fe2+-Mg) = 0.25), and equilibrium Ca-Na distribution between plagioclase-liquid (Grove et al. 1992) were maintained at each step of the cal- culation. In stage 1, the melt moved toward the Ol-Cpx side- bar in the Ol-Cpx-Qz projection following the constraints of Yang et al. (1996) that define the intersection of the Ol- Pl-Cpx cotectic with the Ol-Pl cotectic. In stage 2, the melt moved toward the olivine apex in the Ol-Cpx-Qz projection and toward the Ol-Pl sidebar in the Ol-Pl-Cpx projection. In stage 3, the melt moved toward the olivine apex in both projections. The phase proportions and the switching points between stages 1–2 and 2–3 were adjusted so that the melt moved toward its spinel lherzolite MSP (SL-MSP) at high pressures predicted by Till et al.’s (2012) parameterized expressions of experimental data. At the end of the calcula- tion, the melt was in equilibrium with Fo90 olivine, and it plotted exactly on its SL-MSP at a specific high pressure. The result was unique, as any deviation from the phase pro- portions and switching points between stages would result in a melt not on its lherzolite MSP at any pressure, though it may show equilibrium with Fo90 olivine (Fig. S1). The trace elements were modeled using the mineral-melt parti- tion coefficients listed in Table S3. Calculation method: reverse FC model Spera and Bohrson (2001) considered energy-constrained AFC (EC-AFC) within an adiabatically sealed system that can be described in terms of a set of coupled ordinary dif- ferential equations expressing conservation of energy (enthalpy), total mass, and trace element abundances and isotopic ratios. In this study, the primary magmas were mod- eled by incorporating the EC-AFC formulation of Spera and Bohrson (2001), and Bohrson and Spera (2001) in the reverse fractionation calculations (Reverse EC-AFC). As in the Reverse FC model, the low-pressure fractionation path was modeled backward by adding Ol + Pl + Cpx (stage 1), Ol + Pl (stage 2) and Ol-only (stage 3) in small steps while maintaining equilibrium Fe2+-Mg distribution between oli- vine-liquid and Cpx-liquid, and equilibrium Ca-Na distribu- tion between plagioclase-liquid. In addition, a partial melt of the upper crustal assimilant was subtracted in each reverse fractionation step as described below. The EC-AFC calculations were carried out using the updated RK07A_2011_1.xlsm spreadsheet (Spera and Bohrson 2001; Bohrson and Spera 2001). This is a forward modeling approach that requires knowledge of the initial composition and temperature of the magma (i.e., tempera- ture of the primary magma after rising to the upper crust, Tm 0), and the initial temperature of the upper crustal assim- ilant (Ta 0). These parameters for each sample were deter- mined through an iterative approach using the K content of the magma. In the first iteration, the input K content of the primary magma was calculated through the Reverse FC method. The bulk compositions of this primary magma and the sample were used to determine Tm 0 of the primary magma and Tm of the sample with Eq. 16 of Putirka (2008). The starting value of Ta 0 was 300 °C. The Ma/Mc values (ratio of mass of assimilated crustal melt to mass crystal- lized) as a function of temperature obtained from the EC- AFC forward model were used to subtract the appropriate amounts of partial melt of crustal assimilant in each step of the reverse fractionation. This yielded a tentative estimate of the primary magma considering EC-AFC, whose K con- tent, a recalculated Tm 0 (which changed slightly), and an adjusted Ta 0 were used as input in the second iteration. Sample selection 2014a for ENE- to NE-trending dikes, French and Heaman 2010, Kumar et al. 2012b, and Srivastava et al. 2014b for N- to NNW-trending dikes, Srivastava et al. 2015 for NW- to WNW- trending dikes, and Chatterjee and Bhattacharji 1998, and Anand et al. 2003 for Vempalle volcanics and Tadpatri sills within Cud- dapah Basin), a Total alkali versus silica after Le Bas et al. (1986), subalkalic-alkalic boundary from Macdonald and Katsura (1964), the contamination occurred before the primary magma crystallized. with the upper crust through an AFC-type process. However, contamination of the primitive melts during passage through lithosphere containing fusible crustal material acquired by previous subduction cannot be ruled out. In this scenario, 1 3 Contributions to Mineralogy and Petrology (2023) 178:28 28 Page 14 of 25 28 Page 14 of 25 28 Page 14 o Calculation method: reverse FC model The iterations were continued until the K contents of the sample (at Tm of the sample) and the melt in each step of the reverse fractionation matched the K contents predicted by the EC- AFC forward model (Fig. S2). The constraints of the reverse fractionation method ensured that the primary magma plot- ted on its SL-MSP and the evolving magma remained on its Calculation method: Reverse EC‑AFC model In AFC, the magma mass changes through mass gained by assimilation of partial melt of wall rock and mass lost by crystallization of the magma. The masses of crystallized and assimilated material depend on the heat budget, which is bal- anced by heat lost by the magma through cooling and crys- tallization when the temperature drops below the liquidus, and heat gained by the wall rock through heating and partial melting when the temperature exceeds the solidus. The mass and heat balance constraints are combined together in the AFC formulation (e.g. Thompson et al. 2002). The efficiency of assimilation critically depends on the initial temperature of the crust. Thompson et al. (2002) showed that a picritic 3 3 Contributions to Mineralogy and Petrology (2023) 178:28 Page 15 of 25 2 28 28 fractionation path. Although the K content was used in the modeling, the Rb contents (where available for the sample) also showed a good match with the Rb contents predicted by the EC-AFC forward model (Fig. S2). The calculations were carried out with an average CDC or EDC TTG gneiss (Jayananda et al. 2018) (Table S2), depending on sample location, as the upper crustal assimilant (Table S2). Partial melts of the assimilant were subtracted in each step using the Ma/Mc value that varies according to energy constraints (in contrast to a constant “r” ratio, DePaolo 1981) during fractionation. In the calculations, the solidus and liquidus temperatures of the upper crustal assimilant were 900 °C and 1000 °C, and the energy parameters were: crystallization enthalpy, ∆hcry = 396 kJ/kg, isobaric specific heat of magma, Cp,m = 1.484 kJ/kg per K, fusion enthalpy, ∆hfus = 270 kJ/ kg, and isobaric specific heat of assimilant, Cp,a = 1.37 kJ/ kg per K (Table 1 of Bohrson and Spera 2001). The equi- libration temperature (Teq) was assumed as 1000 °C. The gneiss-melt partition coefficients of the trace elements were estimated from their contents in average TTG gneiss and anatectic melt in Jayananda et al. (2018) (Table S2, S3). The basalt-melt elemental partition coefficients were calculated from the estimated proportions of fractionating phases and mineral-melt elemental partition coefficients (Table S2, S3). fractionated, and they were last equilibrated with spinel lherzolite at P–T of 10–12.5 kbar and 1290–1314 °C. Calculation method: Reverse EC‑AFC model The NW- to WNW-trending dike samples from the ~ 2.21 Ga old Anantapur-Kunigal swarm are 50–74% fractionated, and they were last equilibrated with spinel lherzolite at P–T of 10.5–13 kbar and 1297–1323 °C. All of these results signifi- cantly overlap considering their uncertainties (see below), and there are no systematic differences among the different swarms. It is concluded that the samples were last equili- brated with metasomatized spinel lherzolite in the P–T range of 8–13 kbar/1266–1323 °C. Reverse EC‑AFC modeling results The estimated compositions and P–T conditions of equi- librium of the primary magmas with lherzolite and the amounts of crustal contamination during fractionation for the Reverse EC-AFC models are provided in Tables 4 and S2, and shown in Figs. 5, 6, and 7. The primary magmas are high-Mg basalts and picrites with MgO contents ranging 10.7–13.2 wt%. The fractionation trends for all samples are similar. The basalts evolved by fractionating and assimilat- ing crustal melt with maximum Ma/Mc ratios between 0.16 and 0.64. Stage 1, i.e., the last stage of fractionation, was the longest during which the melts fractionated by an average of 38.1% (from 24.1 to 62.2%, Table S2). In this stage, phases were added in average proportions of Ol:Pl:Cpx = 13:49:38 that resulted in an increase in the average Mg# from 44 to 65 with concomitant decrease in SiO2 and Na2O, and increase in CaO and Al2O3 (Fig. 6, Table S2). During Stage 2 (intermediate stage), the melts fractionated by an average of 19.2% (from 4.9% to 24.1%). Phases were added in aver- age proportions of Ol:Pl = 31:69, resulting in an increase in the average Mg# from 65 to 70, decrease in SiO2, CaO and Na2O, and increase in Al2O3 (Fig. 6). During Stage 3, i.e., the earliest stage of fractionation, the primary magmas fractionated by an average of 4.9%. Only olivine was added, and the average Mg# increased from 70 to 73 as the SiO2, Al2O3, CaO and Na2O contents decreased (Fig. 6). The trace element variations of the melts are shown in Fig. 7. The compatible trace element Ni increased with increasing MgO contents (Fig. 7a). The incompatible trace elements Rb and the REE decreased with increasing MgO as also observed for TiO2 and K2O (Figs. 6b,h, 7b-d). Reverse FC modeling results 5 Ol-Pl-Cpx and Ol-Cpx-Qz pseudoternary projections from Qz and Pl showing compositions of the selected CDC/EDC and Cud- dapah Basin basalts and their primary magmas estimated through Reverse EC-AFC calculations. Also shown are the average fractiona- bulk (corrected) Primary magma MSP, PL MSP, SL Cpx40 Pl77.5 Ol42.5 9 15 21 .001 10 Cpx80 Ol75 Qz55 9 15 21 2 4 6 8 10 .001 Cpx Pl Ol Cpx Ol Qz Fig. 5 Ol-Pl-Cpx and Ol-Cpx-Qz pseudoternary projections from Qz and Pl showing compositions of the selected CDC/EDC and Cud- dapah Basin basalts and their primary magmas estimated through Reverse EC-AFC calculations. Also shown are the average fractiona- tion path (black lines, only the average is shown for clarity) and pla- gioclase lherzolite (PL) MSPs at 1 bar-10 kbar in 2 kbar intervals for the average basalt, and the spinel lherzolite (SL) MSPs at 9–21 kbar in 3 kbar intervals for its primary magma Cpx80 Ol75 Qz55 9 15 21 2 4 6 8 10 .001 Ol Cpx Ol Qz tion path (black lines, only the average is shown for clarity) and pla- gioclase lherzolite (PL) MSPs at 1 bar-10 kbar in 2 kbar intervals for the average basalt, and the spinel lherzolite (SL) MSPs at 9–21 kbar in 3 kbar intervals for its primary magma Cpx80 Ol75 Qz55 9 15 21 2 4 6 8 10 .001 Ol Cpx Ol Qz tion path (black lines, only the average is shown for clarity) and pla- gioclase lherzolite (PL) MSPs at 1 bar-10 kbar in 2 kbar intervals for the average basalt, and the spinel lherzolite (SL) MSPs at 9–21 kbar in 3 kbar intervals for its primary magma bulk (corrected) Primary magma MSP, PL MSP, SL Cpx40 Pl77.5 Ol42.5 9 15 21 .001 10 Cpx Pl O Cpx Cpx80 Cpx Cpx40 Ol Ol7 Pl7 Fig. 5 Ol-Pl-Cpx and Ol-Cpx-Qz pseudoternary projections from Qz and Pl showing compositions of the selected CDC/EDC and Cud- dapah Basin basalts and their primary magmas estimated through Reverse EC-AFC calculations. Also shown are the average fractiona- tion path (black lines, only the average is shown for clarity) and pla- gioclase lherzolite (PL) MSPs at 1 bar-10 kbar in 2 kbar intervals for the average basalt, and the spinel lherzolite (SL) MSPs at 9–21 kbar in 3 kbar intervals for its primary magma kbar and 1297–1363 °C. Reverse FC modeling results They are 58–73% fractionated and 7–30% contaminated with crust that was initially at a tem- perature of 665–808 °C, and assimilation started when the magma was at 1138–1194 °C. The N- to NNW-trending dike samples from the ~ 2.22 Ga old Kandlamadugu swarm were last equilibrated with spinel lherzolite at P–T of 13.5–15.5 kbar and 1333–1352 °C. They are 51–71% fractionated and 13–20% contaminated with crust that was initially at a tem- perature of 710–760 °C, and assimilation started when the magma was at 1147–1177 °C. The NW- to WNW-trending dike samples from the ~ 2.21 Ga old Anantapur-Kunigal swarm were last equilibrated with spinel lherzolite at P–T of 12–16.5 kbar and 1315–1366 °C. They are 48–74% fraction- ated and 5–18% contaminated with crust that was initially at a temperature of 680–778 °C, and assimilation started when the magma was at 1153–1165 °C. Reverse FC modeling results The estimated compositions and P–T conditions of equilib- rium of the primary magmas with lherzolite for the Reverse FC models are provided in Tables 4 and S2. The primary magmas are high-Mg basalts with MgO contents ranging 10–12 wt%. The fractionation trends for all samples are similar. Stage 1, i.e., the last stage of fractionation, was the longest during which the melts fractionated by an average of 39.9% (from 23.5 to 63.4%, Table S2). Phases were added in average proportions of Ol:Pl:Cpx = 12:49:39 that resulted in an increase in the average Mg# from 44 to 66 (Table S2). During Stage 2 (intermediate stage), the melts fractionated by an average of 21.2% (from 2.3 to 23.5%). Phases were added in average proportions of Ol:Pl = 29:71, resulting in an increase in the average Mg# from 66 to 71. During Stage 3, i.e., the earliest stage of fractionation, the primary mag- mas fractionated by an average of 2.3%. Only olivine was added, and the average Mg# increased from 70 to 73. The calculations show that the ~ 1.89 Ga old samples from inside the Cuddapah Basin were last equilibrated with spinel lherzolite at P–T of 10–15.5 kbar and 1291–1359 °C (Table 4, S2). They are 56–72% fractionated and 6–23% contaminated with crust that was initially at a temperature of 757–801 °C, and assimilation started when the magma was at 1159–1224 °C. The ENE- to NE-trending dike samples from the ~ 2.37 Ga old Bangalore-Karimnagar swarm were last equilibrated with spinel lherzolite at P–T of 10.5–16 The calculations show that the ~ 1.89 Ga old samples from inside the Cuddapah Basin are 57–72% fractionated, and they were last equilibrated with spinel lherzolite at P–T of 8–12 kbar and 1266–1315 °C (Table 4, S2). The ENE- to NE-trending dike samples from the ~ 2.37 Ga old Bangalore- Karimnagar swarm are 58–73% fractionated, and they were last equilibrated with spinel lherzolite at P–T of 8–12.5 kbar and 1268–1320 °C. The N- to NNW-trending dike samples from the ~ 2.22 Ga old Kandlamadugu swarm are 53–72% 1 3 Contributions to Mineralogy and Petrology (2023) 178:28 28 Page 16 of 25 28 Page 16 of 25 bulk (corrected) Primary magma MSP, PL MSP, SL Cpx40 Pl77.5 Ol42.5 9 15 21 .001 10 Cpx Pl Fig. Discussion of multiple saturation are larger if the primary magma is allowed to equilibrate with olivine compositions between Fo88 and Fo92. In this case, the average uncertainties in pres- sure and temperature are ± 15% and ± 3%, respectively (e.g., 15 ± 2.3 kbar, 1350 ± 40 °C), and the average uncertainty in the estimated crustal assimilation is ± 20% (e.g., 10 ± 2%). Uncertainties The uncertainties in the estimated compositions and P–T of primary magmas arise from the uncertainties in the equi- librium mineral-melt KD(Fe2+-Mg) coefficients, and the mantle olivine composition that may vary between Fo88 and Fo92. If the primary magma is allowed to equilibrate with olivine compositions between Fo88 and Fo92 using fixed KD(Fe2+-Mg) values, the uncertainty in the Mg# of the pri- mary magma is ± 6% (Mg# range: 68.5–77.5), and the uncer- tainties in the MgO and FeO contents of the primary magma are ± 12% and ± 10% (represented by error bars in Figs. 5–7). These uncertainties are larger than the uncertainties calcu- lated by changing the KD(Fe2+-Mg) values. For example, a ± 10% change in the Ol-melt and Cpx-melt KD(Fe2+-Mg) values results in a ± 3% change in MgO and ± 6% change in FeO of the primary magma for the most fractionated (73%) sample EDD09/14. For less fractionated samples such as GD24 (51% fractionated), a ± 10% change in the Ol-melt and Cpx-melt KD(Fe2+-Mg) values results in a ± 2% change in MgO and ± 3% change in FeO of the primary magma. Using higher values of KD(Fe2+-Mg) results in an increase in FeO and decrease in MgO, and the primary magma (Mg# 71.2–71.9) equilibrates with Fo88.2–88.6 olivine. Using lower values of KD(Fe2+-Mg) results in a decrease in FeO and increase in MgO, and the primary magma (Mg# 74.7–74.0) equilibrates with Fo91.6–91.3 olivine. All of these results significantly overlap considering their uncertainties (see below), and there are no system- atic differences among the different swarms. Considering all samples, the P–T range for last equilibration with spi- nel lherzolite was 10–16.5 kbar/1291–1366 °C. These P–T conditions are higher than the P–T estimated through the Reverse FC models. The samples are mostly ≤ 20% con- taminated except for three samples, one from the Vempalle volcanics of Cuddapah Basin and two from the ENE- to NE- trending ~ 2.37 Ga old Bangalore-Karimnagar dikes, that are 23–30% contaminated. The upper crust was already at high temperatures (665–808 °C) at the time the dikes, sills and volcanics were emplaced. Multiple regression of lherzolite saturated melt composi- tions in experiments indicate that the P–T values at SL-MSP are accurate within ± 1.5 kbar and ± 11 °C (Till et al. 2012; Krein et al. 2021). Uncertainties However, the uncertainties in the P–T 1 3 Contributions to Mineralogy and Petrology (2023) 178:28 Page 17 of 25 28 Page 17 of 25 28 Contributions to Mineralogy and Petrology (2023) 178:28 Page 17 of 25 28 of multiple saturation are larger if the primary magma is allowed to equilibrate with olivine compositions between Fo88 and Fo92. In this case, the average uncertainties in pres- sure and temperature are ± 15% and ± 3%, respectively (e.g., Discussion Crustal contamination 6 8 10 12 14 FeO (wt%) (d) 9 10 11 12 13 CaO (wt%) (e) 1.0 1.5 2.0 2.5 3.0 3 5 7 9 11 13 15 Na2O (wt%) MgO (wt%) (g) 45 47 49 51 53 SiO2 (wt%) (a) 13 14 15 16 17 18 Al2O3 (wt%) (c) 0.5 1.0 1.5 2.0 2.5 TiO2 (wt%) bulk, corrected First Cpx First Pl Primary magma (b) 0.0 0.5 1.0 3 5 7 9 11 13 15 K2O (wt%) MgO (wt%) (h) 0.6 0.7 0.8 0.9 CaO/Al2O3 (f) Fig. 6 Bivariate plots showing the variation of major oxides and CaO/Al2O3 with MgO for the selected CDC/EDC and Cuddapah Basin basalts and their parental and primary magmas estimated through Reverse EC-AFC calculations. The fractionation path of each basalt is distinct but similar. For clarity, only an average fractionation path (black lines) is shown. Melts labeled “First Pl” and “First Cpx” correspond to initiation of plagioclase and Cpx crystallization (a) (d) (c) 0.6 0.7 0.8 0.9 CaO/Al2O3 (f) (f) (e) 0.0 0.5 1.0 3 5 7 9 11 13 15 K2O (wt%) MgO (wt%) (h) 1.0 1.5 2.0 2.5 3.0 3 5 7 9 11 13 15 Na2O (wt%) MgO (wt%) (g) (g) (h) 15 Fig. 6 Bivariate plots showing the variation of major oxides and CaO/Al2O3 with MgO for the selected CDC/EDC and Cuddapah Basin basalts and their parental and primary magmas estimated through Reverse EC-AFC calculations. The fractionation path of each basalt is distinct but similar. For clarity, only an average fractionation path (black lines) is shown. Melts labeled “First Pl” and “First Cpx” correspond to initiation of plagioclase and Cpx crystallization Crustal contamination Thus, the crust was at high temperatures ~ 65 Myr (at least ~ 10 Myr considering uncertainties) before the emplacement of the ~ 2.37 Ga old dikes. The CDC/EDC crust was also thermally primed by the intrusion of the Ippaguda-Dhiburahalli swarm (~ 2.25 Ga) ~ 5 Myr before the emplacement of the Kandlamadugu swarm (~ 2.22 Ga), and the Anantpur-Kunigal swarm (~ 2.21 Ga) was emplaced another ~ 10 Myr later. The prevailing high temperatures of the crust is evident from the presence of anatectic granites such as the 2221 ± 99 Ma old Yelagatti granitoid in northern EDC (Rogers et al. 2007). The EDC crust was also probably thermally primed by the intrusion of the Devarabanda swarm at ~ 2.08 Ga before the eruption of the Vempalle volcanics (> 1.89 Ga) and intrusion of the Tadpatri sills (~ 1.89 Ga) within Cuddapah Basin. EDC dikes also show 5–30% upper crustal contamination. An important result of the Reverse EC-AFC modeling is that the upper crust was already heated to high tempera- tures (665–808 °C) during emplacement of the various dike swarms. Such thermal priming of the crust has been attrib- uted to persistent magmatism for the Steens basalts in the Columbia River flood basalt province (Moore et al. 2018, 2020). According to field observations, the ~ 2.37 Ga old Bangalore-Karimnagar dikes cross-cut an older set of mafic dikes (Padmakumari and Dayal 1987; Kumar and Bhalla 1983). The CDC crust was probably pre-heated to high tem- peratures by the intrusion of the older dikes. Furthermore, there is evidence of a major thermal pulse at ~ 2.5 Ga fol- lowed by slow cooling to ~ 2.4 Ga in the CDC/EDC (Jay- ananda et al. 2011, 2013b; Peucat et al. 2013). For example, in the southern part of CDC, garnet dated at 2439 ± 36 Ma and 2435 ± 56 Ma in metapelite and calc-silicate gneiss grew under granulite facies conditions (Jayananda et al. 2013b). Thus, the crust was at high temperatures ~ 65 Myr (at least ~ 10 Myr considering uncertainties) before the emplacement of the ~ 2.37 Ga old dikes. The CDC/EDC crust was also thermally primed by the intrusion of the Ippaguda-Dhiburahalli swarm (~ 2.25 Ga) ~ 5 Myr before Crustal contamination Application of EC-AFC in the reverse fractionation mod- eling shows that the amount of upper crustal contamination in the Cuddapah Basin basalts is 6–23% (Table 4). This is in good agreement with the model mixing of 10–35% gra- nitic crust with the Cuddapah Basin primary melts based on 1 3 3 Contributions to Mineralogy and Petrology (2023) 178:28 28 Page 18 of 25 28 Page 18 of 25 0 200 400 600 800 3 5 7 9 11 13 15 Ni (ppm) MgO (wt%) bulk, corrected First Cpx First Pl Primary magma (a) 0 5 10 15 20 25 3 5 7 9 11 13 15 La (ppm) MgO (wt%) (c) 0 10 20 30 40 50 60 3 5 7 9 11 13 15 Rb (ppm) MgO (wt%) (b) 0 10 20 30 40 50 3 5 7 9 11 13 15 Ce (ppm) MgO (wt%) (d) Fig. 7 Bivariate plots showing the variation of trace elements with MgO for the selected CDC/EDC and Cuddapah Basin basalts and their parental and primary magmas estimated through Reverse EC- AFC calculations. The fractionation path of each basalt is distinct but similar. For clarity, only an average fractionation path (black lines) is shown. Melts labeled “First Pl” and “First Cpx” correspond to initia- tion of plagioclase and Cpx crystallization 0 10 20 30 40 50 60 3 5 7 9 11 13 15 Rb (ppm) MgO (wt%) (b) 0 10 20 30 40 50 3 5 7 9 11 13 15 Ce (ppm) MgO (wt%) (d) similar. For clarity, only an average fractionation path (black lines) is shown. Melts labeled “First Pl” and “First Cpx” correspond to initia- tion of plagioclase and Cpx crystallization 0 10 20 30 40 50 60 3 5 7 9 11 13 15 Rb (ppm) MgO (wt%) (b) 0 10 20 30 40 50 3 5 7 9 11 13 15 Ce (ppm) MgO (wt%) (d) similar. For clarity, only an average fractionation path (black lines) is shown. Crustal contamination Melts labeled “First Pl” and “First Cpx” correspond to initia- tion of plagioclase and Cpx crystallization 0 10 20 30 40 50 60 3 5 7 9 11 13 15 Rb (ppm) MgO (wt%) (b) 0 200 400 600 800 3 5 7 9 11 13 15 Ni (ppm) MgO (wt%) bulk, corrected First Cpx First Pl Primary magma (a) 0 10 20 30 40 50 3 5 7 9 11 13 15 Ce (ppm) MgO (wt%) (d) 0 5 10 15 20 25 3 5 7 9 11 13 15 La (ppm) MgO (wt%) (c) (c) (d) Fig. 7 Bivariate plots showing the variation of trace elements with MgO for the selected CDC/EDC and Cuddapah Basin basalts and their parental and primary magmas estimated through Reverse EC- AFC calculations. The fractionation path of each basalt is distinct but similar. For clarity, only an average fractionation path (black lines) is shown. Melts labeled “First Pl” and “First Cpx” correspond to initia- tion of plagioclase and Cpx crystallization La/Nb and Ce/Y ratios by Anand et al. (2003). The CDC/ EDC dikes also show 5–30% upper crustal contamination. La/Nb and Ce/Y ratios by Anand et al. (2003). The CDC/ EDC dikes also show 5–30% upper crustal contamination. An important result of the Reverse EC-AFC modeling is that the upper crust was already heated to high tempera- tures (665–808 °C) during emplacement of the various dike swarms. Such thermal priming of the crust has been attrib- uted to persistent magmatism for the Steens basalts in the Columbia River flood basalt province (Moore et al. 2018, 2020). According to field observations, the ~ 2.37 Ga old Bangalore-Karimnagar dikes cross-cut an older set of mafic dikes (Padmakumari and Dayal 1987; Kumar and Bhalla 1983). The CDC crust was probably pre-heated to high tem- peratures by the intrusion of the older dikes. Furthermore, there is evidence of a major thermal pulse at ~ 2.5 Ga fol- lowed by slow cooling to ~ 2.4 Ga in the CDC/EDC (Jay- ananda et al. 2011, 2013b; Peucat et al. 2013). For example, in the southern part of CDC, garnet dated at 2439 ± 36 Ma and 2435 ± 56 Ma in metapelite and calc-silicate gneiss grew under granulite facies conditions (Jayananda et al. 2013b). Potential temperatures The NW- to WNW-trending dike samples from the ~ 2.21 Ga old Anan- tapur-Kunigal swarm show a restricted range of Na2O and CaO/Al2O3, indicating 15–20% batch melting at pressures between ~ 15 kbar and > 20 kbar. Most of the ENE- to NE- trending dike samples from the ~ 2.37 Ga old Bangalore- Karimnagar swarm are similar to the NW- to WNW-trending Anantapur-Kunigal dike samples. Hence, their pressure and degree of batch melting are probably similar. The Cuddapah Basin samples show a negative correlation between FeO and Na2O, and positive correlations between SiO2 and Na2O, and between FeO and CaO/Al2O3. These trends also sug- gest 10% to > 20% batch melting at pressures between ~ 12 kbar and > 20 kbar. However, all CDC/EDC and Cuddapah Basin primary magmas estimated above probably represent pooled melts as their average compositions are similar to a pooled melt generated by polybaric incremental melting at pressures between 26 and 9 kbar along a 1450 °C adiabat (Behn and Grove 2015). Assuming ~ 10–20% melting, TP may be ~ 60–130 °C higher than the estimated TP for nar- row to fully pooled melts (Table S2 of Krein et al. 2021). Thus, the range of TP may be 1293–1515 °C. The estimated TP values of all samples are similar to pre- dicted ambient mantle temperatures in the Paleoproterozoic according to various thermal history models of the Earth (see Fig. 10 of Herzberg 2022). Even the highest estimated TP (1515 °C) is not higher than the Paleoproterozoic ambi- ent mantle temperature predicted by the model of Korenaga 1 1.25 1.50 1.75 2.00 2.25 2.50 6 7 8 9 Na2O (wt%) FeO (wt%) (a) 10% 20% 15% 20 kb 15 kb 0.65 0.70 0.75 0.80 6 7 8 9 CaO/Al2O3 FeO (wt%) ENE to NE 2.37 Ga N to NNW 2.22 Ga NW to WNW 2.21 Ga Vempalle >1.9 Ga Tadpatri 1.9 Ga (c) 10% 20% 15% 1.25 1.50 1.75 2.00 2.25 2.50 46 47 48 49 50 Na2O (wt%) SiO2 (wt%) (b) 10% 20% 15% Fig. 8 Compositions of the CDC/EDC and Cuddapah Basin primary magmas compared with model melts of spinel lherzolite (Behn and Grove 2015). Potential temperatures The apparent potential temperatures (TP*, Krein et al. 2021) for the origin of the CDC/EDC and Cuddapah Basin pri- mary magmas, estimated from the P–T of multiple satura- tion with spinel lherzolite with the Reverse EC-AFC models (10–16.5 ± 2.3 kbar, 1291–1366 ± 40 °C) and an adiaba- tic slope (dT/dP) of 1.5 °C/kbar, are 1233–1385 °C. The Reverse FC models indicate lower P–T of multiple satu- ration with spinel lherzolite, reflecting equilibrium with a metasomatized mantle. The Reverse FC results were not used to estimate potential temperatures. The true poten- tial temperature (TP) depends on the style and degree of 1 3 3 Page 19 of 25 Contributions to Mineralogy and Petrology (2023) 178:28 5 28 28 melting (Krein et al. 2021). To assess the approximate degree of melting, the compositions of the calculated CDC/ EDC and Cuddapah Basin primary magmas based on the Reverse EC-AFC models are compared with model melts of spinel lherzolite (Behn and Grove 2015) (Fig. 8). The N- to NNW-trending dike samples from the ~ 2.22 Ga old Kandlamadugu swarm show a restricted range of FeO and SiO2, indicating ~ 10–20% melting at 15–20 kbar pressure according to the isobaric batch melting models. The NW- to WNW-trending dike samples from the ~ 2.21 Ga old Anan- tapur-Kunigal swarm show a restricted range of Na2O and CaO/Al2O3, indicating 15–20% batch melting at pressures between ~ 15 kbar and > 20 kbar. Most of the ENE- to NE- trending dike samples from the ~ 2.37 Ga old Bangalore- Karimnagar swarm are similar to the NW- to WNW-trending Anantapur-Kunigal dike samples. Hence, their pressure and degree of batch melting are probably similar. The Cuddapah Basin samples show a negative correlation between FeO and melting (Krein et al. 2021). To assess the approximate degree of melting, the compositions of the calculated CDC/ EDC and Cuddapah Basin primary magmas based on the Reverse EC-AFC models are compared with model melts of spinel lherzolite (Behn and Grove 2015) (Fig. 8). The N- to NNW-trending dike samples from the ~ 2.22 Ga old Kandlamadugu swarm show a restricted range of FeO and SiO2, indicating ~ 10–20% melting at 15–20 kbar pressure according to the isobaric batch melting models. Potential temperatures Dashed lines: isobaric batch melts at 10, 15 and 20 kbar; red dot in a and b: pooled polybaric incremental melt generated along a 1450 °C adiabat 1.25 1.50 1.75 2.00 2.25 2.50 46 47 48 49 50 Na2O (wt%) SiO2 (wt%) (b) 10% 20% 15% 1.25 1.50 1.75 2.00 2.25 2.50 6 7 8 9 Na2O (wt%) FeO (wt%) (a) 10% 20% 15% 20 kb 15 kb (a) 9 0.65 0.70 0.75 0.80 6 7 8 9 CaO/Al2O3 FeO (wt%) (c) 10% 20% 15% (c) kbar; red dot in a and b: pooled polybaric incremental melt generated along a 1450 °C adiabat Fig. 8 Compositions of the CDC/EDC and Cuddapah Basin primary magmas compared with model melts of spinel lherzolite (Behn and Grove 2015). Dashed lines: isobaric batch melts at 10, 15 and 20 Contributions to Mineralogy and Petrology (2023) 178:28 28 Page 20 of 25 28 Page 20 of 25 28 Page 20 Contributions to Mineralogy and Petrology (2023) 17 28 Page 20 of 25 1 10 100 La Ce Pr Nd Sm Eu Gd Tb Dy Ho Er Tm Yb ENE to NE ~2.37 Ga (a) 1 10 100 La Ce Pr Nd Sm Eu Gd Tb Dy Ho Er Tm Yb ENE to NE ~2.37 Ga Primary magma 1 10 100 La Ce Pr Nd Sm Eu Gd Tb Dy Ho Er Tm Yb N to NNW ~2.22 Ga (b) 1 10 100 La Ce Pr Nd Sm Eu Gd Tb Dy Ho Er Tm Yb N to NNW ~2.22 Ga Primary magma 1 10 100 La Ce Pr Nd Sm Eu Gd Tb Dy Ho Er Tm Yb NW to WNW ~2.21 Ga (c) 1 10 100 La Ce Pr Nd Sm Eu Gd Tb Dy Ho Er Tm Yb NW to WNW ~2.21 Ga Primary magma 1 10 100 La Ce Pr Nd Sm Eu Gd Tb Dy Ho Er Tm Yb Cuddapah Basin ~1.89 Ga (d) 1 10 100 La Ce Pr Nd Sm Eu Gd Tb Dy Ho Er Tm Yb Cuddapah Basin ~1.89 Ga Primary magma Fig. 9 Chondrite-normalized (McDonough and Sun 1995) REE pat- terns of basalts and their primary magmas (Reverse EC-AFC models) from a ENE- to E-trending, b N- to NNW-trending, and c NW- to WNW-trending dikes of the CDC/EDC, and d sills and flows w the Cuddapah Basin. The range for the Mackenzie dikes (Bar et al. Potential temperatures 1996) is shown by two dashed lines 1 10 100 La Ce Pr Nd Sm Eu Gd Tb Dy Ho Er Tm Yb ENE to NE ~2.37 Ga (a) 1 10 100 La Ce Pr Nd Sm Eu Gd Tb Dy Ho Er Tm Yb ENE to NE ~2.37 Ga Primary magma ENE to NE ~2.37 Ga Primary magma Sm Eu Gd Tb Dy Ho Er Tm Yb 1 10 100 La Ce Pr Nd Sm Eu Gd Tb Dy Ho Er Tm Yb N to NNW ~2.22 Ga (b) 1 10 100 La Ce Pr Nd Sm Eu Gd Tb Dy Ho Er Tm Yb N to NNW ~2.22 Ga Primary magma (b) 10 1 10 100 La Ce Pr Nd Sm Eu Gd Tb Dy Ho Er Tm Yb NW to WNW ~2.21 Ga (c) 1 10 100 La Ce Pr Nd Sm Eu Gd Tb Dy Ho Er Tm Yb NW to WNW ~2.21 Ga Primary magma (c) 100 Sm Eu Gd Tb Dy Ho Er Tm Yb La Ce Pr Nd Sm Eu Gd Tb Dy Ho Er Tm Yb 1 10 100 La Ce Pr Nd Sm Eu Gd Tb Dy Ho Er Tm Yb Cuddapah Basin ~1.89 Ga (d) 1 10 100 La Ce Pr Nd Sm Eu Gd Tb Dy Ho Er Tm Yb Cuddapah Basin ~1.89 Ga Primary magma 1 10 100 La Ce Pr Nd Sm Eu Gd Tb Dy Ho Er Tm Yb Cuddapah Basin ~1.89 Ga Primary magma WNW-trending dikes of the CDC/EDC, and d sills and flows within the Cuddapah Basin. The range for the Mackenzie dikes (Baragar et al. 1996) is shown by two dashed lines (d) 100 Cuddapah Basin ~1.89 Ga Primary magma Fig. 9 Chondrite-normalized (McDonough and Sun 1995) REE pat- terns of basalts and their primary magmas (Reverse EC-AFC models) from a ENE- to E-trending, b N- to NNW-trending, and c NW- to WNW-trending dikes of the CDC/EDC, and d sills and flows within the Cuddapah Basin. The range for the Mackenzie dikes (Baragar et al. 1996) is shown by two dashed lines are evidently derived from a mid-Proterozoic mantle plume (Ernst and Baragar 1992; Baragar et al. 1996). (2008). Thus, there is no indication of a mantle plume based on the above estimates of TP. Potential temperatures However, magma may lose heat during its passage through the lithosphere, especially in dike swarms where lateral flow may be important (Ernst and Baragar 1992; Ernst et al. 2019). In this case, the above TP estimates may be lower than the actual TP. To further assess the origin of the CDC/EDC and Cuddapah Basin basalts, their REE patterns are compared in the following section with the Mackenzie dikes of Canadian Shield that (2008). Thus, there is no indication of a mantle plume based on the above estimates of TP. However, magma may lose heat during its passage through the lithosphere, especially in dike swarms where lateral flow may be important (Ernst and Baragar 1992; Ernst et al. 2019). In this case, the above TP estimates may be lower than the actual TP. To further assess the origin of the CDC/EDC and Cuddapah Basin basalts, their REE patterns are compared in the following section with the Mackenzie dikes of Canadian Shield that Plume origin? But when they are corrected for fractionation and upper crus- tal assimilation (primary magmas with Reverse EC-AFC, Table S2), their LaN/SmN ratios decrease while their DyN/ YbN ratios remain similar (Figs. 9, 10, S3). High LaN/SmN ratios of basalts may result from upper crustal contamination or through clinopyroxene-dominated fractional crystalliza- tion occurring at high pressures. Since the thermobarometric calculations do not show evidence of high-pressure crystal- lization, the elevated LaN/SmN ratios of the CDC/EDC and Cuddapah Basin basalts are probably due to upper crustal contamination. Fig. 10 Bivariate plot of chondrite-normalized (McDonough and Sun 1995) REE ratios for the CDC/EDC dikes, Cuddapah Basin sills and flows, and Mackenzie dikes (Baragar et al. 1996). The filled symbols represent primary magmas (Reverse EC-AFC models) basalts (Fig. 10). Thus, there is no indication from the REE data of a plume-related origin for any of the CDC/EDC and Cuddapah Basin basalts. The non-plume origin for all CDC/EDC dikes and Cud- dapah Basin sills and volcanics agrees with the studies of Anand et al. (2003), Sheppard et al. (2017), Shellnutt et al. (2018), and Söderlund et al. (2019), whose conclusions are based on petrological modeling, and geological, isotopic, trace element, and field structural data. The ~ 1300–1500 °C potential temperatures estimated in this study (Reverse EC- AFC models) and Anand et al. (2003) are lower than the estimated temperatures (> 1575 °C) for the late Archean komatiitic amphibolites from Kolar schist belt (CDC-EDC boundary, Rajamani et al. 1985) that may have originated from a mantle plume. Herzberg (2022) also concluded that Archean and Paleoproterozoic komatiites with temperatures of ~ 1700 °C may have originated from mantle plumes, but the ~ 1550 °C temperatures estimated for the ~ 1.88 Ga tholeiitic intrusives of the Circum-Superior LIP indicate that a plume was not involved in their origin. Dike emplacement is controlled by factors such as crustal stress, crustal hetero- geneity, magma viscosity and intrusion rates (Rivalta et al. 2015; Kjøll et al. 2019). A discussion of these mechanisms is beyond the scope this paper. Compared to the CDC/EDC and Cuddapah Basin basalts, the DyN/YbN ratios of the Mackenzie basalts are distinctly higher (Fig. 10), indicating a deeper origin from a garnet- bearing source (Baragar et al. 1996). Plume origin? The Mackenzie dikes are composed of subalkaline tholei- itic basalts with Mg# ranging 41–50 (averages in Table 6 of Baragar et al. 1996), similar to the CDC/EDC and Cud- dapah Basin basalts considered above (Mg# 37–55). They 1 3 Contributions to Mineralogy and Petrology (2023) 178:28 Page 21 of 25 28 Page 21 of 25 28 28 0 0.5 1 1.5 2 2.5 3 3.5 0.9 1.1 1.3 1.5 LaN/SmN DyN/YbN ENE to NE 2.37 Ga N to NNW 2.22 Ga NW to WNW 2.21 Ga Cuddupah ~1.89 Ga Mackenzie dikes Fig. 10 Bivariate plot of chondrite-normalized (McDonough and Sun 1995) REE ratios for the CDC/EDC dikes, Cuddapah Basin sills and flows, and Mackenzie dikes (Baragar et al. 1996). The filled symbols represent primary magmas (Reverse EC-AFC models) also consist of plagioclase, pyroxene, and Fe-Ti oxides with rare olivine, and their estimated P–T of emplacement are ≤ 5 kbar/ ~ 1025–1225 °C (Baragar et al. 1996). Thus, the major element compositions and emplacement conditions of the Mackenzie dikes are similar to the CDC/EDC dikes and Cuddapah Basin sills. 0 0.5 1 1.5 2 2.5 3 3.5 0.9 1.1 1.3 1.5 LaN/SmN DyN/YbN ENE to NE 2.37 Ga N to NNW 2.22 Ga NW to WNW 2.21 Ga Cuddupah ~1.89 Ga Mackenzie dikes p The incompatible trace element patterns of the Macken- zie basalts show LILE enrichment relative to HFSE, LREE enrichment relative to HREE, enriched Nb-Ce plateaus, and negative anomalies for Sr, Ti and K (Baragar et al. 1996). Their εNd(t) values are mostly between 0 and + 2, indicat- ing little contamination with the continental crust. By com- parison, all CDC/EDC and Cuddapah Basin basalts show prominent negative anomalies for Nb, but not for K (Sriv- astava et al. 2015), and they show evidence of upper crustal contamination in incompatible trace element ratio diagrams (Fig. 4d, e, f). Their εNd(t) values (–10 to + 1) also suggest variable degrees of contamination (Anand et al 2003; Kumar et al. 2012b; Liao et al. 2019). Compared to the Mackenzie basalts, the REE abundances of the CDC/EDC and Cud- dapah Basin basalts are lower, but they also show LREE enrichment relative to HREE (Figs. 9, S3). When the basalts are corrected only for fractionation (primary magmas with Reverse FC, Table S2), their chondrite-normalized LaN/SmN and DyN/YbN ratios remain largely unchanged (Fig. S3). Plume origin? By contrast, the lower DyN/YbN ratios of all CDC/EDC and Cuddapah Basin basalts and their primary magmas, and the flat REE patterns of most of the primary magmas (Reverse EC-AFC models) (Figs. 9, S3) indicate melting of a primitive spinel lherzo- lite source (see modeling by Shellnutt et al. 2018). Some of the primary magmas even show LaN/SmN < 1 (Fig. 10), indicating the possibility of a depleted source. This does not preclude origin from a plume because plumes are chemi- cally heterogeneous, often containing both enriched and depleted components (White 2010). However, neither the CDC/EDC and Cuddapah Basin basalts nor their primary magmas exhibit a deep melting signature such as the high DyN/YbN ratios observed in the plume-derived Mackenzie Conclusions Mineral thermobarometry indicates that the CDC/EDC dikes around Cuddapah Basin crystallized at upper crus- tal P–T conditions of ~ 1–6 kbar and ~ 1120–1210 °C. Spi- nel and ilmenite equilibrated at subsolidus temperatures 1 3 Contributions to Mineralogy and Petrology (2023) 178:28 Page 22 of 25 28 comments and editorial handling by Othmar Müntener also helped to improve the presentation of the manuscript. (~ 460–660 °C) and oxygen fugacity values below the fayalite-magnetite-quartz buffer (∆FMQ values of -0.5 to -3) that correspond to bulk Fe3+/∑Fe ratios of 0.06–0.16. Thirty selected CDC/EDC and Cuddapah Basin basalts of different ages from the literature (with minor correction in bulk com- position for crystal accumulation) plot on their Ol-Pl-Cpx cotectic boundaries at 1 bar-6.5 kbar pressures, consistent with the pressure estimates from mineral thermobarometry. Primary magmas modeled through reverse fractionation calculations incorporating crustal assimilation (Reverse EC-AFC) show that they were last equilibrated with spinel lherzolite at P–T conditions of 10–16.5 kbar (± 2.3 kbar) and 1291–1366 °C (± 40 °C). Considering EC-AFC, the models show that the basalts are mostly ≤ 20% contaminated with the upper crust except for three samples that are 23–30% contaminated. The upper crust was thermally primed to high temperatures (665–808 °C) at the time of emplacement of the different dike swarms. A comparison with model melts of spinel lherzolite (Behn and Grove 2015) shows that basalts can be generated by ~ 10% to > 20% batch melting at ~ 12–25 kbar pressures. The estimated primary magmas all basalts probably represent polybaric incremental pooled melts generated along a ~ 1450 °C adiabat. The estimated range of mantle potential temperatures is 1293–1515 °C. These potential temperatures are not higher than ambient mantle temperatures in the Paleoproterozoic according to different thermal history models of the Earth, and are incon- sistent with an origin of the basalts from mantle plumes. The incompatible element and REE patterns of the basalts are distinct from the plume-derived mid-Proterozoic Mac- kenzie basalts of the Canadian Shield, the latter showing higher chondrite-normalized DyN/YbN ratios indicative of melting of a garnet-bearing mantle source. By contrast, the lower DyN/YbN ratios of all CDC/EDC and Cuddapah Basin basalts and flat REE patterns of their primary magmas indi- cate origin from a shallower, spinel-bearing mantle source. Conclusions The basalts show LREE enrichment over HREE, and their LaN/SmN ratios are higher than the LaN/SmN ratios of the primary magmas, which can be attributed to upper crustal contamination. The estimated low potential temperatures, melting in the spinel lherzolite stability field, and the REE characteristics of the basalts and their primary magmas do not support an origin of the CDC/EDC and Cuddapah Basin basalts from mantle plumes. Funding Open Access funding provided by the MIT Libraries. Funding Open Access funding provided by the MIT Libraries. Data availability All data are available in the tables of the main text and in the electronic supplementary material. Data availability All data are available in the tables of the main text and in the electronic supplementary material. Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, 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/. References In: Saha AK (ed.) Geological evolution of Peninsular India - petrological and structural aspects, Recent Researches in Geology. 13:1–15 Ernst RE, Srivastava RK (2008) India’s place in the Proterozoic world: constraints from the Large Igneous Province (LIP) record. In: Sivaji Ch, Chalapathi Rao NV (eds) Srivastava RK. Geochem, Geophys Geochron, Narosa Publ House Pvt Ltd, New Delhi, India pp, pp 41–56 gy Bindeman IN, Davis AM, Drake MJ (1998) Ion microprobe study of plagioclase-basalt partition experiments at natural concen- tration levels of trace elements. Geochim Cosmochim Acta 62(7):1175–1193 Ernst RE, Liikane DA, Jowitt SM, Buchan KL, Blanchard JA (2019) A new plumbing system framework for mantle plume-related continental large igneous provinces and their mafic-ultramafic intrusions. J Volcanol Geotherm Res 384:75–84 Blundy J, Melekhova E, Ziberna L, Humphreys MCS, Cerantola V, Brooker RA, McCammon CA, Pichavant M, Ulmer P (2020) Effect of redox on Fe–Mg–Mn exchange between olivine and melt and an oxybarometer for basalts. Contrib Mineral Pet 175:103 French JE, Heaman LM (2010) Precise U-Pb dating of Paleoprotero- zoic mafic dyke swarms of the Dharwar craton, India: implica- tions for the existence of the Neoarchean supercraton Sclavia. Precamb Res 183:416–441 French JE, Heaman LM, Chacko T, Srivastava RK (2008) 1891–1883 Ma Southern Bastar-Cuddapah mafic igneous events, India: a newly recognised large igneous province. Precamb Res 160:308–322 Bohrson WA, Spera FJ (2001) Energy-constrained open-system mag- matic processes II: Application of energy-constrained assimi- lation-fractional crystallization (EC-AFC) model to magmatic systems. J Petrol 42:1019–1041 Friend CRL, Nutman AP (1991) SHRIMP U-Pb geochronology of the Closepet granite and peninsular gneiss, Karnataka, South India. J Geol Soc India 38:357–368fi y Chadwick B, Vasudev VN, Hegde GV (2000) The Dharwar craton, southern India, interpreted as the result of late Archean oblique convergence. Precamb Res 99:91–111 GERM (2022) Geochemical earth reference model partition coefficient (Kd) database. Earth Ref. org. https://kdd.earthref.org/KdD/ Chardon D, Jayananda M, Peucat J-J (2011) Lateral constrictional flow of hot orogenic crust: insights from the Neoarchean of south India, geological and geophysical implications for oro- genic plateaux. Geochem Geophys Geosyst 12:Q02005 Ghosh JG, DeWit MJ, Zartman RE (2004) Age and tectonic evolution of Neoproterozoic ductile shear zones in the Southern Granulite Terrain of India, with implications for Gondwana studies. Tec- tonics 23(3):1–38. https://doi.org/10.1029/2002TC001444 Chatterjee N (2021) Origin of the primitive, strongly SiO2-undersaturated alkalic rocks from the Deccan Traps by low-degree mantle melting and high-pressure fractional crys- tallization. References Aigner-Torres M, Blundy J, Ulmer P, Pettke T (2007) Laser Ablation ICPMS study of trace element partitioning between plagioclase and basaltic melts: an experimental approach. Contrib Mineral Petrol 153:647–667 Anand M, Gibson SA, Subbarao KV, Kelly SP, Dickin AP (2003) Early Proterozoic melt generation processes beneath the intrac- ratonic Cuddapah Basin, southern India. J Petrol 44:2139–2171 p Andersen DJ, Lindsley DH (1988) Internally consistent solution models for Fe-Mg-Mn-Ti spinels: Fe-Ti oxides. Amer Mineral 73:714–726f Annen C, Sparks RSJ (2002) Effects of repetitive emplacement of basaltic intrusions on thermal evolution and melt generation in the crust. Earth Planet Sci Lett 203:937–955 Armstrong JT (1995) CITZAF–a package for correction programs for the quantitative electron microbeam x-ray analysis of thick polished materials, thin-films and particles. Microbeam Anal 4:177–200 Babechuk MG, Widdowson M, Kamber BS (2014) Quantifying chemical weathering intensity and trace element release from two contrasting basalt profiles, Deccan Traps, India. Chem Geol 363:56–75 Baragar WRA, Ernst RE, Hulbert L, Peterson T (1996) Longitudinal petrochemical variation in the Mackenzie dyke swarm, north- western Canadian Shield. J Petrol 37:317–359fi Beattie P, Ford C, Russell D (1991) Partition coefficients for olivine- melt and orthopyroxene-melt systems. Contrib Mineral Petrol 109:212–224 Behn MD, Grove TL (2015) Melting systematics in mid-ocean ridge basalts: application of a plagioclase-spinel melting model to global variations in major element chemistry and crustal thick- ness. J Geophys Res Solid Earth 120:4863–4886. https://doi. org/10.1002/2015JB011885 Supplementary Information The online version contains supplemen- tary material available at https://doi.org/10.1007/s00410-023-02012-0. Belica ME, Piispa EJ, Meert JG, Pesonen LJ, Plado J, Pandit MK, Kamenov GD, Celestino M (2014) Paleoproterozoic mafic dyke swarms from the Dharwar craton; paleomagnetic poles for India from 2.37 to 1.88 Ga and rethinking the Columbia supercontinent. Precamb Res 244:100–122 Acknowledgements This study is dedicated to late Prof. Somdev Bhattacharji who graciously provided the dike samples for this study. The samples were collected during a UNDP sponsored field trip. This manuscript greatly benefitted from detailed critical reviews by Wendy Bohrson, Richard Ernst and an anonymous reviewer. The construc- tive comments of the reviewers are greatly appreciated. Thoughtful Bhattacharji S, Singh RN (1984) Thermomechanical structure of the southern part of the Indian shield and its relevance to Precam- brian basin evolution. Tectonophys 105:103–120 1 3 Contributions to Mineralogy and Petrology (2023) 178:28 Page 23 of 25 28 Bhattacharji S (1987) Lineaments and igneous episodes in the evolu- tion of intracratonic Proterozoic basins on the Indian Shield. References Contrib Mineral Petrol 176:31 Grove TL (1993) Corrections to expressions for calculating mineral components in “Origin of calc-alkaline series lavas at medi- cine lake volcano by fractionation, assimilation and mixing” and “Experimental petrology of normal MORB near the kane fracture zone: 22°–25°N, Mid-Atlantic Ridge.” Contrib Min- eral Petrol 114:422–424 Chatterjee N, Bhattacharji S (1998) Formation of Proterozoic tholei- ite intrusives in and around Cuddapah Basin, south India and their Gondwana counterparts in east Antarctica: and composi- tional variation in their mantle sources. Neues Jb Miner Abh 174(1):79–102 Grove TL, Holbig ES, Barr JA, Till CB, Krawczynski MJ (2013) Melts of garnet lherzolite: experiments, models and compari- son to melts of pyroxenite and carbonated lherzolite. Con- trib Mineral Petrol 166:887–910. https://doi.org/10.1007/ s00410-013-0899-9 Chatterjee N, Bhattacharji S (2001) Petrology, geochemistry and tec- tonic settings of the mafic dykes and sills associated with the evolution of the Proterozoic Cuddapah Basin of south India. Proc Indian Acad Sci (earth Planet Sci) 110:433–453 Grove TL, Kinzler R, Bryan W (1992) Fractionation of mid-ocean ridge basalt (MORB). In: Phipps Morgan J, Blackman D, Sinton J (eds.) Mantle Flow and Melt Generation at Mid-Ocean Ridges. Geophys Monogr 71, AGU, Washington, DC, pp 281–310 Chatterjee N, Sheth H (2015) Origin of the Powai ankaramite, and the composition, P-T conditions of equilibration and evolution of the primary magmas of the Deccan tholeiites. Contrib Mineral Petrol 169:32 phys Monogr 71, AGU, Washington, DC, pp 281–310 GSI (1998) Geological Map of India. In: Dasgupta A, Chakravorty K (eds) Acharyya SK. Geol Surv India, Hyderabad Clark C, Collins AS, Timms NE, Kinny PD, Chetty TRK, Santosh M (2009) SHRIMP U-Pb age constraints on magmatism and high- grade metamorphism in the Salem Block, southern India. Gond Res 16(1):27–36 Gupta S, Rai SS, Prakasam KS, Srinagesh D, Bansal BK, Chadha RK, Priestley K, Gaur K (2003) The nature of the crust in southern India: implications for Precambrian crustal evolution. Geophys Res Lett. https://doi.org/10.1029/2002GL016770i Collins AS, Patranabis-Deb S, Alexander E, Bertram CN, Falster GM, Gore RJ, Mackintosh J, Dhang PC, Saha D, Payne JL, Jourdan F, Backé G, Halverson GP, Wade BP (2015) Detrital mineral age, radiogenic isotopic stratigraphy and tectonic significance of the Cuddapah Basin, India. Gond Res 28:1294–1309 Halls HC (1982) The importance and potential of mafic dyke swarms in studies of geodynamic processes. References Geosci Canada 9:145–154 Halls HC, Kumar A, Srinivasan R, Hamilton MA (2007) Paleomag- netism and U-Pb geochronology of eastern trending dykes in the Dharwar craton, India: feldspar clouding, radiating dyke swarms and the position of India at 2.37 Ga. Precamb Res 155:47–68 p Condie KC (2005) High field strength element ratios in Archean basalts: a window to evolving sources of mantle plumes? Lithos 79:491–504 Herzberg C (2022) Understanding the Paleoproterozoic Circum-Supe- rior Large Igneous Province constrains the thermal properties of Earth’s mantle through time. Precamb Res 375:106671 Das R, Saikia U, Rai SS (2015) The deep geology of South India inferred from Moho depth and Vp/Vs ratio. Geophys J Int 203:910–926. https://doi.org/10.1093/gji/ggv351 Herzberg C, Asimow PD, Arndt N, Niu Y, Lesher CM, Fitton JG, Cheadle MJ, Saunders AD (2007) Temperatures in ambient man- tle and plumes: constraints from basalts, picrites and komatiites. Geochem Geophys Geosyst 8:Q02006. https://doi.org/10.1029/ 2006GC001390 p g gj gg Davies GF (1999) Dynamic Earth: Plates. Cambridge University Press, Plumes and Mantle Convection, p 458f DePaolo DJ (1981) Trace element and isotopic effects of combined wall rock assimilation and fractional crystallization. Earth Planet Sci Lett 53:189–202 Herzberg C, Condie K, Korenaga J (2010) Thermal history of the Earth and its petrological expression. Earth Planet Sci Lett 292:79–88 Ernst RE, Baragar WRA (1992) Evidence from magnetic fabric for the flow pattern of magma in the Mackenzie giant radiating dyke swarm. Nature 356:511–513 Higgins O, Sheldrake T, Caricchi L (2022) Machine learning thermo- barometry and chemometry using amphibole and clinopyroxene: a window into the roots of an arc volcano (Mount Liamuiga, 1 3 28 Page 24 of 25 Contributions to Mineralogy and Petrology (2023) 178:28 28 Saint Kitts). Contrib Mineral Petrol 177:10. https://doi.org/10. 1007/s00410-021-01874-6 Manikyamba C, Kerrich R (2012) Eastern Dharwar craton, India: con- tinental lithosphere growth by accretion of diverse plume and arc terranes. Geosci Front 3:225–240 Jayananda M, Banerjee M, Pant NC, Dasgupta S, Kano T, Mahesha N, Mahableswar B (2011) 262 Ga high-temperature metamorphism in the central part of the Eastern Dharwar Craton: implications for late Achaean tectonothermal history. Geol J. https://doi.org/ 10.1002/gj.1308 McDonough WF, Sun S-S (1995) Composition of the Earth. Chem Geol 120:223–253. https://doi.org/10.1016/0009-2541(94) 00140-4 McKenzie D, Bickle MJ (1988) The volume and composition of melt generated by extension of the lithosphere. References J Petrol 29:625–679 Jayananda M, Peucat JJ, Chardon D, Krishna Rao B, Corfu F (2013a) Neoarchean greenstone volcanism, Dharwar craton, Southern India: constraints from SIMS zircon geochronology and Nd iso- topes. Precamb Res 227:55–76 Mishra DC (2015) Plume and Plate Tectonics Model for formation of some Proterozoic Basins of India along Contemporary Mobile Belts: Mahakoshal – Bijawar, Vindhyan and Cuddapah Basins. J Geol Soc India 85:525–536 Jayananda M, Tsutsumi Y, Miyazaki Y, Gireesh RV, Kapfo K-U, Tush- ipokla HH, Kano T (2013b) Geochronologic constraints on Meso and Neoarchean regional metamorphism and magmatism in the Dharwar craton, southern India. J Asian Earth Sci 78:18–38 Mohanty S (2011) Palaeoproterozoic assembly of the Napier Complex, Southern India and Western Australia: Implications for the evolu- tion of the Cuddapah basin. Gond Res 20:344–361. https://doi. org/10.1016/j.gr.2011.03.009 Jayananda M, Santosh M, Aadhiseshan KR (2018) Formation of Archean (3600–2500 Ma) continental crust in the Dharwar Cra- ton, southern India. Earth-Sci Rev 181:12–42 Moore NE, Grunder AL, Bohrson WA (2018) The three-stage pet- rochemical evolution of the Steens Basalt compared to large igneous provinces and layered mafic intrusions. Geosphere 14(6):2505–2532. https://doi.org/10.1130/GES01665.1 Johnson KTM, Kinzler RJ (1989) Partitioning of REE, Ti, Zr, Hf, and Nb between clinopyroxene and basaltic liquid: an ion microprobe study. Eos 70:1388 Moore NE, Grunder AL, Bohrson WA, Carlson RW, Bindeman IN (2020) Changing mantle sources and the effects of crustal pas- sage on the Steens Basalt SE Oregon chemical and isotopic con- straints. Geochem Geophys Geosyst. https://doi.org/10.1029/ 2020GC008910 Jorgenson C, Higgins O, Petrelli M, Bégué F, Caricchi L (2022) A machine learning-based approach to clinopyroxene thermo- barometry: Model optimization and distribution for use in Earth sciences. J Geophys Res. https://doi.org/10.1029/2021JB022904 Morgan WJ (1971) Convection plumes in the lower mantle. Nature 230:42–43 Kinzler R, Grove TL (1992a) Primary magmas of mid-ocean ridge basalts, 1. Exp Res J Geophys Res 97:6885–6906. https://doi. org/10.1029/91JB02840 Murty YGK, Babu Rao V, Guptasarma D, Rao JM, Rao MN, Bhat- tacharji S (1987) Tectonic, petrochemical and geophysical stud- ies of mafic dyke swarms around the Proterozoic Cuddapah Basin, south India. In: Halls HC, Fahrig W (eds.) Mafic Dyke Swarms. Geol Assoc Canada Spl Paper 34:303–316 g Kinzler RJ, Grove TL (1992b) Primary magmas of mid-ocean ridge basalts 2. Applications J Geophys Res 97:6907–6926 Kjøll HJ, Galland O, Labrousse L, Andersen TB (2019) Emplacement mechanisms of a dyke swarm across the brittle-ductile transition and the geodynamic implications for magma-rich margins. References Earth Planet Sci Lett 518:223–235 Nagaraja Rao BK, Rajurkar ST, Ramlingaswamy G, Ravindra Babu B (1987) Stratigraphy, structure and evolution of the Cuddapah basin. In: Radhakrishna BP (ed.) Purana Basins of Peninsular India (Middle to Late Proterozoic). Mem Geol Soc India 6:33–86 Korenaga J (2008) Urey ratio and the structure and evolution of Earth’s mantle. Rev Geophys. https://doi.org/10.1029/2007RG000241 Nagaraju E, Parashuramulu V, Ramesh Babu N, Narayana AC (2018a) A 2207 Ma radiating mafic dyke swarm from eastern Dharwar craton, Southern India: drift history through Paleoproterozoic. Precamb Res 317:89–100 Krein SB, Molitor ZJ, Grove TL (2021) ReversePetrogen: A Mul- tiphase dry reverse fractional crystallization-mantle melting ther- mobarometer applied to 13589 mid-ocean ridge basalt glasses. J Geophys Res 126:e2020JB021292 Nagaraju E, Parashuramulu V, Kumar A, Srinivas Sarma D (2018b) Paleomagnetism and geochronological studies on a 450 km long 2216 Ma dyke from the Dharwar craton, southern India. Phys Earth Planet Inter 274:222–231 Kumar A, Bhalla MS (1983) Palaeomagnetics and igneous activity of the area adjoining the southwestern margin of the Cuddapah basin. India Geophys J R Astron Soc 73:27–37 Kumar A, Hamilton MA, Halls HC (2012a) A Paleoproterozoic giant radiating dyke swarm in the Dharwar Craton, southern India. Geochem Geophys Geosyst 7:Q02011 Naqvi SM, Rogers JJW (1987) Precambrian Geology of India. Oxford Univ Press Inc 223 p. Kumar A, Nagaraju E, Besse J, Bhaskar Rao YJ (2012b) New age, geochemical and paleomagnetic data on a 2.21 Ga dyke swarm from southern India: constraints on Paleoproterozoic reconstruc- tion. Precamb Res 220–221:123–138 Nesbitt HW, Young GM (1982) Early Proterozoic climates and plate motions inferred from major element chemistry of lutites. Nature 299:715–717 NGRI (1978) Gravity map series of India. National Geophysical Research Institute, Hyderabad, India, NGRI-GPH/2, 1 sheet 1:5000000 Kumar A, Parashuramulu V, Nagaraju E (2015) A 2082 Ma radiat- ing dyke swarm in the Eastern Dharwar Craton, southern India and its implications to Cuddapah basin formation. Precamb Res 266:490–505 Padmakumari VM, Dayal AM (1987) Geochronological studies of some mafic dykes around the Cuddapah basin, south India. In: Radhakrishna BP (Ed.) Purana Basins of Peninsular India (Mid- dle to Late Proterozoic). Geol Soc India Memoir 6, pp. 369–373i Le Bas MJ, Le Maitre RW, Streckeisen A, Zanettin B (1986) A chemi- cal classification of volcanic rocks based on the total alkali-silica diagram. References J Petrol 27:745–750 Pearce JA (2008) Geochemical fingerprinting of oceanic basalts with applications to ophiolite classification and the search for Archean oceanic crust. Lithos 100:14–48 Liao AC-Y, Shellnutt JG, Hari KR, Denyszyn SW, Vishwakarma N, Verma CB (2019) A petrogenetic relationship between 2.37 Ga boninitic dyke swarms of the Indian Shield: evidence from the central Bastar Craton and the NE Dharwar Craton. Gond Res 69:193–211 Peucat J-J, Jayananda M, Chardon D, Capdevila R, Fanning Marc C, Paquette J-L (2013) The lower crust of Dharwar craton, south India: patchwork of Archean granulitic domains. Precamb Res 227:4–29 Macdonald GA, Katsura T (1964) Chemical composition of Hawaiian lavas. J Petrol 5:82–133 Poornachandra Rao GVS (2005) Orthogonal dykes around the Cud- dapah basin - a paleomagnetic study. J Indian Geophys Union 9:1–11 1 3 Contributions to Mineralogy and Petrology (2023) 178:28 Page 25 of 25 28 Presnall DC, Gudfinnsson GH, Walter MJ (2002) Generation of mid- ocean ridge basalts at pressures from 1 to 7 GPa. Geochim Cos- mochim Acta 66:2073–2090 Spera FJ, Bohrson WA (2001) Energy-constrained open-system mag- matic processes I: general model and energy-constrained assim- ilation and fractional crystallization (EC-AFC) formulation. J Petrol 42:999–1018. https://doi.org/10.1093/petrology/42.5.999 Putirka KD (2008) Thermometers and barometers for volcanic systems. Rev Mineral Geochem 69:61–120 Srivastava RK, Jayananda M, Gautam GC, Gireesh V, Samal AK (2014a) Geochemistry of an ENE–WSW to NE–SW trending ∼2.37 Ga mafic dyke swarm of the Eastern Dharwar Craton India: does it represent a single magmatic event? Chem Erde Geochem. 74:251–265 Putirka KD (2016a) Rates and styles of planetary cooling on earth, moon, mars, and vesta, using new models for oxygen fugacity, ferric-ferrous ratios, olivine-liquid Fe-Mg exchange, and mantle potential temperature. Amer Mineral 101:819–840 Putirka KD (2016b) Amphibole thermometers and barometers for igne- ous systems and some implications for eruption mechanisms of felsic magmas at arc volcanoes. Amer Mineral 101:841–858 Srivastava RK, Jayananda M, Gautam GC, Samal AK (2014b) ∼2.21– 2.22 Ga N-S to NNW–SSE trending Kunigal mafic dyke swarm from Eastern Dharwar Craton, India: implications for Paleoprote- rozoic large igneous provinces and supercraton Superia. Mineral Petrol 109:695–711 Putirka K, Johnson M, Kinzler R, Walker D (1996) Thermobarometry of mafic igneous rocks based on clinopyroxene-liquid equilibria, 0–30 kbar. Contrib Mineral Petrol 123:92–108i Srivastava RK, Samal AK, Gautam GC (2015) Geochemical charac- teristics and petrogenesis of four Palaeoproterozoic mafic dyke swarms and associated large igneous provinces from the eastern Dharwar craton, India. References Intern Geol Rev 57:1462–1484 Putirka KD, Perfit M, Ryerson FJ, Jackson MG (2007) Ambient and excess mantle temperatures, olivine thermometry, and active vs. passive upwelling. Chem Geol 241:177–206 Radhakrishna T, Joseph M (1996) Proterozoic paleomagnetism of the mafic dyke swarms in the high grade region of southern India. Precamb Res 76:31–46 Stark JC, Wang X-C, Denyszyn SW, Li Z-X, Rasmussen B, Zi J-W, Sheppard S, Liu Y (2019) Newly identified 1.89 Ga mafic dyke swarm in the Archean Yilgarn Craton, Western Australia sug- gests a connection with India. Precamb Res 329:156–169 Rajamani V, Shivkumar K, Hanson GN, Shirey SB (1985) Geochem- istry and petrogenesis of amphibolites, Kolar Schist Belt, South India: Evidence for komatiitic magma derived by low percent- ages of melting of the mantle. J Petrol 26(1):92–123 Swami Nath J, Ramakrishnan M, Viswanatha MN (1976) Dharwar stratigraphic model and Karnataka craton evolution. Geol Surv India Records 107:149–175 Taylor SR, McLennan SM (1995) The geochemical evolution of the continental crust. Rev Geophys 33:241–265 Rao JM, Bhattacharji S, Rao MN, Hermes OD (1995) 40Ar-39Ar ages and geochemical characteristics of dolerite dykes around the Proterozoic Cuddapah basin, south India. Mem Geol Soc India 33:307–328 Thompson AB, Matile L, Ulmer P (2002) Some thermal constraints on crustal assimilation during fractionation of hydrous, mantle- derived magmas with examples from central Alpine batholiths. J Petrol 43:403–422 Rivalta E, Taisne B, Bunger AP, Katz RF (2015) A review of mechani- cal models of dike propagation: schools of thought, results and future directions. Tectonophys 638:1–42 Till CB (2017) A review and update of mantle thermobarometry for primitive arc magmas. Amer Mineral 102:931–947 p y Roeder PL, Emslie RF (1970) Olivine liquid equilibrium. Contrib Min- eral Petrol 29:275–289 Till CB, Grove TL, Krawczynski MJ (2012) A melting model for vari- ably metasomatized plagioclase and spinel lherzolite. J Geophys Res 117:B06206. https://doi.org/10.1029/2011JB009044 Rogers AJ, Kolb J, Meyer FM, Armstrong RA (2007) Tectono-mag- matic evolution of the Hutti-Maski greenstone belt, India: con- strained using geochemical and geochronological data. J Asian Earth Sci 31:55–70 Till CB, Grove TL, Carlson RW, Fouch MJ, Donnelly-Nolan JM, Wagner LS, Hart WK (2013) Depths and temperatures of <10.5 Ma mantle melting and the lithosphere-asthenosphere boundary below southern Oregon and northern California. Geochem Geo- phys Geosyst 14:864–879. https://doi.org/10.1002/ggge.20070 Samal AK, Srivastava RK, Ernst RE (2021) An appraisal of mineral systems associated with Precambrian large igneous provinces of the Indian shield. References Ore Geol Rev 131:104009 Tormey DR, Grove TL, Bryan WB (1987) Experimental petrology of normal MORB near the Kane fracture zone: 22–25°N, mid- Atlantic Ridge. Contrib Mineral Petrol 96:121–139 Shellnutt JG, Hari KR, Liao AC-Y, Denyszyn SW, Vishwakarma N (2018) A 1.88 Ga giant radiating mafic dyke swarm across south- ern India and Western Australia. Precamb Res 308:58–74 Watson S, McKenzie D (1991) Melt generation by plumes; a study of Hawaiian volcanism. J Petrol 32:501–537 Sheppard S, Rasmussen B, Zi J-W, Somasekhar V, Sarma DS, Ram Mohan M, Krapež B, Wilde SA, McNaughton NJ (2017) Sedi- mentation and magmatism in the Paleoproterozoic Cuddapah Basin, India: consequences of lithospheric extension. Gond Res 48:153–163 White WM (2010) Oceanic island basalts and mantle plumes: the geo- chemical perspective. Annu Rev Earth Planet Sci 38:133–160 Yang HJ, Kinzler RJ, Grove TL (1996) Experiments and models of anhydrous, basaltic olivine-plagioclase-augite saturated melts from 0.001 to 10 kbar. Contrib Mineral Petrol 124:1–18 Singh AP, Mishra DC, Gupta SB, Rao MRKP (2004) Crustal structure and domain tectonics of the Dharwar Craton (India): insight from new gravity data. J Asian Earth Sci 23:141–152 Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Sleep NH (1990) Hotspots and mantle plumes: some phenomenology. J Geophys Res 95:6715–6736 Söderlund U, Bleeker W, Demirer K, Srivastava RK, Hamilton M, Nils- son M, Pesonen LJ, Samal AK, Jayananda M, Ernst RE, Srinivas M (2019) Dharwar craton, India: Implications for paleorecon- structions and support for a ∼30° change in dyke trends from south to north. Precamb Res 329:26–43 1 3 1 3
https://openalex.org/W4385769349	https://www.researchsquare.com/article/rs-3242162/latest.pdf	English	null	CovMedCare: Confluence of Internet of Things, Blockchain and Machine Learning for Remote Monitoring System of Pandemic Patients	Research Square (Research Square)	2,023	cc-by	6,529	Abstract Pandemics like Covid-19 necessitate the need for continuous remote monitoring of patients. The scarcity of an efficient and secure remote monitoring system for patients led to surge in work pressure for medical staff in hospitals globally. In this paper, we propose a secure and robust decentralized patient monitoring model called CovMedCare for monitoring the Covid-19 in-patients utilizing the confluence the Machine Learning, Internet of Things and Blockchain technologies. In the proposed model, sensor data from WBAN is integrated with previous health records of the patients to predict deteriorating health condition of the patient over a blockchain network. The accuracy of the proposed model is assessed on a dataset with five medical sensor attributes and twelve previous health records attributes. The experimental results exhibit that CovMedCare achieves an accuracy of 99% in identifying the patient whose health condition is deteriorating, which is higher than that of other traditional machine learning models. Research Article 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 Page 1/38 Page 1/38 1. Introduction Pandemics have troubled the human race since time immemorial. The ongoing Covid-19 disease pandemic is the most current threat to international health. It appeared for the first time in December 2019. It is caused by new coronavirus that produces severe acute respiratory syndrome is. Middle East respiratory syndrome (MERS) (2012 to the present) and SARS (2002 and 2003), the two previous pandemics in the last 18 years, are comparable to this one [1]. This pandemic has raised grave challenges for the medical communities, research, and public health. As of August 2022, 606,129,697 cases and 6,488,808 deaths were reported worldwide [2]. To combat the spread of SARS-CoV-2, many countries took drastic steps such as imposing restrictions, from complete lockdowns to severe movement restrictions, affecting the stability and growth of nations. As the cases rose exponentially, the healthcare systems of countries crumpled. In the post-Covid-19 era, the global focus is to build a robust, secure, and scalable smart healthcare system to tackle future healthcare crises effectively. Technological advancement in the medical field has aided in developing such a system. In the case of contagious diseases like Covid-19, continuous remote monitoring of a patient's health is a grave challenge that must be handled. Due to the widespread outbreak and colossal increase in number of cases, continuous monitoring of Covid-19 patients is a challenging task in the healthcare sector. The disease is highly contagious; remote health monitoring can be a boon for healthcare professionals during emergencies. In order that healthcare professionals can intrude at the earliest sign of suffering, warrants the need for continuous patient monitoring. The primary objective of continuous patient monitoring is to provide timely medical care. Constant surveillance of hospitalized patients gives insights on patient's condition without risking the lives of nurses and doctors. The unit consists of noninvasive sensors to collect the heart rate, health temperature, oxygen saturation level, respiration rate, blood pressure etc. readings of the Covid-19 patients. The system also dramatically reduces the risk of infection in healthcare workers, which would scale down the growing demand for PPE (personnel protection equipment) kits and logistics. Page 2/38 Wearable devices with embedded sensors (such as temperature sensors) can be used for continuous patient monitoring. Nowadays, to monitor covid-19 patients, Wireless Body Area Networks (WBAN) sense the patient's vitals regularly through various medical sensors (MSs). 1. Introduction One of the challenging tasks in WBAN is to utilize patients' previous health records (PHRs) along with Monitored Sensor Data (MSD) to analyze patients' health. Lately, numerous researches using latest technology have been proposed to monitor patients. Nonetheless the current models miss the mark to produce a model to manage several medical sensors along with patients' previous medical records for patient monitoring. Various models have been proposed to analyze patients' data stored on centralized servers. These storage systems suffer from the drawbacks of single-point failure and the threat of being hacked. Blockchain can be used to overcome the storage issues in traditional storage systems. The proposed integrated system aids in the early identification of sudden deterioration in patient's conditions, and professionals can be alerted timely. It also enables professionals to remotely monitor the health status of many patients in the hospital using a smartphone without exposure to the infection. It helps in continuous surveillance around the clock. The remaining paper is structured as follows: a brief history of the technologies utilized to create the suggested model is provided in Section 2. The associated works are discussed in Section 3. The suggested model is described in great length in Section 4. The discussion of the CovMedCare model's performance assessments may be found in Section 5. The conclusion and potential areas for further research are offered in Section 6. 2. Related Work This section presents the current patient monitoring models using the Wireless Body Area Network (WBAN), blockchain, as well as several standard classification models. WBAN is crucial in keeping track of the patient's vitals for severity prediction risk [3, 4]. Various authors have proposed many disease prediction algorithms using several machine learning algorithms [5–7]. These classification models, such as Support Vector Machine (SVM), Linear Regression (LR), Decision Tree (DT), Gaussian Naive Bayes (GN), and Random Forest (RF), were applied to several healthcare datasets for disease prediction [8–10]. Researchers in [11] analyzed the standard classification models. They combined the results to enhance the performance further using a gradient-boosting classifier. A supervised machine learning algorithm for Covid-19 disease has been proposed in [12], which validated the identification of patients utilizing an epidemiology-labeled dataset. Most of these works are used to identify a Covid-19 patient. Also, these algorithms have utilized centralized servers to store data. Centralized storage systems have various drawbacks, including single-point failure problems and privacy issues. Some researchers have adopted a decentralized blockchain network as a storage platform to overcome these issues. Blockchain provides immutability, trust and eliminates the need for intermediaries or a dependence on centralized entity for transaction control [13] [14]. Various researchers have demonstrated effective applications of blockchain in the healthcare domain [22–25]. Yet, at the time this article was being written, no similar work or publicly available research was found similar to our proposed work. Though companies like IBM Page 3/38 Page 3/38 utilized its project Hyperledger, to market the usage of blockchain to the healthcare domain and Internet of Things [24], no publications describing their operational model was found. Remote Patient Monitoring (RPM) systems aim to improve disease outcomes [26] using wireless sensor networks. Nonetheless, the major focus of blockchain applications in healthcare has been to maintain Electronic Health Records to aid in interoperability between organizations. MIT has developed a prototype called MedRec to facilitate the sharing of EHRs using blockchain [23]. Researchers are creating an EHR management system using blockchain as the fundamental structure, i.e., data stored in EHR is treated as a transaction grouped into blocks. A chain of blocks forms a blockchain. For example, a company called the ICO for Medical Chain, is creating the first blockchain- based EHR, which is patient-centric [27]. In one research, authors have developed a blockchain-based platform, BHEEM [15], to store and disseminate EHRs efficiently. 2. Related Work It solved the security and privacy issues of conventional storage mechanisms. Further, authors in [16] proposed a biometric-based validation system for secure storage of healthcare records. These existing models demonstrate the absence of works related to continuous monitoring of inpatients, the lack of clinical datasets, the absence of both previous medical records and sensor data, the unavailability of a secure end-to-end model, and the lack of secure and robust database to store data collected from sensors. Motivated by these issues, we have proposed an IoT-enabled and Blockchain- based Smart Healthcare System for COVID-19 patient monitoring using an ensemble Random Forest classification model. This paper proposes a novel smart and secure health monitoring system, CovMedCare, for Covid-19 inpatient monitoring and risk prediction using IoT, blockchain and machine learning. Initially, data of a Covid-19 patient is gathered in two different modes, i.e., Monitored Sensor Data and Previous Health Records. Then this data is stored securely on a blockchain network. Finally, an ensemble Random Forest classifier is applied to train CovMedCare model to predict disease severity and alert the professionals in case of emergency. The following is a list of the main contributions of the proposed CovMedCare: A smart model for continuous monitoring of patients called the CovMedCare is developed with the Random Forest classifier for predicting the disease severity of patients admitted to the hospital and alerting the medical professionals and relatives in case of emergency. WBAN is integrated with a decentralized blockchain network to store data and facilitate interoperability and security. The prediction achieves accuracy as high as 99%, which is the highest compared to other standard classifiers. 3. The Proposed Approach These devices format, aggregate and send the data collected from the sensors to the blockchain network using the internet. They also act as a gateway. Wireless Sensor nodes send data to the Central Control Unit (CCU). The CCU then forwards data to the remote PC’s for storage or analysis. Figure 3 demonstrates the generalized architecture of the WBAN system proposed in [1]. Each hospital has several devices registered to it which are identified by their MAC address. These devices are the coordinator nodes or master nodes or central control units in the Wireless Body Area Network (WBAN). These devices format, aggregate and send the data collected from the sensors to the blockchain network using the internet. They also act as a gateway. Wireless Sensor nodes send data to the Central Control Unit (CCU). The CCU then forwards data to the remote PC’s for storage or analysis. Figure 3 demonstrates the generalized architecture of the WBAN system proposed in [1]. Figure 4 shows the detailed architecture of the WBAN system of multi-patient monitoring in a hospital. To detect signals from the human body, Medical Implant Communication System (MICS), Industrial, Scientific and Medical (ISM), and Ultra-wideband (UWB) bands is used. Communication technologies like ZigBee, Bluetooth, Wi-Fi or WTMS is used for communicating sensors with the remote CCU. Gateway or local PC is connected to the remote CCU through Ethernet or USB cable. Gateway transmits data to the remote server or blockchain nodes. 3. The Proposed Approach In this section, we explain the proposed CovMedCare model at length. We discuss the mechanism of continuous monitoring using various vital parameters with previous health records using the ensemble Page 4/38 RF technique. The idea of a novel health monitoring system for handling pandemics inpatients has been proposed in this section. Figure 1 shows the layered architecture. The architecture comprises three layers: RF technique. The idea of a novel health monitoring system for handling pandemics inpatients has been proposed in this section. Figure 1 shows the layered architecture. The architecture comprises three layers: 1. Data Collection and aggregation layer: This layer consists of the WBAN system. The health records of the patients along with sensor data are formatted and aggregated and sent to the next layer. 2. Storage Layer: This is the blockchain layer used for securely storing hea 3. Data accessibility and machine learning layer: The data stored on blockchain can be fed to machine learning algorithms for better decision making or can be accessed directly by certified stakeholders only. The proposed system consists of several stakeholders such as certified doctors, patients, patient’s relatives, hospitals, and researchers. It is the confluence of the three technologies i.e. machine learning, blockchain and wireless body area network. Figure 2 shows the interactions between WBAN, stakeholders and Hospitals. The proposed system consists of several stakeholders such as certified doctors, patients, patient’s relatives, hospitals, and researchers. It is the confluence of the three technologies i.e. machine learning, blockchain and wireless body area network. Figure 2 shows the interactions between WBAN, stakeholders and Hospitals. 1. Each hospital acts as a blockchain node. 1. Each hospital acts as a blockchain node. 2. Each government hospital acts as a certificate authority which issues digital certificates to verified and authentic parties only. 3. Only stakeholders with digital certificates are allowed to access data stored on the blockchain. 4. Each hospital consists of a WBAN which consists of several patients. 5. Government hospitals may act as miners in the blockchain network. 6. Stakeholders can access data on their mobiles or personal computers only after verifying their identity through digital certificates. Each hospital has several devices registered to it which are identified by their MAC address. These devices are the coordinator nodes or master nodes or central control units in the Wireless Body Area Network (WBAN). 4. Architecture of the CovMedCare The architecture of the proposed CovMedCare model using distributed ledger technology is shown in Fig. 5. CovMedCare model comprises two sources of data, i.e., the data from the sensors of wireless body Page 5/38 area network (WBAN) and the Previous Health Records (PHRs) of the patients (shown as HR1, HR2, ….. HRk). CovMedCare utilizes a set of medical sensors (MSs) consisting of body temperature (MS1), respiration rate (MS2), blood pressure (MS3), oxygen saturation (MS4), and heart rate (MS5) of the patients to gather physiological data for monitoring and predicting patient's health, that is transferred to the processing devices via gateway or local edge device using a Wi-Fi connection as shown in action 1 and 2 in Fig. 5. Then, the proposed model comprises of components including local edge devices (denoted as E= {E1, E2,…, En}) and a blockchain network (BN) which consists of blocks which stores transactions (denoted as T={T1, T2,…, Tm}). (Action 3 in Fig. 5). Blockchain network is being used to implement the storage mechanism for the proposed system. After preprocessing the collected medical data, the local edge devices transmit the data to the decentralized BN for permanent storage. At first, the PHRs and the MSD of the patients are stored securely in the BN, called Patients Data Storage (PDS) (Action 3 in Fig. 5). Data is fetched using Data Fetching Unit from the blockchain (Action 4 in Fig. 5). Then, the prediction system initiates the prediction of the covid disease using PHRs and MSD of the patients (Action 5 in Fig. 5). It includes two major parts: 1) Data fusion and Preprocessing of data and 2) Disease prediction classification model. Initially, the data are preprocessed for missing-data filtering and to remove the noise using different machine learning algorithms (Action 6 in Fig. 5). Lastly, the refined data are given as inputs to the ensemble Random Forest classification model for the severity prediction of Covid-19 patient (Action 6 in Fig. 5). Finally, the result is used to alert the professionals in case of emergencies. (Action 7 in Fig. 5). Figure 6. Structure of Transaction 4.2 Data Storage A unique blockchain may be developed by defining the block structure. The essential entries of the transactions are stored on the block. In the proposed blockchain network, there are two types of transactions. During patient registration, the first type of transaction is executed to keep the previous health records of the patients along with their details. After the patient's registration using the first transaction, a unique PID is assigned to the patient. The second type of transaction stores the sensor data collected corresponding to the PID at regular intervals. The different attributes of the transactions are shown in Fig. 6. In transactional context, the transferred data by the patient is processed. The block created by the selected miner using the Proof of stake consensus algorithm is sent to other nodes for validation. After the mining process, a final consensus is reached. The output of the consensus algorithm decides if the block is to be added or discarded in the blockchain network. Page 6/38 class Transaction: senderPublicKey type timestamp pid data signature class PreviousMedicalRecords: heart_disease asthma diabetes bronchitis chronic_kidney_disease lung_cancer pneumonia pregnant_women emphysema taste_smell_disorder viral_infection_in_throat neurological_disorder class sensorData: pid timestamp body_temperature oxygen_saturation repiration_rate Page 7/38 signature class PreviousMedicalRecords: heart_disease asthma diabetes bronchitis chronic_kidney_disease lung_cancer pneumonia pregnant_women emphysema taste_smell_disorder viral_infection_in_throat neurological_disorder class sensorData: pid timestamp body_temperature oxygen_saturation repiration_rate heart_rate blood_pressure Figure 6. Structure of Transaction Figure 6. Structure of Transaction Page 7/38 To hash the block, the cryptographic algorithm SHA256 is used. Previous block’s hash is stored in the next block. The symbols and their corresponding meanings utilized in our work have been listed in Table 1. T is a set that stores the details of the transaction, PHR is a set that stores the attributes of transaction type 1, and Ȿ stores the attributes of transaction type 2, as shown below: T is a set that stores the details of the transaction, PHR is a set that stores the attributes of transaction type 1, and Ȿ stores the attributes of transaction type 2, as shown below: PHR = {Ƿ1, Ƿ2, Ƿ3, Ƿ4, Ƿ5, Ƿ6, Ƿ7, Ƿ8, Ƿ9, Ƿ10, Ƿ11, Ƿ12} (2) PHR = {Ƿ1, Ƿ2, Ƿ3, Ƿ4, Ƿ5, Ƿ6, Ƿ7, Ƿ8, Ƿ9, Ƿ10, Ƿ11, Ƿ12} (2) = {ρ, } (3) α, β, γ, δ, ϵ Both sets are secured using the patient's digital signature which is calculated by utilizing the SHA256 algorithm. 4.2 Data Storage Both sets are secured using the patient's digital signature which is calculated by utilizing the SHA256 algorithm. Hash_PHR = SHA256(EHR) Hash_Ȿ =SHA256(Ȿ) (4) The patient uses his private key to sign, the sets EHR and Ȿ with their respective hashes. Ppr and Ppu denote the patient's private and public keys. RSA algorithm is used to obtain the digital signature as follows: Digital signature 1 = RSA {Ppr, Hash_PHR, PHR} (5) Digital signature 2 = RSA {Ppr, Hash_Ȿ, Ȿ} (6) Digital signature 1 = RSA {Ppr, Hash_PHR, PHR} (5) Digital signature 2 = RSA {Ppr, Hash_Ȿ, Ȿ} (6) Miners validate the same after the digital signature has been obtained. Thus, the complete transaction denoted by set by Tx as: Tx= {Ppu, Ty, Ґ, ρ, Data, Sign} (7) Tx= {Ppu, Ty, Ґ, ρ, Data, Sign} (7) Tx= {Ppu, Ty, Ґ, ρ, Data, Sign} (7) where Data = EHR if Ty = 1 or Data is Ȿ if Ty = 2 and Sign = Digital signature 1 if Ty = 1 or Digital Signature 2 if Ty = 2 where Data = EHR if Ty = 1 or Data is Ȿ if Ty = 2 and where Data = EHR if Ty = 1 or Data is Ȿ if Ty = 2 and Sign = Digital signature 1 if Ty = 1 or Digital Signature 2 if Ty = 2 These transactions are added to the transaction pool. The miners use these transaction pools to create the next block. Page 8/38 Table 1 Acronym/Symbol used & their Description Acronym/Symbol Description Body Temperature Oxygen Saturation Respiration Rate Heart Rate Blood Pressure Ƿ 1 Heart disease Ƿ 2 Asthma Ƿ 3 Diabetes Ƿ 4 Chronic Kidney Disease Ƿ 5 Lung Cancer Ƿ 6 Pregnant Women Ƿ 7 Emphysema Ƿ 8 Taste and smell disorder Ƿ 9 Viral throat infection Ƿ 10 Neurological disorder Ƿ 11 Pneumonia Ƿ 12 Bronchitis Ρ Patient's id Ty Type of transaction Ґ Timestamp valuation of CovMedCare α β γ δ ϵ Page 9/38 5. Performance Evaluation of CovMedCare 5. Performance Evaluation of CovMedCare Page 9/38 The proposed CovMedCare model has been implemented using Python language. The decentralized blockchain network has been developed using InterPlanetary File System (IPFS) in python language on Ubuntu system. We have employed the decentralized network utilizing hardware and software, as shown in Table 2. 4.2 Data Storage In this section, we elaborate the analysis of CovMedCare at the system level using various classifiers for patient severity prediction. One important task for developing classification is to examine the accuracy of the proposed classifier to find an accurate classification model having high prediction accuracy. To demonstrate the eminence of the proposed CovMedCare model, different standard classifiers, i.e., Support Vector Machine (SVM), Linear Regression (LR), Random Forest (RF), Naïve Bayes (NB) and K-Nearest Neighbor (KNN) are considered and evaluated based on the several metrics. The outcomes illustrate that the Random Forest algorithms perform best among the traditional classifiers for patient severity prediction with 99% accuracy. After training, we saved the model in pickle format and deployed it to give predictions. Table 2 Details of Hardware and Software used Hardware • Intel(R) Core (TM) i7-8750H CPU @ 2.20GHz 2.21 GHz • RAM: 8.00 GB DDR4 Memory • GPU: NVIDIA GeForce 1650 Software version (including tools and standard) • Operating System: Ubuntu • Python 3.10.4 • IPFS 0.5.0 • Visual Studio Code • Storage: 4150 GB • 1 GbE Dara Rate per Port Application • Covid-19 patient monitoring system Nodes • 4 Implementation Parameters Block size Block hash size • 6 MB • 256 Bit Table 2 Details of Hardware and Software used Hardware • Intel(R) Core (TM) i7-8750H CPU @ 2.20GHz 2.21 GHz • RAM: 8.00 GB DDR4 Memory • GPU: NVIDIA GeForce 1650 Software version (including tools and standard) • Operating System: Ubuntu • Python 3.10.4 • IPFS 0.5.0 • Visual Studio Code • Storage: 4150 GB • 1 GbE Dara Rate per Port Application • Covid-19 patient monitoring system Nodes • 4 Implementation Parameters Block size Block hash size • 6 MB • 256 Bit Table 2 Details of Hardware and Software used 5.1. Experimental dataset The proposed CovMedCare was tested using a Covid-19 dataset [28]. The ranges of patients' vitals and comorbidities and other symptoms were validated by a healthcare professional from the Moti Lal Nehru Page 10/38 Medical College, Prayagraj. Vital signs were inserted into the dataset to create a huge dataset for patient severity prediction. The data of each patient is collected at intervals of 5 minutes for a period of 3 and half hours. It comprises two types of data, i.e., measured sensor data that includes 40000 entries of five vital signs of patients assigned a numeric value and fifteen Previous Health Records assigned a binary number representing presence (i.e., 1) or absence (i.e., 0) of the disease. The feature descriptions of the dataset are tabulated in Table 3. 5.1. Experimental dataset Table 3 Feature Description of Covid-19 datasets Monitored Sensor Data Feature Label Name of the Feature Feature Description Range of Data Data Type M1 Body Temperature (BT) Patient's Body Temperature in F 97–103 F Numeric M2 Respiration Rate (RR) Patient’s respiration rate 8–40 Breaths per minute Numeric M3 Blood Pressure (BP) Diastolic Blood Pressure in mmHg < 120 mmHG Numeric M4 Oxygen Saturation (OS) Level of Blood Saturation 65–100% Numeric M5 Heart Rate (HR) Heart Rate Range in bpm 50–140 beats per minute Numeric Table 3 Feature Description of Covid-19 datasets Feature Description of Covid-19 datasets Page 11/38 Page 11/38 Previous Health Records Feature Label Name of Feature Feature Description (0 is Absent, 1- Present) Range of Data Data Type H1 Patient's ID (PID) Patient's unique Identity Number 1-5000 Numeric H2 Age (AGE) Patient’s Age 4-100 Numeric H3 Gender (GEN) Patient’s Gender (0-Male, 1- Female) 0, 1 Binary H4 Heart Diseases (HD) Medical history of Heart Disease 0, 1 Binary H5 Asthma (AST) Medical history of asthma 0, 1 Binary H6 Diabetes (SUG) Chronic blood sugar level 0, 1 Binary H7 Bronchitis (BRO) Medical history of Bronchitis 0, 1 Binary H8 Chronic Kidney Disease (CKD) Medical history of Chronic Kidney Disease 0, 1 Binary H9 Lung Cancer (LC) Lung Cancer history 0, 1 Binary H10 Pneumonia (PNE) Medical history of Pneumonia 0, 1 Binary H11 Pregnant Women (PW) Pregnancy status 0, 1 Binary H12 Emphysema (EMP) Medical history of Emphysema 0, 1 Binary H13 Taste and Smell Disorders (TSD) Medical history of Taste and Smell Disorder 0, 1 Binary H14 Viral Infection in Throat (VIT) Medical history of viral throat infection 0, 1 Binary H15 Neurological Disorder (ND) Medical history of Neurological Disorder 0, 1 Binary 5.2 Blockchain performance We have utilized python to develop the customized blockchain that enables to use various consensus algorithms and mining approaches. Different platforms exist to develop blockchain applications, such as Hyperledger fabric, Ethereum, Coinbase, etc. All these platforms have their security and consensus protocols. Python offers flexibility to the developers to design their consensus and mining protocols to be used for various applications. Each miner node in the blockchain network, has a unique address. The blockchain network is a decentralized swarm-based Peer-to-Peer network, i.e., the network does not have a single server that it communicates to, but it communicates with all nodes that are parts of the same network. The proposed blockchain-based P2P swarm network is built over the IPFS. IPFS provides various functionalities like decentralization, content-addressable data sharing and Peer-to-Peer network. Page 12/38 Page 12/38 IPFS makes the network more scalable as compared to other state-of-the-art approaches. Using IPFS as off-chain storage leads to the development of lightweight blockchain nodes which are highly scalable. IPFS makes the network more scalable as compared to other state-of-the-art approaches. Using IPFS as off-chain storage leads to the development of lightweight blockchain nodes which are highly scalable. In our customized blockchain, we have used the Proof of Stake (PoS) consensus algorithm. It chooses validators according to the stakes miners have in the network. This algorithm has numerous advantages over the traditional proof-of-work algorithm, such as less power consumption. Energy-efficient PoS is more beneficial for stakeholders. As PoS requires negligible computations, it is an environment-friendly consensus protocol. It does not require special hardware to execute. For experimental purpose, we have developed three blockchains with 40000 transactions. The details of the blockchain are tabulated in Table 4. Table 4 Details of the blockchain Blockchain No. of Transactions per Blockchain No. of Blocks in the Blockchain Blockchain 1 1000 40 Blockchain 2 2000 20 Blockchain 3 4000 10 Figure: Impact of number of records fetched on performance. Figure: Impact of number of records fetched on performance. Figure: Impact of number of records fetched on performance. Figure 7, Fig. 8 and Fig. 9 shows comparison between the minimum, maximum and average read latency of the three blockchains, respectively. As seen from the Figs. 7–9, blockchain 1 outperforms the other two blockchains. It takes the lowest time for querying the blockchain. Blockchain 3 takes the highest time for querying the blockchain. 5.2 Blockchain performance It may be concluded that performance of a blockchain is inversely proportional to the number of transactions per block in the blockchain. 5.3. Machine Learning performance metrics This section discusses the various validation metrics utilized to evaluate the classification models. Various validation parameters, including accuracy, F1-score, recall, and precision, are computed to determine the efficiency of the standard classifiers for patient disease severity prediction. The algorithms are also assessed based on training and testing time. Figure 10 shows the number of samples in each class. As it can be concluded the dataset is a balanced dataset. The validation results for the proposed CovMedCare model using Random Forest (RF) classifier algorithm along with the four other standard classification models are tabulated in Table 5, which enlists the accuracy, recall, precision, and F1-Score of the classification models. After combining and preprocessing the dataset, we utilized the RF classification technique and other standard classification models. The RF classification model acquires the highest accuracy of 99%, whereas the LR classification model obtains the lowest of 92%. As seen from the table, RF algorithm performs better in terms of all the Page 13/38 Page 13/38 performance parameters i.e., precision, recall, f1-score, training accuracy and testing accuracy with a value of 0.99 when compared with other classification models. Table 5 Validation results of different standard classification models Validation Metrics \Models Logistic Regression Support Vector Machine K-Nearest Neighbor Random Forest Naive Bayes Precision 0.77 0.87 0.873 0.99 0.77 Recall 0.82 0.84 0.886 0.99 0.82 F1-Score 0.79 0.86 0.88 0.99 0.79 Training accuracy 0.91 0.91 0.95 0.99 0.87 Testing Accuracy 0.92 0.92 0.93 0.99 0.87 Figure 12 represents the training and testing time of various machine learning algorithms in milliseconds. Support vector machine takes the highest time for training and prediction. Random forest takes time for training but testing time is less. Hence it the most suitable for this application. Figure 13–17 shows the confusion matrix of various machine learning algorithms. Figure 18–22 show the learning curves of the machine learning algorithms. Figure 18–22 show the learning curves of the machine learning algorithms. A comparison of our work with other similar work has been shown in Table 4. As observed, our proposed work is scalable, customizable and it is a confluence of the three technologies blockchain, internet of things and machine learning to monitor Covid-19 inpatient hospitalization. 5.3. Machine Learning performance metrics Page 14/38 Page 14/38 Page 14/38 Table 4 Comparision of CovMedCare with other similar works [17] [18] [19] [20] [21] Our Work Blockchain Platform Ethereum Not Mentioned Ethereum Ethereum Hyperledger Own blockchain using Python IoT Data Yes No No Yes No Yes Off-Chain Data Storage IPFS Not used IPFS Not used Not used IPFS Consensus Algorithm Proof of Authority Not mentioned Proof of Work Not mentioned Hybrid consensus mechanism Proof-of- Stake Objective Continuous monitoring of diabetes patients Covid-19 detection. Covid-19 detection. Remote monitoring of generic patients EMR storage. Continuous monitoring of Covid-19 inpatients Machine Learning Algorithms used None Federated Learning LR, KNN, SVM, RF, ANN None None LR, KNN, SVM, RF, ANN Integration of PMR and Sensor data No No No No No Yes Integration of IoT, Blockchain and Machine Learning No No No No No Yes Scalable Yes No Yes No No Yes References 1. Rabaan, A.A., Al-Ahmed, S.H., Haque, S., Sah, R., Tiwari, R., Malik, Y.S., …, Rodriguez-Morales, A.J.: SARS-CoV-2, SARS-CoV, and MERS-CoV: A comparative overview. Infezioni in Medicina. (2020) 2. https://www.worldometers.info/coronavirus 2. https://www.worldometers.info/coronavirus 3. Hassen, H.B., Ayari, N., Hamdi, B.: A home hospitalization system based on the Internet of Things, fog computing and cloud computing. Inf. Med Unlocked. 20, 100368 (2020) 4. Taiwo, O., Ezugwu, A.E.: Smart healthcare support for remote patient monitoring during COVID-19 quarantine. Inf. Med Unlocked. 20, 100428 (2020) 5. Binkhonain, M., Zhao, L.: A review of machine learning algorithms for identification and classification of non-functional requirements. Expert Syst. Appl. X. 1, 1–13 (2019) 6. Uddin, S., Khan, A., Hossain, M.E., Moni, M.A.: Comparing different supervised machine learning algorithms for disease prediction. BMC Med. Inform. Decis. Mak. 19(1), 1–16 (2019) 6. Uddin, S., Khan, A., Hossain, M.E., Moni, M.A.: Comparing different supervised machine learning algorithms for disease prediction. BMC Med. Inform. Decis. Mak. 19(1), 1–16 (2019) 7. Abbas, S.A., Rehman, A.U., Majeed, F., Majid, A., Malik, M.S.A., Kazmi, Z.H., Zafar, S.: Performance analysis of classification algorithms on birth dataset. IEEE Access. 8, 102146–102154 (2020) 7. Abbas, S.A., Rehman, A.U., Majeed, F., Majid, A., Malik, M.S.A., Kazmi, Z.H., Zafar, S.: Performance analysis of classification algorithms on birth dataset. IEEE Access. 8, 102146–102154 (2020) 8. Sen, P.C., Hajra, M., Ghosh, M.: Supervised classification algorithms in machine learning: a survey and review. In: Emerging Technology in Modelling and Graphics, pp. 99–111. Springer (2020) 8. Sen, P.C., Hajra, M., Ghosh, M.: Supervised classification algorithms in machine learning: a survey and review. In: Emerging Technology in Modelling and Graphics, pp. 99–111. Springer (2020) 9. Abdar, M., Zomorodi-Moghadam, M., Das, R., Ting, I.-H.: Performance analysis of classification algorithms on early detection of liver disease. Expert Syst. Appl. 67, 239–251 (2017) 9. Abdar, M., Zomorodi-Moghadam, M., Das, R., Ting, I.-H.: Performance analysis of classification algorithms on early detection of liver disease. Expert Syst. Appl. 67, 239–251 (2017) 10. Dogan, N., Tanrikulu, Z.: A comparative analysis of classification algorithms in data mining for accuracy, speed and robustness. Inf. Technol. Manag. 14(2), 105–124 (2013) 10. Dogan, N., Tanrikulu, Z.: A comparative analysis of classification algorithms in data mining for accuracy, speed and robustness. Inf. Technol. Manag. 14(2), 105–124 (2013) 11. AlJame, M., Ahmad, I., Imtiaz, A., Mohammed, A.: Ensemble learning model for diagnosing COVID-19 from routine blood tests. Inf. Med Unlocked. 21, 100449 (2020) 11. 6. Conclusion This paper presents a smart decentralized healthcare monitoring and prediction system for covid-19 patients called CovMedCare using blockchain and ensemble Random Forest model. WBAN network is used to collect data from sensors. Blockchain network is used for secure data storage. Lastly, the Random Forest classifier is utilized for training the CovMedCare model for disease severity prediction. It is compared with other traditional classifiers using several performance metrics to demonstrate its efficiency. The RF classifier attained 99% accuracy, much higher than the traditional classifiers. This system is highly useful for medical professionals, patients, and their relatives. In the future, we intend to explore deep learning techniques for the prediction of disease severity. Page 15/38 Page 15/38 References AlJame, M., Ahmad, I., Imtiaz, A., Mohammed, A.: Ensemble learning model for diagnosing COVID-19 from routine blood tests. Inf. Med Unlocked. 21, 100449 (2020) 12. Muhammad, L., Algehyne, E.A., Usman, S.S., Ahmad, A., Chakraborty, C., Mohammed, I.: Supervised machine learning models for prediction of COVID-19 infection using epidemiology dataset. SN Comput. Sci. 2(1), 1–13 (2021) 13. Mistry, I., Tanwar, S., Tyagi, S., Kumar, N.: "Blockchain for 5g enabled iot for industrial automation: A systematic review, solutions, and challenges," Mechanical Systems and Signal Processing, vol. 135, p. 106382, (2020) 14. Kabra, N., Bhattacharya, P., Tanwar, S., Tyagi, S.: Mudrachain: Blockchain-based framework for automated cheque clearance in financial institutions. Future Generation Computer Systems. 102, 574–587 (2020) 15. Vora, J., Nayyar, A., Tanwar, S., Tyagi, S., Kumar, N., Obaidat, M.S., Rodrigues, J.J.: "Bheem: A blockchain-based framework for securing electronic health records," in 2018 IEEE Globecom Workshops (GC Wkshps), pp. 1–6, IEEE, (2018) 16. Hathaliya, J.J., Tanwar, S., Tyagi, S., Kumar, N.: Securing electronics healthcare records in healthcare 4.0: a biometric-based approach. Comput. Electr. Eng. 76, 398–410 (2019) Page 16/38 Page 16/38 17. Azbeg, K., Ouchetto, O., Andaloussi, S.J.: BlockMedCare: A healthcare system based on IoT, Blockchain and IPFS for data management security. Egypt. Inf. J. (2022) 18. Kumar, R., Khan, A.A., Kumar, J., Golilarz, N.A., Zhang, S., Ting, Y., Zheng, C., Wang, W.: Blockchain- federated-learning and deep learning models for covid-19 detection using ct imaging. IEEE Sens. J. 21(14), 16301–16314 (2021) 19. Mistry, C., Thakker, U., Gupta, R., Obaidat, M.S., Tanwar, S., Kumar, N., Rodrigues, J.J.: June. MedBlock: An AI-enabled and blockchain-driven medical healthcare system for COVID-19. In ICC 2021-IEEE International Conference on Communications (pp. 1–6). IEEE. (2021) 20. Griggs, K.N., Ossipova, O., Kohlios, C.P., Baccarini, A.N., Howson, E.A., Hayajneh, T.: Healthcare blockchain system using smart contracts for secure automated remote patient monitoring. J. Med. Syst. 42(7), 1–7 (2018) 21. Fan, K., Wang, S., Ren, Y., Li, H., Yang, Y.: Medblock: Efficient and secure medical data sharing via blockchain. J. Med. Syst. 42(8), 1–11 (2018) 22. Dubovitskaya, A., Xu, Z., Ryu, S., Schumacher, M., Wang, F.: Secure and trustable electronic medical records sharing using blockchain. (2017). arXiv:1709.06528, 22. Dubovitskaya, A., Xu, Z., Ryu, S., Schumacher, M., Wang, F.: Secure and trustable electronic medical records sharing using blockchain. (2017). arXiv:1709.06528, 23. References Ekblaw, A., Azaria, A., Halamka, J.D., Lippman, A.: A case study for blockchain in healthcare: “medrec” prototype for electronic health records and medical research data. In: Proceedings of IEEE Open & Big Data Conference, Vol. 13, p. 13, (2016) 23. Ekblaw, A., Azaria, A., Halamka, J.D., Lippman, A.: A case study for blockchain in healthcare: “medrec” prototype for electronic health records and medical research data. In: Proceedings of IEEE Open & Big Data Conference, Vol. 13, p. 13, (2016) 24. Unleashed, I.B.: Blockchain is good for your health, and your business. (2017). https://www.ibm.com/blogs/blockchain/2017/12/ blockchain-good-health-business/, 24. Unleashed, I.B.: Blockchain is good for your health, and your business. (2017). https://www.ibm.com/blogs/blockchain/2017/12/ blockchain-good-health-business/, 25. Yue, X., Wang, H., Jin, D., Li, M., Jiang, W.: Healthcare data gateways: found healthcare intelligence on blockchain with novel privacy risk control. J. Med. Syst. 40(10), 218 (2016) 25. Yue, X., Wang, H., Jin, D., Li, M., Jiang, W.: Healthcare data gateways: found healthcare intelligence on blockchain with novel privacy risk control. J. Med. Syst. 40(10), 218 (2016) 26. Noah, B., Keller, M.S., Mosadeghi, S., Stein, L., Johl, S., Delshad, S., Tashjian, V.C., Lew, D., Kwan, J.T., Jusufagic, A.: Impact of remote patient monitoring on clinical outcomes: an updated meta-analysis of randomized controlled trials. npj Digit. Med. 1(1), 2 (2018) 26. Noah, B., Keller, M.S., Mosadeghi, S., Stein, L., Johl, S., Delshad, S., Tashjian, V.C., Lew, D., Kwan, J.T., Jusufagic, A.: Impact of remote patient monitoring on clinical outcomes: an updated meta-analysis of randomized controlled trials. npj Digit. Med. 1(1), 2 (2018) 27. Medical chain:, Medical chain whitepaper 2.1. Tech. rep. Medical chain, (2018) 27. Medical chain:, Medical chain whitepaper 2.1. Tech. rep. Medical chain, (2018) 27. Medical chain:, Medical chain whitepaper 2.1. Tech. rep. Medical chain, ( 28. link: Covid_Dataset/DATA.csv at main · rebecca-6/Covid_Dataset 28. link: Covid_Dataset/DATA.csv at main · rebecca-6/Covid_Dataset 28. link: Covid_Dataset/DATA.csv at main · rebecca-6/Covid_Dataset 29. Yuce, M.R.: Implementation of wireless body area networks for healthcare systems. Sens. Actuators: Phys. 162(1), 116–129 (2010) 29. Yuce, M.R.: Implementation of wireless body area networks for healthcare systems. Sens. Actuators: Phys. 162(1), 116–129 (2010) 30. Yuce, M.R.: Implementation of wireless body area networks for healthcare systems. Sens. Actuators A: Phys. 162(1), 116–129 (2010) Figures Figures Page 17/38 Figure 1 Page 18/38 Figure 2 Interactions between WBAN, Stakeholders and Hospitals Figure 2 Interactions between WBAN, Stakeholders and Hospitals Page 19/38 Page 19/38 Page 19/38 Page 20/38 Figure 3 General WBAN network proposed in [29,30] Figure 3 General WBAN network proposed in [29,30] Page 20/38 Figure 4 WBAN system for Multi-Patient Monitoring in a Hospital WBAN system for Multi-Patient Monitoring in a Hospital Page 21/38 Page 21/38 Figure 5 Architecture of Proposed System Architecture of Proposed System Figure 6 Structure of Transaction Page 22/38 Figure 7 Minimum Time Comparison Page 23/38 Figure 7 Minimum Time Comparison Figure 7 Minimum Time Comparison Figure 7 Minimum Time Comparison Page 23/38 Page 24/38 Figure 8 Average Time Comparison Figure 8 Average Time Comparison Figure 8 Average Time Comparison Page 24/38 Figure 9 Maximum Time Comparison Page 25/38 Figure 9 Maximum Time Comparison Figure 9 Maximum Time Comparison Figure 9 Maximum Time Comparison Page 25/38 Figure 10 Bar plot of the target class Bar plot of the target class Page 26/38 Figure 11 Comparison of various machine learning algorithms Page 27/38 Figure 12 Figure 12 Training and Testing time of various algorithm Training and Testing time of various algorithm Page 28/38 Page 28/38 Figure 13 Confusion Matrix of Logistic Regression Page 29/38 Figure 14 Confusion Matrix of Support Vector Machine Figure 14 Confusion Matrix of Support Vector Machine Confusion Matrix of Support Vector Machine Page 30/38 Page 30/38 Figure 15 Confusion Matrix of K-Nearest Neighbor Figure 15 Confusion Matrix of K-Nearest Neighbor Page 31/38 Page 31/38 Figure 16 Confusion Matrix ofNaïve Bayes Page 32/38 P 33/38 Figure 17 Confusion Matrix of Random Forest Figure 17 Confusion Matrix of Random Forest Page 33/38 Figure 18 Learning Curve of Logistic Regression Figure 18 Page 34/38 Figure 18 Learning Curve of Logistic Regression Figure 18 Figure 18 Learning Curve of Logistic Regression Learning Curve of Logistic Regression Page 34/38 Figure 19 Learning Curve of SVC Page 35/38 Figure 20 Figure 20 Page 36/38 Page 36/38 Figure 21 Learning Curve of K- Nearest Neighbor Figure 21 Learning Curve of K- Nearest Neighbor Learning Curve of K- Nearest Neighbor Page 37/38 Figure 22 Learning Curve of Random Forest Figure 22 Learning Curve of Random Forest Page 38/38 Page 38/38

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